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Biomedical subjects

R A Coleman

Publications and source records attributed to R A Coleman.

At least 55 records · Page 3Linked to original sources
PubMed1997

Picumeterol: dissociation of improvement in lung function and reduction of airways hyperresponsiveness in asthmatics.

AIMS: The new potent and selective beta 2-adrenoceptor agonist, GR 114297A (picumeterol) is the R-enantiomer of the racemic form, GR 63411B. Picumeterol has been shown to produce long-lasting relaxation of airways smooth muscle both in vitro and in vivo. We assessed the intrinsic activity of picumeterol by increasing intracellular levels of c-AMP and compared this with isoprenaline and salbutamol. METHODS: In human atopic asthmatics, we have investigated the duration of action and efficacy of picumeterol and GR 63411B with regard to improvement in resting lung function (i.e. FEV1) and airways responsiveness (i.e. PC20) to methacholine (MCh). The study design consists of two clinical parts each for one drug. Different asthmatics participated in the two studies, seven in the first part and eight in the second part. In human bronchial smooth muscle cells in vitro, we have investigated the intrinsic activity of picumeterol in increasing intracellular levels of cyclic AMP and compared it with isoprenaline and salbutamol. RESULTS: In vivo, both drugs caused bronchodilatation with similar potency, but, their effects were short-lasting. Despite their bronchodilator activity, neither drug improved PC20, when compared with placebo. In vitro, picumeterol was found have intrinsic activity lower than the other beta 2-adrenoceptor agonists tested. CONCLUSIONS: In the clinical studies, the bronchodilator potencies of picumeterol and GR 63411B were similar. However, both drugs were short-acting, which is at odds with their activity in vitro. Our data suggest that these compounds display dissociation between bronchodilator activity and protection against MCh-induced bronchoconstriction. These findings may be explained by low intrinsic activity and need further conformation.

Adrenergic beta-2 Receptor Agonists↗
PubMed1997

Role of IgE in hyperresponsiveness induced by passive sensitization of human airways.

Incubation of airways from nonatopic patients with serum from patients with high IgE levels confers responsiveness to "specific" (allergen) and hyperresponsiveness to "nonspecific" (histamine) stimuli. We have tested the hypothesis that the level of IgE determines the degree of specific and nonspecific responsiveness. Bronchial rings from nonatopic patients were sensitized overnight with serum containing high levels of allergen-specific IgE, or with an allergen-specific chimeric IgE (JW8) in physiologic buffer. In vitro responsiveness to allergen and histamine was evaluated and compared with non-sensitized tissues from the same patients. Responses to specific allergen were demonstrated in all tissues sensitized with atopic serum or chimeric IgE, but not in nonsensitized tissues. Allergen responses were specific, since tissues sensitized using serum containing high Dermatohagoides farinae-specific IgE only, did not respond to either horse or dog allergens. The potency and magnitude of the maximal contraction to histamine was significantly (p < 0.05) increased in tissues sensitized using atopic serum with high total IgE concentrations compared with nonsensitized preparations, but was unchanged in tissues sensitized using chimeric IgE or serum with low total IgE levels. Therefore, specific IgE determines allergen responsiveness in passively sensitized human airways, but histamine hyperresponsiveness is independent of specific IgE and appears to be related to some other factor associated with serum containing high concentrations of total IgE.

Aged↗
PubMed1997

Leptin directly alters lipid partitioning in skeletal muscle.

