Characterization of RO4583298 as a novel potent, dual antagonist with in vivo activity at tachykinin NK1 and NK3 receptors.

BACKGROUND AND PURPOSE: Clinical results of osanetant and talnetant (selective-NK3 antagonists) indicate that blocking the NK3 receptor could be beneficial for the treatment of schizophrenia. The objective of this study was to characterize the in vitro and in vivo properties of a novel dual NK1/NK3 antagonist, RO4583298 (2-phenyl-N-(pyridin-3-yl)-N-methylisobutyramide derivative). EXPERIMENTAL APPROACH: RO4583298 in vitro pharmacology was investigated using radioligand binding ([³H]-SP, [³H]-osanetant, [³H]-senktide), [³H]-inositol-phosphate accumulation Schild analysis (SP- or [MePhe7]-NKB-induced) and electrophysiological studies in guinea-pig substantia nigra pars compacta (SNpc). The in vivo activity of RO4583298 was assessed using reversal of GR73632-induced foot tapping in gerbils (GFT; NK1) and senktide-induced tail whips in mice (MTW; NK3). KEY RESULTS: RO4583298 has a high-affinity for NK1 (human and gerbil) and NK3 (human, cynomolgus monkey, gerbil and guinea-pig) receptors and behaves as a pseudo-irreversible antagonist. Unusually it binds with high-affinity to mouse and rat NK3, yet with a partial non-competitive mode of antagonism. In guinea-pig SNpc, RO4583298 inhibited the senktide-induced potentiation of spontaneous activity of dopaminergic neurones with an apparent non-competitive mechanism of action. RO4583298 (p.o.) robustly blocked the GFT response, and inhibited the MTW. CONCLUSIONS AND IMPLICATIONS: RO4583298 is a high-affinity, non-competitive, long-acting in vivo NK1/NK3 antagonist; hence providing a useful in vitro and in vivo pharmacological tool to investigate the roles of NK1 and NK3 receptors in psychiatric disorders.

Importance of phenylalanine 107 in agonist recognition by the 5-hydroxytryptamine(3A) receptor.

The 5-hydroxytryptamine (5-HT)(3) receptor is a member of the ligand-gated ion channel receptor family with significant homology to the nicotinic acetylcholine, gamma-aminobutyric acid(A), and glycine receptors. In this receptor class, the agonist binding site is formed by parts of the extracellular amino-terminal region. This study examines the effects of altering phenylalanine 107 (F107) of the 5-HT(3AL) subunit, obtained from NG108-15 cells, using site-directed mutagenesis. The wild-type (WT) and mutant receptors were expressed in HEK 293 cells and characterized using both whole-cell patch-clamp and radioligand binding. The tyrosine mutant F107Y exhibits a significantly lower affinity for the agonist 5-HT (K(i) = 203 versus 15.6 nM) and an increase of similar magnitude in the EC(50) value (10.6 versus 1.2 microM) compared with WT. The activation kinetics of the maximal currents generated by 5-HT with this mutant were markedly slower than those of the WT receptor, but application of supramaximal concentrations of the agonist markedly decreased the time to half-peak. The asparagine mutant F107N displayed a significantly higher affinity for 5-HT than the WT receptor (1.62 versus 15.6 nM), which was mirrored in direction and magnitude by changes in the EC(50) value for this agonist (0.2 versus 1.2 microM). In contrast to the WT receptor, the mutant F107N was activated by acetylcholine (EC(50) = 260 microM). The response to acetylcholine was blocked by the 5-HT(3) receptor antagonist renzapride with a similar IC(50) value as that determined against currents generated by 5-HT in the WT receptor. These data suggest that F107 is an important determinant of agonist recognition at the 5-HT(3) receptor.

Studies with recombinant Saccharomyces cerevisiae CaaX prenyl protease Rce1p.

