Peter C. Barber

Studies on the innervation of human renal allografts

Kidneys are innervated by a plexus of nerves around the renal artery, which is disrupted by transplantation. This is a report of a comparison of the nerves in human renal allografts and normal kidneys. There were many sympathetic ganglia around normal renal arteries but none around transplanted vessels, although equal numbers of ganglia were present in hilar tissues of normal and transplanted kidneys. An immunohistological study with an antibody to synaptophysin showed that the number of synapses in transplanted ganglia was severely reduced. Immunohistological studies on allograft kidneys using antibodies to various neurofilament proteins and the Schwann cell marker S100 showed a marked reduction in neurofilament proteins shortly after transplantation with subsequent partial recovery, and a lesser reduction in S100. Renal allografts have structurally abnormal innervation but are not completely denervated.

Autoradiographic distribution of [3H]-(S)-zacopride-labelled 5-HT3 receptors in human brain

Autoradiographic binding studies using the 5-HT3 (5-hydroxytryptamine3) receptor radioligand, [3H]-(S)-zacopride (0.5 nM), identified a heterogeneous distribution of specific binding sites (defined by granisetron, 1 microM) throughout the human brain. Highest radiolabelled 5-HT3 receptor densities were detected in discrete nuclei within the brainstem (nucleus tractus solitarius, area postrema, spinal trigeminal nerve nucleus; 50-200 fmol/mg tissue equivalent) with more modest levels of expression in the forebrain (e.g. hippocampus, nucleus accumbens, putamen, caudate; 4-17 fmol/mg tissue equivalent). Within the hippocampal formation, radiolabelled 5-HT3 receptors were differentially distributed with highest levels in the granule cell layer of the dentate gyrus. Saturation studies with [3H]-(S)-zacopride (0.05-16 nM; non-specific binding defined by granisetron, 10 microM) binding to homogenates of human putamen indicated that [3H]-(S)-zacopride (0.05-16 nM; non-specific binding defined by granisetron, 10 microM) binding to homogenates of human putamen indicated that [3H]-(S)-zacopride labelled an apparently homogenous population of binding sites (Bmax = 72 + 7 fmol mg-1 protein, pKd = 8.69 +/- 0.09, Hill coefficient = 0.99 +/- 0.06, mean +/- SEM, n = 4). The pharmacological profile of [3H]-(S)-zacopride binding to homogenates of putamen indicated the selective labelling of the human variant of the 5-HT3 receptor. The marked differences, however, in the pharmacology (e.g. low affinity for D-tubocurarine) and relative distribution (e.g. presence of 5-HT3 receptors in the human extrapyramidal system) of 5-HT3 receptors in the human forebrain when compared with other species further necessitates caution in predicting clinical responses based on data generated in animal models of disease.

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.

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.

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−ls−l 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]‐BRL46470 4.05 × 10−3±2.53 × 10−3 s−1 and 5.83 × 10−5 ± 0.91 × 10−5 s−1; [3H]‐granisetron 3.20 × 10−3± 1.70 × 10−3 s−1 and 18.58 × 10−5±4.19 × 10−5 s−1: sub‐Kd concentration; [3H]‐BRL46470 2.47 × 10−3± 1.18 × 10−3 s−1 and 9.30 × 10−5 ± 2.59 × 10−5 s−1; [3H]‐granisetron 65.91 × 10−3±22.14 × 10−3 s−1 and 49.96 × 10−5± 12.26 × 10−5 s−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‐HT3 receptor 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, rat ileum, NG108‐15 cells and HEK‐5‐HT3As cells.

Hepatic localisation of rat cysteine dioxygenase

BACKGROUND/AIM Cysteine dioxygenase (CDO, E.C. 1.13.11.20) is the main catabolic enzyme of cysteine, metabolising cysteine to cysteinesulphinic acid. CDO abnormality has been implicated in a number of neurological and non-neurological diseases, with CDO deficiency possibly leading to excitotoxic damage to the brain and impaired Phase II metabolism in the liver. METHODS Two novel anti-CDO antibodies raised against linear synthetic peptides corresponding to two distinct epitopes on the 22 kDa gene product of the CDO-I gene were used for immunohistochemistry and Western blotting. These antibodies were characterised by their ability to both block and precipitate CDO enzyme activity as well as the ability of the respective antigenic peptides to absorb the antibodies and prevent the immunodetection of CDO. RESULTS The antibodies were found to detect the presence of a 68 kDa protein, which was subsequently shown to be CDO. Distribution was found to be centrilobular and did not alter when CDO was induced with cysteine or methionine; however, the intensity of staining increased, indicating an increase in the levels of CDO in that region. CONCLUSIONS These results suggest that the 68 kDa Type II is the predominant isoform in vitro and in vivo and that its centrilobular localisation may allow CDO to initiate the production of sulphate and taurine for Phase II conjugation in the liver.

Identification of angiotensin II receptor subtypes in human brain.

The present study assessed the binding characteristics of [125I]angiotensin II to slices of human cerebellum adhered to glass slides using quantitative receptor autoradiography. Specific [125I]angiotensin II binding, defined by the inclusion of unlabelled angiotensin II (1.0 microM), was detected in the molecular layer of the cerebellum (0.09 +/- 0.02 fmol/mg tissue equivalent, mean +/- s.e.m., n = 3). The angiotensin II-2 receptor subtype selective ligand, PD123177, competed for approximately 65% of the specific binding in the molecular layer whilst the remainder of the specific binding was displaced by the angiotensin II-1 receptor subtype selective ligand, DuP753. It is concluded that angiotensin II receptor subtypes exist in human brain tissue and provide potential therapeutic sites of action.

Cysteine dioxygenase: regional expression of activity in rat brain

The levels of expression of cysteine dioxygenase (CDO) protein and activity were investigated in nine functionally distinct regions of the rat brain before and after induction with methionine by Western analysis and an activity assay. Activity expression ranged from no activity in the brain-stem to high activity expression in the olfactory bulb and basal ganglia. Upon exposure to 400 mg/l methionine for 5 days, significant induction of expression was observed in the basal ganglia, brain-stem, cerebellum, hippocampus, midbrain and olfactory bulb. Protein expression appeared to correlate with activity expression when levels before and after induction were compared. This non-uniformity of expression may reflect different physiological functions of CDO in these areas.