Leptin, an adipocyte-derived hormone that directly regulates both adiposity and energy homeostasis, decreases food intake and appears to partition metabolic fuels toward utilization and away from storage. Because skeletal muscle expresses the leptin receptor and plays a major role in determining energy metabolism, we studied leptin's effects on glucose and fatty acid (FA) metabolism in isolated mouse soleus and extensor digitorum longus (EDL) muscles. One muscle from each animal served as a basal control. The contralateral muscle was treated with insulin (10 mU/ml), leptin (0.01-10 microg/ml), or insulin plus leptin, and incorporation of [14C]glucose or [14C]oleate into CO2 and into either glycogen or triacylglycerol (TAG) was determined. Leptin increased soleus muscle FA oxidation by 42% (P < 0.001) and decreased incorporation of FA into TAG by 35% (P < 0.01) in a dose-dependent manner. In contrast, insulin decreased soleus muscle FA oxidation by 40% (P < 0.001) and increased incorporation into TAG by 70% (P < 0.001). When both hormones were present, leptin attenuated both the antioxidative and the lipogenic effects of insulin by 50%. Less pronounced hormone effects were observed in EDL muscle. Leptin did not alter insulin-stimulated muscle glucose metabolism. These data demonstrate that leptin has direct and acute effects on skeletal muscle.

Animals↗
PubMed1996

Sustained activation of a G protein-coupled receptor via "anchored" agonist binding. Molecular localization of the salmeterol exosite within the 2-adrenergic receptor.

An inherent therapeutic limitation of many G protein-coupled receptor agonists is a short duration of action due to rapid dissociation from receptors. Salmeterol is a modified beta-adrenergic receptor (betaAR) agonist that has a long duration of action at the beta2AR (but not the beta1AR) both in vitro and in vivo and that is persistent despite extensive washout of the agonist. It has been proposed that salmeterol binds not only to the active site of the beta2AR (localized to receptor transmembrane spanning domains (TMDs) 3 and 5) but also to another site (termed the "exosite") that anchors it to the receptor and provides for repetitive active-site binding events. To identify the location of this exosite, we used site-directed mutagenesis to replace beta2AR amino acids 149-173 (within TMD4) with beta1AR sequence. The resulting constructs were then expressed in COS-7 cells for radioligand binding studies. Using this approach, when this domain was replaced with the analogous beta1AR sequence, the ability of salmeterol to persist at the receptor under washout conditions was reduced by 67%. The results from more selective mutants (S-(149-166), S-(164-173), and S-(149-158)) indicated that a limited 10-amino acid region (beta2AR residues 149-158), localized at the interface of the cytoplasm and the transmembrane domain, contains a critical determinant for exosite binding. Whereas CHW cells stably expressing wild-type beta2AR displayed persistent salmeterol-promoted cAMP accumulation despite agonist washout, substitution of beta2AR residues 149-158 with beta1AR sequence resulted in a 56% attenuation of salmeterol-promoted cAMP accumulation under identical washout conditions. A reverse chimera was also studied, which consisted of a substitution of beta2AR residues 152-156 into the beta1AR. This substitution was found to confer exosite binding to the beta1AR. None of these mutations decreased the affinity of salmeterol for the receptor at the active site as assessed in competition binding studies. Anchored binding to this motif thus represents a novel mechanism by which agonists like salmeterol can repetitively activate receptors. Conceivably, with other G protein-coupled receptors that have similar motifs, anchored ligands can be designed to provide for long durations of action by this mechanism.

Adrenergic beta-Agonists↗
PubMed1996

Fatty acids and anionic phospholipids alter the palmitoyl coenzyme A kinetics of hepatic monoacylglycerol acyltransferase in Triton X-100 mixed micelles.