Eukaryotic proteins with carboxyl-terminal CaaX motifs undergo three post-translational processing reactions-protein prenylation, endoproteolysis, and carboxymethylation. Two genes in yeast encoding CaaX endoproteases, AFC1 and RCE1, have been identified. Rce1p is solely responsible for proteolysis of yeast Ras proteins. When proteolysis is blocked, plasma membrane localization of Ras2p is impaired. The mislocalization of undermodified Ras in the cell suggests that Rce1p is an attractive target for cancer therapeutics. Homologous expression of plasmid-encoded Saccharomyces cerevisiae RCE1 under the control of the GAL1 promoter gave a 370-fold increase in endoprotease activity over an uninduced control. Yeast Rce1p was detected by Western blotting with a yRce1p antibody or with an anti-myc antibody to Rce1p bearing a C-terminal myc-epitope. Membrane preparations were examined for their sensitivity to a variety of protease inhibitors, metal ion chelators, and heavy metals. The enzyme was sensitive to cysteine protease inhibitors, Zn(2+), and Ni(2+). The substrate selectivity of yRce1p was determined for a variety of prenylated CaaX peptides including farnesylated and geranylgeranylated forms of human Ha-Ras, Ki-Ras, N-Ras, and yeast Ras2p, a-mating factor, and Rho2p. Six site-directed mutants of conserved polar and ionic amino acids in yRce1p were prepared. Four of the mutants, H194A, E156A, C251A, and H248A, were inactive. Results from the protease inhibition studies and the site-directed mutagenesis suggest that Rce1p is a cysteine protease.

Analysis of the ligand binding site of the 5-HT3 receptor using site directed mutagenesis: importance of glutamate 106.

The 5-HT3 receptor is a ligand-gated ion channel with significant structural similarity to the nicotinic acetylcholine receptor. Several regions that form the ligand binding site in the nicotinic acetylcholine receptor are partially conserved in the 5-HT3 receptor, presumably reflecting the conserved signal transduction mechanism. Specific amino acid differences in these regions may account for their distinct ligand recognition properties. Using site-directed mutagenesis, we have replaced one of these residues, glutamate 106 (E106), with aspartate (D), asparagine (N), alanine (A) or glutamine (Q) and characterized the ligand-binding and electrophysiological properties of the mutant receptors after transient expression in HEK-293 cells. The affinity for the selective 5-HT3 receptor antagonist [3H]GR65630 was decreased 14-fold in the mutant E106D (Kd = 3.69 +/- 0.32 nM) when compared to wildtype (WT, E106) 5-HT3 receptor (0.27 +/- 0.03 nM), while the affinity for E106N was unchanged (0.42 +/- 0.07 nM, means +/- SEM, n = 3-10). Decreased affinities for both E106D and E106N were observed for the antagonists granisetron, ondansetron and renzapride and for the agonists 5-HT (130- and 30-fold) and 2-methyl-5-HT (250- and 20-fold), respectively. Both mutants still formed 5-HT-activatable ion channels, but the high Hill coefficient of the concentration effect curves in wildtype (2.0) was decreased to unity in both cases. The EC50 of 5-HT was increased seven-fold in E106N (8.7 microM) when compared to wildtype (1.2 microM), but unchanged in E106D, and the potency of the antagonist ondansetron for both mutants was decreased. E106A and E106Q expressed poorly preventing a detailed characterization. These data suggest that E106 contributes to the ligand-binding site of the 5-HT3 receptor and may form an ionic or hydrogen bond interaction with the primary ammonium group of 5-HT.

Ability of angiotensin II to modulate striatal dopamine release via the AT1 receptor in vitro and in vivo.

