Sites In Human Brain Research Articles

Labelling of peripheral-type benzodiazepine binding sites in human brain with [ 3H]PK 11195: Anatomical and subcellular distribution

The peripheral-type benzodiazepine binding site, erstwhile characterized in the rodent and feline brain, has now been characterized in post-mortem human brain using[ 3H]PK 11195. The kinetics and pharmacological properties of the binding of this ligand are similar to peripheral-type benzodiazepine binding sites elsewhere. The potency of RO5-4864 for this site in human brain is close to that seen in ruminate and carnivore tissues but considerably lower than in rodent tissues. The regional distribution of these binding sites would suggest a neuronal rather than a glial localization. [ 3H]PK 11195 bound in a similar fashion to slide-mounted sections of human brain, thus allowing quantitative studies of the regional distribution of peripheral-type benzodiazepine binding sites to be made. The binding sites were distributed heterogeneously, but were restricted to the grey matter. Highest densities of binding sites were found in forebrain structures. The localization was not limited to any functional system, nor did it resemble any previously described transmitter system. The similarities between peripheral-type benzodiazepine binding sites in human and in feline brain in terms of their pharmacological characteristics and their regional and subcellular distribution suggest that the cat, rather than the rat, may be the better model for studying a possible role for this site in human cerebral function.

Serum protein leakage in aged human brain and inhibition of ligand binding at alpha2‐ adrenergic and cholinergic binding sites

Serum proteins are known to extravasate into the brain parenchyma in senile and presenile dementia (Glenner: Hum. Pathol. 16:433-435, 1986; Wisniewski and Kozlowski: Ann. NY Acad. Sci. 396:119-129, 1982). We have recently demonstrated that human serum Cohn fraction IV (alpha-globulin enriched) inhibits ligand binding at putative dopamine and serotonin2 receptors labeled by [3H]spiroperidol in human brain (Andorn, Pappolla, Fox, Klemens, and Martello: Proc. Natl. Acad. Sci. USA 83:4572-4575, 1986). We now demonstrate that serum proteins can be identified in the neuropil and in neuronal cell bodies in normal aged brain, that alpha-globulin-enriched fractions inhibit ligand binding at alpha 2-adrenergic and muscarinic binding sites in human brain as well, and that serum proteins can be identified within neuronal cytoplasm and axons.

The regional distribution of dopamine and serotonin uptake and transmitter concentrations in the human brain

The regional distribution of the dopamine and serotonin uptake sites in human brain have been assessed and compared with the distribution of the transmitters and their metabolites measured in the same brains and also with a limited regional distribution of the uptake sites in rat and sheep brain. The affinity of the uptake sites for both transmitters was determined and found to be c. 0.2 ? M in all 3 species. Most dopamine uptake in all species was in caudate and putamen samples. Many regions of the human brain showed no dopamine uptake and little dopamine uptake was seen in sheep cortex or nigral preparations. Dopamine and metabolite concentrations were highest in the caudate, putamen and substantia nigra. Most serotonin uptake was seen in the hypothalamus in all 3 species; less was observed in the striatal regions; the cortical and nigral preparations of sheep brain showed little serotonin uptake though cortical preparations of rat brain had high levels of uptake. In the human brain, other regions did not show serotonin uptake. Highest concentrations of serotonin were found in the substantia nigra and medulla, intermediate concentrations in the putamen, globus pallidus, hypothalamus, olfactory tubercle and thalamus; very low concentrations of serotonin were found in other regions. The use of the human uptake site for pharmacological studies and as a marker for monoaminergic afferents in human health and disease is discussed.