In order to gain a better understanding of the kinetics of activation and inhibition of hepatic monoacylglycerol acyltransferase (MGAT) (EC 2.3.1.22) by fatty acid, we examined the effect of fatty acid with respect to MGAT's long-chain acyl-CoA substrate in Triton X-100 mixed micelles. At concentrations between 2.5 and 5.3 mol %, oleic acid stimulated MGAT activity 2-fold, whereas oleic acid inhibited MGAT at concentrations higher than 7.5 mol %. The dependence on palmitoyl-CoA was highly cooperative with a Hill constant of greater than 2.4. When present at less than 3 mol%, oleic acid eliminated the lag in the dependence curve. When concentrations of oleic acid were higher than 3 mol %, Michaelis-Menton kinetics were observed with an apparent k(m) value of about 54 microM for palmitoyl-CoA but with progressively decreasing Vmax values. This effect was not observed with octanoic acid, suggesting that the medium-chain fatty acid is unable to associate stably with the mixed micelle and, thus, cannot substantially alter substrate affinity. When anionic phospholipids were tested, phosphatidic acid, lysophosphatidic acid, phosphatidylserine, and phosphatidylinositol eliminated some of the lag in activation by palmitoyl-CoA. At high molar concentrations of the anionic lipid activators, apparent k(m) values ranged from 77 microM for phosphatidic acid to 196 microM for phosphatidylinositol. Zwitterionic phospholipids had no effect, nor did the non-phospholipid activators bovine serum albumin or sn-1,2-diacylglycerol. CaCl2, but not neomycin or KC1, could overcome the inhibitory effect of oleic acid; thus, the inhibitory effect of fatty acid did not appear to occur by electrostatic interactions. These blockers did not change the effects observed with the anionic phospholipid activators or with the inhibitor, sphingosine. An altered k(m) for palmitoyl-CoA in the presence of fatty acid or anionic phospholipid suggests that both long-chain fatty acids and phospholipid cofactors may induce a conformational change in MGAT, thereby altering the enzyme's affinity for its long-chain acyl-CoA substrate. These data further support the hypothesis that the synthesis of glycerolipids via the monoacylglycerol pathway may be highly regulated via a variety of lipid second messengers such as phosphatidic acid and diacylglycerol, as well as by the influx of fatty acids derived from high-fat diets, or from the hydrolysis of adipocyte triacylglycerol during fasting or diabetes.

Acyltransferases↗
PubMed1996

Acylglycerol recycling from triacylglycerol to phospholipid, not lipase activity, is defective in neutral lipid storage disease fibroblasts.

Neutral lipid storage disease (NLSD) is an autosomal recessive disorder in which excess triacylglycerol (TG) accumulates in most cells. Although it has been hypothesized that the TG accumulation is caused by a functional defect in cytosolic lipase activity, we were able to expose TG hydrolysis in NLSD cells by using triacsin C, an inhibitor of acyl-CoA synthetase that blocks the reincorporation of hydrolyzed fatty acids into glycerolipids. Our data suggest that TG lipolysis in NLSD cells is masked by rapid TG resynthesis, occurring because released acylglycerols cannot be used for phospholipid synthesis. In uptake studies, triacsin C blocked the incorporation of [3H]glycerol into glycerolipids, incorporation of [14C]oleate into TG, but not incorporation of [14C]oleate into phospholipid. Thus, the drug inhibited both de novo synthesis of glycerolipids via the glycerol-3-phosphate pathway and the synthesis of TG from diacylglycerol. The drug did not appear to block reacylation of lysophospholipids. Triacsin C caused a loss of about 60% of the TG mass from both NLSD and oleate-loaded control cells. Rates of TG lipolysis were similar in NLSD cells and oleate-loaded control cells labeled with [6-(7-nitro-2,1,3-benzoxadiazol-4-yl)-amino]hexanoic acid or labeled with [14C]oleate or [3H]glycerol and chased in the presence of triacsin C. During a 96-h chase, [14C]oleate reincorporation into the different phospholipid species increased only in control cells. Similar results were observed when NLSD, and control cells were chased after labeling with [3H]glycerol. These data strongly suggest that normal human fibroblasts mobilize stored TG for phospholipid synthesis and that recycling to PC occurs via a TG-derived mono- or diacylglycerol intermediate. Normal recycling to phosphatidylethanolamine may primarily involve TG-derived acyl groups rather than an acylglycerol precursor. NLSD cells appear to have a block in this recycling pathway with the result that both hydrolyzed fatty acids and the acylglycerol backbone are re-esterified to form TG. Because the NLSD phenotype includes ichthyosis, fatty liver, myopathy, cardiomyopathy, and mental retardation, the recycling pathway appears to be critical for the normal function of skin, liver, muscle, heart, and the central nervous system.

Acylation↗
PubMed1996

Rat renal arcade segment expresses vasopressin-regulated water channel and vasopressin V2 receptor.