1. The ability of angiotensin II to modulate dopamine release from rat striatal slices in vitro and in the intact rat striatum in vivo was assessed by the microdialysis technique. 2. In slices of rat striatum, angiotensin II (0.1-1.0 microM) induced a concentration-related increase in endogenous dopamine release which was maximal (approximately 250% above basal levels) within the first 2-4 min of agonist application and subsequently declined to near basal values. The angiotensin II-induced increase in dopamine release was Ca(2+)-dependent and was completely antagonized by the selective AT1 receptor antagonist, losartan (1.0 microM). In contrast, the AT2 receptor antagonist, PD123177 (1.0 microM) failed to modify the angiotensin II-induced response. Neither antagonist alone modified basal dopamine release from striatal slices. 3. In freely moving rats, angiotensin II (1.0-10 microM; administered via the microdialysis probe) induced a concentration-related increase in extracellular levels of dopamine which was maximal (approximately 150% above basal levels) within 20-40 min of agonist application and subsequently declined. The angiotensin II (10 microM)-induced increase in extracellular levels of dopamine was completely antagonized by the AT1 receptor antagonist, losartan (0.1-1.0 microM; administered via the microdialysis probe) but not by the AT2 receptor antagonist, PD123177 (1.0 microM; administered via the microdialysis probe). Neither antagonist alone modified basal extracellular levels of dopamine. 4. Homogenate radioligand binding studies with [125I]-angiotensin II (0.1 nm) identified relatively low levels of specific binding sites in rat striatal homogenates compared to homogenates of pyriform cortex (51.3 +/- 9.2 and 651.3 +/- 55.1 fmol g-1 wet weight, respectively, mean +/- s.e.mean, n = 3; non-specific binding defined by unlabelled angiotensin II). The majority of the specific [125I]-angiotensin II (0.1 nM) binding in the striatal and pyriform cortex homogenates was sensitive to the selective AT1 receptor antagonist, losartan (1.0 microM). 5. In conclusions the present study provides direct evidence that angiotensin II acting via the AT1 receptor subtype facilitates the release of dopamine in the rat striatum in vitro and in vivo. This receptor-mediated response may account for the modulation of dopamine-mediated behavioural responses by antagonists of the AT1 receptor and inhibitors of angiotensin converting enzyme.

Ability of 5-HT4 receptor ligands to modulate rat striatal dopamine release in vitro and in vivo.

1. The ability of 5-HT4 (5-hydroxytryptamine4) receptor ligands to modify dopamine release from rat striatal slices in vitro and in the striatum of freely moving rats was assessed by the microdialysis technique. 2. The release of dopamine from slices of rat striatum continually perfused with Krebs buffer was enhanced by 5-HT4 receptor agonists; 5-HT (10 microM), 5-methoxytryptamine (5-MeOT; 10 microM), renzapride (10 microM) and (S)-zacopride (10 microM) maximally increased dopamine release by 133 +/- 5, 214 +/- 25, 232 +/- 29 and 264 +/- 69%, respectively (mean +/- s.e.mean, n = 3-8). The drug-induced responses were maximal within the first 2 min of drug application, and subsequently declined. The non-selective 5-HT3/5-HT4 receptor antagonist, SDZ205-557 (10 microM), failed to modify basal dopamine release from striatal slices but completely antagonized the (S)-zacopride (10 microM)-induced increase in dopamine release. 3. To allow faster drug application, the modulation of dopamine release from rat striatal slices in a static release preparation was also investigated. The 5-HT4 receptor agonist, renzapride (10 microM) also enhanced dopamine release in this preparation (maximal increase = 214 +/- 35%, mean +/- s.e.mean, n = 14), whilst a lower concentration of renzapride (3 microM) was less effective. The renzapride-induced response was maximal within the first 2 min of drug application, before declining. In this preparation, the stimulation of dopamine release by renzapride (10 microM), was completely antagonized by the selective 5-HT4 receptor antagonist, GR113808 (100 nM). In addition, both the Na+ channel blocker, tetrodotoxin (100 nM) and the non-selective protein kinase A inhibitor, H7 (100 nM) completely prevented the stimulation of dopamine release induced by renzapride (10 microM). 4. In vivo microdialysis studies demonstrated that the 5-HT4 receptor agonists, 5-MeOT (10 microM), renzapride (100 microM) and (S)-zacopride (100 microM) maximally elevated extracellular levels of dopamine in the striatum by 220 +/- 20, 161 +/- 10 and 189 +/- 53%, respectively (mean +/- s.e.mean, n = 5-9). A lower concentration of renzapride (10 microM) was less effective. The elevation of extracellular striatal dopamine levels induced by either renzapride (100 microM) or (S)-zacopride (100 microM) were completely antagonized by the non-selective 5-HT4 receptor antagonist, SDZ205-557 (100 microM). In addition, the elevation of extracellular levels of dopamine induced by either 5-MeOT (10 microM) or renzapride (100 microM) was completely prevented by the selective 5-HT4 receptor antagonist, GR113808 (1 microM) and the renzapride (100 microM)-induced response was also completely prevented by the non-selective protein kinase A inhibitor, H7 (1 microM). In this in vivo preparation, both GR113808 (1 microM) and H7 (1 microM), when perfused alone, reduced extracellular levels of dopamine. 5. In conclusion, the present study provides evidence that the 5-HT4 receptor facilitates rat striatal dopamine release in vitro and in vivo.

Evidence that the atypical 5-HT3 receptor ligand, [3H]-BRL46470, labels additional 5-HT3 binding sites compared to [3H]-granisetron.

1. The radioligand binding characteristics of the 3H-derivative of the novel 5-HT3 receptor antagonist BRL46470 were investigated and directly compared to the well characterized 5-HT3 receptor radioligand [3H]-granisetron, in tissue homogenates prepared from rat cerebral cortex/hippocampus, rat ileum, NG108-15 cells, HEK-5-HT3As cells and human putamen. 2. In rat cerebral cortex/hippocampus, rat ileum, NG108-15 cell and HEK-5-HT3As cell homogenates, [3H]-BRL46470 bound with high affinity (Kd (nM): 1.57 +/- 0.18, 2.49 +/- 0.30, 1.84 +/- 0.27, 3.46 +/- 0.36, respectively; mean +/- s.e. mean, n = 3-4) to an apparently homogeneous saturable population of sites (Bmax (fmol mg-1 protein): 102 +/- 16, 44 +/- 4, 968 +/- 32 and 2055 +/- 105, respectively; mean +/- s.e. mean, n = 3-4) but failed to display specific binding in human putamen homogenates. 3. In the same homogenates of rat cerebral cortex/hippocampus, rat ileum, NG108-15 cells, HEK-5-HT3As cells and human putamen as used for the [3H]-BRL46470 studies, [3H]-granisetron also bound with high affinity (Kd (nM): 1.55 +/- 0.61, 2.31 +/- 0.44, 1.89 +/- 0.36, 2.03 +/- 0.42 and 6.46 +/- 2.58 respectively; mean +/- s.e. mean, n = 3-4) to an apparently homogeneous saturable population of sites (Bmax (fmol mg-1 protein): 39 +/- 4, 20 +/- 2, 521 +/- 47, 870 +/- 69 and 18 +/- 2, respectively; mean +/- s.e. mean, n = 3-4). 4. Competition studies with a range of structurally different 5-HT3 receptor ligands indicated that in both rat cerebral cortex/hippocampus and rat ileum homogenates, [3H]-BRL46470 binding exhibited a pharmacological profile consistent with the labelling the 5-HT3 receptor with compounds competing with Hill coefficients close to unity.5 In HEK-5-HT3As cell homogenates, [3H]-BRL46470 and [3H]-granisetron associated rapidly((3.84+/-0.4)106 M-1S-1 and (5.85+/-0.2)106 M-1S-1, respectively, mean+/-s.e.mean, n=3-4) in an apparently monophasic manner. Following the establishment of equilibrium, both [3H]-BRL46470 and [3H]-granisetron at a saturating concentration ([3H]-BRL46470 approximately 16 nM; [3H]-granisetron approximately 18 nM) and at a sub-Kd concentration (approximately 1 nm for both radioligands)dissociated biphasically in HEK-5-HT3As cell homogenates (saturating concentration; [3H]-BRL464704.05 x 10-3+/-2.53 x I0-3 s-1 and 5.83 x 10-5+0.91 x I0-5 s-1; [3H]-granisetron 3.20 x 10-3+ 1.70 x IO-3 s-1 and18.58 x 10-5 +/- 4.19 x I0-5 s-1: sub-Kd concentration; [3H]-BRL46470 2.47 x 10-3+/- 1.18 x 10-3 s-1 and 9.30x 10-5+/-2.59x 10-5 S-1; [3H]-granisetron 65.91 x 10-3+/-22.14x I0-3 s-1 and 49.96x 10-5+/-12.26x 10-5s- 1 mean+/- s.e.mean, n = 4-8) when induced by a 300 fold dilution in ice-cold Tris/Krebs.6 In conclusion, the present study provides evidence that [3H]-BRL46470 specifically labels the 5-HT3receptor in rat cerebral cortex/hippocampus, rat ileum, NG108-15 cell and HEK-5-HT3As cell homogenates, but fails to label the 5-HT3 receptor expressed in human putamen. Whilst the pharmacological profile of the site labelled by [3H]-BRL46470 is directly comparable to that labelled by [3H]-granisetron, [3H]-BRL46470 consistently labelled approximately twice the density of sites compared to [3H]-granisetron in the same tissue homogenates prepared from rat cortex/hippocampus, ratileum, NG108-15 cells and HEK-5-HT3As cells.