The arcades are long, branched renal tubules which connect deep and mid-cortical nephrons to cortical collecting ducts in the renal cortex. Because they are inaccessible by standard physiological techniques, their functions are poorly understood. In this paper, we demonstrate that the arcades are a site of expression of two proteins, aquaporin-2 (the vasopressin-regulated water channel) and the V2 vasopressin receptor, that are important to regulated water transport in the kidney. Using a peptide-derived polyclonal antibody to aquaporin-2, quantitative ELISA in microdissected segments showed that aquaporin-2 is highly expressed in arcades and that the expression is increased in response to restriction of fluid intake. Immunocytochemistry revealed abundant aquaporin-2 labeling of structures in the cortical labyrinth in a pattern similar to that of the Na(+)-Ca2+ exchanger and kallikrein, marker proteins expressed in arcades but not in cortical collecting ducts. RT-PCR experiments demonstrated substantial aquaporin-2 and V2 receptor mRNA in microdissected arcades. In situ hybridization, using 35S-labeled antisense cRNA probes for the V2 receptor demonstrated strong labeling of both arcades and cortical collecting ducts. Thus, these results indicate that the arcades contain the specific proteins associated with vasopressin-regulated water transport, and may be a heretofore unrecognized site of free water absorption.

Animals↗
PubMed1996

Isolated, electrically-stimulated airway preparations--their use in determining beta-adrenoceptor agonist activity.

We have assessed the suitability of electrically-stimulated superfused preparations of guinea-pig trachea, cat trachea and human bronchus for investigating the relaxant activity of the beta-adrenoceptor agonist, isoprenaline. Superfused strips of guinea-pig trachea, cat trachea and human bronchus all contracted in response to electrical stimulation. Guinea-pig trachea possesses inherent tone, and in its presence, electrical stimulation caused biphasic responses, comprising a modest, transient contraction, usually followed by a longer lasting relaxation. Human bronchus also possesses inherent tone, but responses were variable, generally monophasic, comprising a transient contraction of variable magnitude, but a longer lasting relaxation was occasionally observed after the transient contraction. Cat trachea possesses no inherent tone, and electrical stimulation of this preparation caused simple monophasic contractile responses. On guinea-pig trachea, addition of indomethacin (2.8 microM) abolished the inherent tone, and under these conditions, electrical stimulation caused monophasic contractile responses similar to those observed in cat trachea. On human bronchus, however, indomethacin enhanced inherent tone, which tended to uncover or exaggerate any relaxant component in the responses to electrical stimulation. The 5-lipoxygenase inhibitor, zileuton (10 microM), reduced, but did not abolish, the tone and converted the electrically-induced response to a monophasic contraction. In all preparations in which inherent tone was low or absent, whether naturally (cat trachea) or through pharmacological intervention (guinea-pig trachea with indomethacin, or human bronchus with zileuton), isoprenaline (1-100 nM) inhibited electrically-stimulated contractions in a concentration-related fashion (EC50s: 9-100 nM). In preparations exhibiting inherent tone (guinea-pig trachea with indomethacin or human bronchus with or without indomethacin), this tone was inhibited by isoprenaline. This relaxant activity, on guinea-pig trachea at least, was concentration-related (EC50: 5.4 nM). Such isoprenaline-induced relaxations complicated the analysis of inhibitory effects against electrically-induced contractions. Thus, in such experiments, only at higher concentrations did isoprenaline reliably inhibit these contractions (EC50: 23-119 nM), lower concentrations of isoprenaline often resulting in an apparent enhancement. The enhancement was probably artefactual, resulting from the fact that the electrically-induced contractions originated from a lower baseline. These data suggest that electrically-stimulated airway preparations are suitable for evaluating the relaxant activity of beta-adrenoceptor agonists, but the relaxant potency should be assessed in preparations lacking inherent tone, such as cat trachea, guinea-pig trachea in the presence of cyclo-oxygenase inhibition, or human bronchus in the presence of 5-lipoxygenase inhibition.