The interaction of trichloroethanol with murine recombinant 5-HT3 receptors.

1. The effects of ethanol, chloral hydrate and trichloroethanol upon the 5-HT3 receptor have been investigated by use of electrophysiological techniques applied to recombinant 5-HT3 receptor subunits (5-HT3R-A or 5-HT3R-As) expressed in Xenopus laevis oocytes. Additionally, the influence of trichloroethanol upon the specific binding of [3H]-granisetron to membrane preparations of HEK 293 cells stably transfected with the murine 5-HT3R-As subunit and 5-HT3 receptors endogenous to NG 108-15 cell membranes was assessed. 2. Ethanol (30-300 mM), chloral hydrate (1-30 mM) and trichloroethanol (0.3-10 mM), produced a reversible, concentration-dependent, enhancement of 5-HT-mediated currents recorded from oocytes expressing either the 5-HT3R-A, or the 5-HT3R-As subunit. 3. Trichloroethanol (5 mM) produced a parallel leftward shift of the 5-HT concentration-response curve, reducing the EC50 for 5-HT from 1 +/- 0.04 microM (n = 4) to 0.5 +/- 0.01 microM (n = 4) for oocytes expressing the 5-HT3R-A. A similar shift, from 2.1 +/- 0.05 microM (n = 11) to 1.3 +/- 0.1 microM (n = 4), was observed in oocytes expressing the 5-HT3R-As subunit. Trichloroethanol (5 mM) had little or no effect upon the maximum current produced by 5-HT for either recombinant receptor. 4. Trichloroethanol (5 mM) similarly reduced the EC50 for 2-methyl-5-HT from 13 +/- 0.4 microM (n = 4) to 4.6 +/- 0.2 microM (n = 4) and from 15 +/- 2 microM (n = 4) to 5 +/- 0.4 microM (n = 4) for oocytes expressing the 5-HT3R-A and 5-HT3R-As subunit respectively. Additionally, trichloroethanol (5 mM) produced a clear enhancement of the maximal current to 2-methyl-5-HT (expressed as a percentage of the maximal current to 5-HT) from 63 +/- 0.7% (n = 4) to 101 +/- 1.6% (n = 4) and from 9 +/- 0.2% (n = 4) to 74 +/- 2% (n = 4) for oocytes expressing the 5-HT3R-A and 5-HT3R-As subunit respectively. 5. Trichloroethanol (2.5 mM) had no effect upon the Kd, or Bmax, of specific [3H]-granisetron binding to membrane homogenates of NG 108-15 cells or HEK 293 cells. Similarly, competition for [3H]-granisetron binding by the 5-HT3 receptor antagonists ondansetron and tropisetron was unaffected. However, competition for [3H]-granisetron binding by the 5-HT3 receptor agonists, 5-HT, 2-methyl-5-HT and phenylbiguanide was enhanced by trichloroethanol (2.5 mM). 6 Unexpectedly, the competition for [3H]-granisetron binding by the 5-HT3 receptor antagonist,quipazine, was enhanced by 2.5 mM trichloroethanol. Quipazine (1 nM-0.3 microM) antagonized 5-HT evoked currents recorded from oocytes expressing the 5-HT3R-A subunit with an IC50 of 18 +/- 3 nM(n = 4). Additionally, quipazine (30 nM-0.3 microM) produced a small inward current which was greatly enhanced by 5 mM trichloroethanol and antagonized by 100 nM ondansetron. Collectively, these observations suggest that quipazine may act as a partial agonist.7. The demonstration that a recombinant homo-oligomeric receptor, expressed in a foreign membrane,retains a sensitivity to alcohols, together with the sequencing of alcohol-insensitive 5-HT3 receptor subunits, may lead to a better definition of the alcohol binding site(s).