Adrenergic beta-2 Receptor Agonists↗
PubMed1996

Immunolocalization of the secretory isoform of Na-K-Cl cotransporter in rat renal intercalated cells.

Two bumetanide-sensitive ion cotransporters that carry Na+, K+, and Cl- in a coupled fashion have been identified. One type, the "absorptive" isoform, carries these ions across the apical plasma membrane of the thick ascending limb of Henle's loop. Another isoform, the "secretory" cotransporter, has been identified in a number of epithelial tissues by physiological means, but its sites of expression in the kidney have not been fully characterized. Complementary DNA believed to code for the secretory isoform (called "BSC2" or "NKCC1") have recently been cloned. This study used a specific affinity-purified antipeptide antibody to this protein for immunolocalization in the rat kidney. Immunoblot studies using this antibody show abundant immunoreactivity against bands of 140-190 and 120 kd in the parotid gland, colon, and stomach, sites where the secretory form of the cotransporter has been identified by physiological techniques. This distribution supports the hypothesis that this isoform represents the secretory form of the cotransporter. Studies in the kidney revealed that the same bands are associated with membrane fractions chiefly in the outer medulla. Immunolocalizations show that immunoreactivity is selectively and intensely localized to the basolateral plasma membrane of a subfraction of outer medullary collecting duct cells. An independently produced monoclonal antibody (T4) specific for Na-K-Cl cotransporter displays the same localization. Dual localizations of cotransporter antibody with respect to antibody specific for principal cells (aquaporin-2) and intercalated cells (band 3 and H(+)-ATPase) show that cotransporter immunoreactivity is localized to alpha-intercalated cells of the outer medullary collecting duct in the rat. This distinctive localization suggests that the secretory form of the cotransporter may play a role in renal NH4+ and/or acid secretion by this cell type.

Amino Acid Sequence↗
PubMed1996

Exosites: their current status, and their relevance to the duration of action of long-acting beta 2-adrenoceptor agonists.

The four beta 2-adrenoceptor agonists, clenbuterol, bambuterol, formoterol and salmeterol, are all long-acting bronchodilators, that have distinct mechanisms for their extended durations of action. Various theories have been put forward in an attempt to explain these mechanisms. In this respect, there is strong evidence for the existence of specific additional binding sites (exosites) for salmeterol and related compounds, and that exosites exist on non-ligand recognition regions of the beta 2-adrenoceptor protein. Here, Robert Coleman and colleagues compare and contrast the profiles of action of these long-acting beta 2-adrenoceptor agonists, particularly as they relate to the role of exosites.

Adrenergic alpha-Agonists↗
PubMed1995

Sphingosine inhibits rat hepatic monoacylglycerol acyltransferase in Triton X-100 mixed micelles and isolated hepatocytes.

Hepatic monoacylglycerol acyltransferase (MGAT), a developmentally-regulated microsomal activity that catalyzes the synthesis of sn-1,2-diacylglycerol, is regulated by anionic phospholipids and sn-1,2-diacylglycerol in Triton X-100 mixed micelles. Spingomyelin stimulated MGAT activity, whereas sphingosine, sphinganine, phytosphingosine, and stearylamine were inhibitors (IC50 of 9, 5.5, 5, and 6 mol %, respectively). Since ceramide and octylamine had relatively little effect, inhibition appears to require a free amino group and a long-chain hydrocarbon. Inhibition by sphingosine was competitive with respect to phosphatidic acid, phosphatidylinositol, or phosphatidylserine, suggesting that anionic phospholipids may activate MGAT at a specific site that is competitively blocked by sphingolipids. Both sphingosine and sphinganine inhibited MGAT activity in cultured hepatocytes from 10-day-old rats in a dose-dependent manner. Stimulation of MGAT activity by diacylglycerol was specific for sn-1,2-stereoisomers that contained two long fatty acyl chains. The diacylglycerol analogs phorbol 12-myristyl 13-acetate and ceramide had no effect. The highly cooperative activation of MGAT by sn-1,2-diacylglycerol was also inhibited by sphingosine. It is unlikely that activation of MGAT by low molar concentrations of anionic phospholipids is solely due to electrostatic interactions between the enzyme and negatively charged lipids because high ionic strength, neomycin, and Ca2+ had similar effects on enzyme activity irrespective of the presence or absence of phosphatidic acid. These data suggest that MGAT activity may be regulated physiologically by specific intermediates of glycerolipid metabolism and that, in neonatal rat liver, signal transduction may be linked to the synthesis of complex lipids via the monoacylglycerol pathway.

Acyltransferases↗
PubMed1995

Dimerization of the TATA binding protein.

The TATA binding protein (TBP) is a central component of all eukaryotic transcription machineries. The recruitment of TBP to the promoter is slow and possibly rate limiting in transcription complex assembly. In an effort to understand the nature of this potential rate-limiting step, we have investigated the physical state of TBP prior to DNA binding. By chemical cross-linking, gel filtration chromatography, and protein affinity chromatography, we find that the conserved carboxyl-terminal DNA binding domain of human TBP dimerizes when not bound to DNA. The data completely support the proposed dimeric structure of plant TBP, previously determined by x-ray crystallography. TBP dimers are quite stable, having an approximate equilibrium dissociation constant (KD) in the low nanomolar range. The dimerization interface appears to be dominated by hydrophobic forces, as predicted by the crystal structure. TBP dimers do not bind DNA, but they must dissociate into monomers before stably binding to the TATA box. Dissociation of TBP dimers appears to be relatively slow, and as such has the potential to dictate the kinetics of DNA binding.

Base Sequence↗
PubMed1995

Evidence for functional binding and stable sliding of the TATA binding protein on nonspecific DNA.

The TATA binding protein (TBP) is required at RNA polymerase I, II, and III promoters that either contain or lack a TATA box. In an effort to understand how TBP might function at such a wide variety of promoters, we have investigated the specific and nonspecific DNA binding properties of human TBP. We show that TBP has less than a 10(3)-fold preference for binding a TATA box (TATAAAAG) than for an average nonspecific site. In contrast to TBP, which binds to the minor groove of DNA, major groove binding proteins typically display binding specificities in the range of 10(6). Once TBP is bound to DNA, whether it be a TATA box or nonspecific DNA, binding is quite stable with a t1/2 of dissociation in the range of 20-60 min for a 300-base pair DNA fragment. In this binding state, TBP appears to be capable of stable one-dimensional sliding along the DNA. Sequence-specific binding can be accounted for, in part, by different rates of sliding. Additional findings demonstrate that specific and nonspecific DNA impart upon TBP an enormous and equivalent degree of thermal stability, suggesting that the TBP-DNA interface on non-specific DNA is not radically different from that on TATA. Consistent with this notion, we find that nonspecifically bound TBP is competent in establishing pol II transcription complexes on DNA. Together, these finding provide a plausible mechanistic explanation for the ability of TBP to function at TATA-containing and TATA-less promoters.

Base Sequence↗
PubMed1995

Beta 2-adrenoceptors mediate a reduction in endothelial permeability in vitro.

The permeability of bovine pulmonary artery endothelial (CPAE) monolayers to Evans blue-labelled albumin (Evans blue-albumin) has been measured in vitro. Thrombin caused a concentration-dependent increase in Evans blue-albumin clearance across endothelial monolayers. Isoprenaline inhibited thrombin-induced Evans blue-albumin clearance in a concentration-dependent manner (EC50 21 nM). This effect was mimicked by the selective beta 2-adrenoceptor agonists salbutamol (EC50 64 nM) and salmeterol (EC50 2.7 nM), but not by the selective beta 1-adrenoceptor agonist, RO-363 ((1-[3',4'-dihydroxyphenoxy]-2-hydroxy-[3",4"- dimethoxyphenethylamino]-propane)oxalate), nor by the selective beta 3-adrenoceptor agonist, CL-316,243 (disodium (R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3- benzodioxole-2,2-dicarboxylate). Isoprenaline, salbutamol and salmeterol, but not RO-363 or CL-316,243 produced small, but significant reductions in Evans blue-albumin clearance across unstimulated endothelial monolayers. Inhibition of the response to thrombin by isoprenaline was antagonised by the selective beta 2-adrenoceptor antagonist, ICI-118,551 ((erythro-DL-1(7-methylindan-4- yloxy)3-isopropylaminobutan-2-ol), pKB 8.4). Salmeterol also inhibited hydrogen peroxide-stimulated Evans blue-albumin clearance. Hence, the widely used beta 2-adrenoceptor agonists, salbutamol and salmeterol, are able to reduce endothelial permeability at nanomolar concentrations.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1995