Distribution and characterization of the [3H]granisetron-labelled 5-HT3 receptor in the human forebrain.

The present study has demonstrated the distribution of [3H]granisetron-labelled 5-HT3 receptors in the human forebrain with relatively high levels of this receptor in homogenates of hippocampus, caudate nucleus, putamen, nucleus accumbens and amygdala. Lower levels of 5-HT3 receptors were found in other brain regions and the cervical vagus nerve. Pharmacological characterization of the labelled 5-HT3 receptor in human putamen homogenates identified a relatively low affinity for d-tubocurarine compared to the 5-HT3 receptor in NG108-15 neuroblastoma-glioma cell homogenates. In contrast, the affinities of 19 other 5-HT3 receptor ligands were not significantly different for the [3H]granisetron-labelled receptor in these two preparations. Such findings indicate that the human putamen 5-HT3 receptor displays a unique pharmacology which may have significance given the reported clinical potential of compounds active at this receptor when assessed in animal models of disease.

Labelling of 5-HT3 receptor recognition sites in the rat brain using the agonist radioligand [3H]meta-chlorophenylbiguanide.

The binding of the tritiated derivative of the 5-HT3 receptor agonist meta-chlorophenylbiguanide ([3H]mCPBG) to rat cortical homogenates and whole rat brain sections was assessed in an attempt to further investigate the binding of agonists to the 5-HT3 receptor. In crude homogenates of rat cortex, no reproducible specific [3H]mCPBG (1.0 nM) binding (defined by either 10 microM granisetron, 100 microM 5-HT or 100 nM 'cold' mCPBG) was detected. Using autoradiographic techniques, in rat hindbrain sections, [3H]mCPBG (1.0 nM) labelled a differential distribution of specific binding sites (defined by the inclusion of granisetron, 1.0 microM). Specific binding was only detected within the dorsal vagal complex (nucleus tractus solitarius, area postrema and dorsal motor nucleus of the vagus nerve). An identical distribution of specific binding was detected in adjacent sections incubated with the selective 5-HT3 receptor radioligand, [3H](S)-zacopride (0.5 nM; non-specific binding defined by the inclusion of granisetron, 1.0 microM). No reproducible specific [3H]mCPBG (1.0 nM) binding (defined by the inclusion of granisetron, 1.0 microM) was detected within the rat forebrain. In contrast, [3H](S)-zacopride (0.5 nM) labelled specific sites (defined by the inclusion of granisetron, 1.0 microM) in some limbic brain structures (e.g. cerebral cortex, hippocampus, amygdala). These studies indicate that [3H]mCPBG labels the 5-HT3 receptor in rat brain tissue. However, the relatively high level of non-specific binding associated with this radioligand appears to mask the specific binding in regions which do not express relatively high densities of the 5-HT3 receptor.