GR159897, a potent non-peptide antagonist at tachykinin NK2 receptors.

GR159897 ((R)-1-[2-(5-fluoro-1H-indol-3-yl)ethyl]-4-methoxy-4- [(phenylsulfinyl)methyl]piperidine) is a novel, highly potent and selective non-peptide antagonist at tachykinin NK2 receptors. GR159897 inhibited binding of the NK2 receptor antagonist radioligand [3H]cyclohexylcarbonyl-Gly-Ala-(D)Trp-Phe-NMe2 ([3H]GR100679) to human ileum NK2 receptors transfected into Chinese hamster ovary cells (pKi 9.5) and to rat colon membranes (pKi 10.0). GR159897 was a competitive antagonist of contractions induced by the NK2 receptor agonist [Lys3,Gly8-R-gamma-lactam-Leu9]neurokinin A-(3-10) (GR64349) in guinea-pig trachea (pA2 8.7), and had negligible activity at human NK1 receptors transfected into Chinese hamster ovary cells (pKi 5.3), NK1 receptors in guinea-pig trachea (pKB < 5) or NK3 receptors in guinea-pig cerebral cortex (pKi < 5). In vivo, in the anaesthetised guinea-pig, GR159897 (0.12 mg.kg-1 i.v.) potently antagonised bronchoconstriction induced by GR64349 (dose-ratio = 28), with a long duration of action (3 h). GR159897 should be a useful tool for studying the physiological and pathophysiological role of tachykinin NK2 receptor activation.

Animals↗
PubMed1995

An experimental model to investigate the dynamics of wound contraction.

An excisional wound model in the Hooded Lister rat is described. The methods used to inflict, measure and characterise the process of wound contraction are comprehensively discussed. The model is shown to be reliable, reproducible and capable of detecting the effect of systemically administered prednisolone.

Animals↗
PubMed1995

Further evidence that tachykinin-induced contraction of human isolated bronchus is mediated only by NK2-receptors.

The tachykinin-receptors mediating contraction of human bronchus have been characterized using both tachykinin-receptor selective agonists and blocking drugs under conditions where tachykinin metabolism by endogenous peptidases has been controlled, and true equilibrium conditions have been established. The findings that neurokinin A (EC50 = 2 nM) is the most potent agonist, and the NK2-receptor selective agonist, GR64349, is only 3-fold weaker, whereas agonists selective for NK1-receptors, substance P methyl ester, or NK3-receptors, senktide, are inactive, suggest that this effect is mediated exclusively by NK2-receptors. This is supported by observations that GR64349 is antagonised by the selective NK2-receptor blocking drugs, MEN10207 (pA2 = 6.7), R396 (pA2 = 6.1), (+/-)SR48968 (pA2 = 8.4) and GR159897 (pA2 = 8.6), but not by the NK1-receptor blocking drug, GR82334 (pA2 < 5). In approximately half of the preparations, the peptidase inhibitors, phosphoramidon (1 microM) and bestatin (100 microM), caused a marked and well-maintained contraction (approximately 20% of neurokinin A maximum), which may indicate a role for endogenous tachykinins in the regulation of tone in this preparation. This is supported by the finding that neurokinin A-immunoreactive nerve fibres are located around intrinsic neurones of local ganglia and within the smooth muscle layer of this preparation.

Aged↗