Reduced levels of 5-HT3 receptor recognition sites in the putamen of patients with Huntington's disease.

The present study assessed 5-HT3 receptor recognition site levels in homogenates of putamen derived from patients with clinically and neurochemically diagnosed Huntington's disease or Parkinson's disease and those from age-, sex- and post-mortem delay-matched neurologically and psychiatrically normal patients to investigate the cellular location of 5-HT3 receptors in the human putamen. Specific [3H]granisetron (0.91 nM) binding (defined by ondansetron, 10 microM) was significantly reduced in putamen homogenates from eight out of ten patients with Huntington's disease compared to similar homogenates from 'control' patients (72 +/- 6 and 39 +/- 8 fmol/g wet weight, mean +/- S.E.M., n = 10 and 8, tissue from 'control' and Huntington's disease patients, respectively, P = 0.004). In contrast, specific [3H]granisetron (1.04 nM) binding levels were similar in putamen homogenates from patients with Parkinson's disease when compared to homogenates from 'control' patients. The present results indicate that at least a proportion of the 5-HT3 receptor population in the human putamen is located on neurones that have their cell bodies within this brain region and that these receptors are not primarily located on dopamine neurone terminals in the human putamen.

A comparison of rat brain amino acid and monoamine content in diazepam withdrawal and after exposure to a phobic stimulus.

1. The content of amino acids (taurine, glycine, glutamic acid, gamma-aminobutyric acid (GABA) and aspartic acid) and monoamines (5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and noradrenaline) in homogenates of rat cortical and hippocampal tissue were measured by high performance liquid chromatography (h.p.l.c.) with fluorescent and electrochemical detection respectively, after two anxiogenic treatments: exposure to a phobic stimulus (cat odour) and withdrawal from chronic diazepam treatment. 2. In neither of the two anxiogenic situations was there a significant change in any amino acid content, in either brain area. 3. In the group withdrawn from chronic diazepam, cortical 5-HT and 5-HIAA levels and hippocampal 5-HT levels were significantly increased. Noradrenaline content was significantly decreased in the hippocampus. 4. The changes in 5-HT and 5-HIAA levels following cat odour exposure were area-specific in that they decreased in the hippocampus, but increased in the cortex. 5. Following cat odour exposure, noradrenaline levels appeared not to change in either area studied. However during exposure to cat odour, it was found that half the animals avoided the odour source and half were indifferent. The animals showing marked avoidance had significantly higher cortical noradrenaline content and this was significantly different from control, whereas hippocampal noradrenaline levels were not dependent upon the differences in avoidance of the odour source. 6. The results show clearly different neurochemical changes in the rat following exposure to a phobic stimulus and withdrawal from diazepam. It is hoped comparative studies such as this will enable better understanding of anxiety states in the rat which could parallel the different classes of anxiety recognised in the clinic

Identification and characterisation of angiotensin II receptor subtypes in human brain.

Autoradiographic and homogenate binding studies using the radioligand, [125I]angiotensin II, identified a heterogeneous distribution of specific binding sites (defined by angiotensin II, 1.0 microM) throughout the human forebrain. Highest AT receptor densities were detected in the paraventricular nucleus, median eminence, substantia nigra, putamen and caudate nucleus (2.4, 1.2, 1.0, 0.30 and 0.24 fmol/mg tissue equivalent, respectively). The AT1 receptor antagonist, losartan (1.0 microM) competed for the majority of the specific binding. [125I]Angiotensin II-specific binding (although not consistently above non-specific binding levels) was also detected in various other brain regions (e.g. amygdala, entorhinal cortex, frontal cortex, hippocampus, inferior colliculus, nucleus accumbens, parietal cortex, periaquaductal grey, superior colliculus, striate cortex, temporal cortex, thalamus). In the presence of losartan (1.0 microM), angiotensin II, saralasin, losartan and PD123177 competed for [125I]angiotensin II binding to membranes prepared from the cerebellum or substantia nigra with a rank order of affinity; angiotensin II = saralasin > PD123177 > losartan. In the presence of PD123177 (1.0 microM), the rank order of affinity of losartan and PD123177 was reversed. These studies indicate the presence of both AT1 and AT2 receptor subtypes within various regions of the human forebrain.

Production and characterization of monoclonal antibodies against non-A1c glycated hemoglobin.

Hybridomas secreting monoclonal antibodies specific for hemoglobin nonenzymatically glycated in the non-A1c position were produced by fusion of SP 2/0 myeloma cells with spleen cells from BALB/c mice immunized with nonenzymatically glycated hemoglobin prepared from human erythrocytes. Wells containing hydridomas secreting antibodies against glycohemoglobin were identified by binding, in an enzyme-linked immunosorbent assay, to purified glycated hemoglobin. The colony designated E85, which secreted antibodies discriminating between glycated versus unglycated hemoglobin, was cloned at least four times by limiting dilution and used for further study, performed with purified monoclonal antibody. Specificity of E85 was demonstrated by immunoblotting and by ELISA, wherein the monoclonal antibody reacted with glycated hemoglobin but not with hemoglobin A1c or with unglycated hemoglobin. Immunoblotting of human plasma with E85 on nitrocellulose yielded no reactive proteins, indicating site specificity for glycated epitopes residing in hemoglobin but not in other nonenzymatically glycated proteins present in plasma. E85 differs from other antibodies raised against glycated hemoglobin and other glycated proteins, which recognize hemoglobin glycated at the N terminal valine of the beta chain (HbA1c) or which recognize glycated residues only after reductive conversion to glucitollysine and which do not discriminate between different glycated proteins. Thus, this report describes the establishment of the first hybridoma secreting monoclonal antibody raised against a physiologic (unreduced) form of non-A1c glycohemoglobin, and for the glycated epitope when it resides in glycohemoglobin but not in other proteins or in hemoglobin A1c.

Purification of glycated hemoglobin free of hemoglobin A1c and its use to produce monoclonal antibodies specific for deoxyfructosyllysine [correction of deoxyfructosyllsine] residues in glycohemoglobin.

Hemoglobin nonenzymatically glycated at E-amino groups of lysine residues was purified from human erythrocyte lysates and used for immunization of BALB/c mice. Hybridomas secreting monoclonal antibodies for glycated hemoglobin were produced by fusion of mouse spleen cells with SP 2/0 myeloma cells. Immunoblotting with purified monoclonal antibody demonstrated specificity for glycated hemoglobin, with no reaction with HbAO. Glycated hemoglobin was effectively separated from other hemoglobins upon application of erythrocyte lysates to an affinity column of monoclonal antibody immobilized onto Sepharose 4B. A small fraction of purified HbA1c adsorbed to the monoclonal antibody affinity column, indicating that glycation can occur at both E-amino lysine and N-terminal valine positions in the same molecule. HbA1c did not react with the antibody after removal by immunoadsorption of molecules containing glycated lysine, confirming specificity of the antibody for deoxyfructosyl-lysine residues. The findings indicate that these monoclonal antibodies are site specific for glycated lysine amino groups in hemoglobin, and can provide rapid and efficient separation and identification of glycated hemoglobin in human erythrocyte lysates.