125I-Bolton Hunter-labelled substance P-binding sites in human brain

role in various functions, e.g. locomotor activity, nociception, arousal and thermoregulation (Griffiths, 1985). By using h.p.1.c. to determine the relative stabilities of the TRH analogues to degradation by a proline endopeptidase and a pyroglutamyl aminopeptidase (in the two subcellular fractions), the following descending order of stability was found: RX77368. CG3509, RX74355. MK771, TRH, [3-methyl-His]TRH, CG3703. It is suggested that both binding-affinity for TRH receptors and stability to degradation may contribute to the central actions of the analogue series studied; methylation of the proline residue (in RX74355 and RX77368) reduced metabolism without seriously influencing receptor binding. Conformational analysis of these analogues has shown a preferred conformer for central TRH actions with the histidyl and prolyl residues in close proximity (Ward et al., 1985), and it may be possible to combine theoretical structural considerations with information on receptor binding and stability to design novel analogues of TRH.

‘Peripheral’ benzodiazepine binding sites in human brain and kidney: Autoradiographic studies

Benzodiazepine (BZ) recognition sites of the ‘peripheral’ type were localized autoradiographically in human postmortem brain and kidney using [ 3H]Ro 5-4864. These sites presented a relatively homogeneous distribution. Areas such as the ependyma, choroid plexus and olfactory bulb, which in the rat are very rich in these binding sites, presented densities in the human brain which were about 1 10 of those seen in the rat. Human tissues presenting gliosis, such as the hippocampi from senile dementia patients, did not show a clear increase in the number of [ 3H]Ro 5-4864 sites, in contrast with the high densities found in rat brain areas presenting neurotoxin-induced gliosis. Intermediate densities of binding were seen in a human glioblastoma tumor. The human kidney also showed lower densities of peripheral BZ binding sites, when compared to the rat kidney. These results indicate that marked species differences exist in the densities of peripheral BZ sites and that caution has to be exerted when extrapolating data from the experimental animal to human.

Studies on muscarinic binding sites in human brain identified with [3H]pirenzepine.

Pirenzepine, a potent antimuscarinic agent with apparent selectivity for a subtype (M1) of muscarinic receptors, was used in tritiated form to characterize its binding to human brain tissue. Specific [3H]pirenzepine binding showed rapid association and dissociation. From kinetic and competitive binding experiments, its KD was 5.5 nM and 9 nM, respectively. Regional distribution of [3H]pirenzepine binding determined in parallel with [3H]quinuclidinyl benzilate binding, a nonselective muscarinic antagonist, indicated a significant correlation for the maximum number of binding sites for the two radioligands in 13 brain regions, with the highest amount of binding for each in the putamen and the least in the cerebellum. Binding for [3H]pirenzepine averaged 57% of that for [3H]quinuclidinyl benzilate, with a range of 20% (cerebellum) to 77% (frontal cortex). Most antidepressants and neuroleptics tested had affinities for [3H]pirenzepine binding sites that were not significantly different from their previously reported values obtained with the use of [3H]quinuclidinyl benzilate.

Similar distribution of monoamine oxidase (MAO) and parkinsonian toxin (MPTP) binding sites in human brain.

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) causes parkinsonism in humans and other species. We found [3H] MPTP binding sites that were saturable, specific, and of high affinity. In autoradiographic studies, the highest binding densities of [3H] MPTP occurred in the hypothalamus, interpeduncular nucleus, and ependymal lining of the ventricles. High to moderate binding was seen in the dentate gyrus, caudate, putamen, substantia nigra, and cingulate cortex. The distribution of [3H] MPTP binding correlated with the distribution of [3H] pargyline binding to MAO. Human substantia nigra contains more MPTP binding sites than rat substantia nigra, and this may explain the sensitivity of humans to the neurotoxic effects of MPTP.

Characterization of [3H]ouabain binding sites in human brain, platelet, and erythrocyte.

[3H]Ouabain binding was investigated in membranes prepared from human brain, erythrocyte, and platelet. Scatchard analysis of [3H]ouabain binding to human hypothalamic membranes revealed a single class of noninteracting binding sites with an apparent affinity constant (KD) of 21 nM. Though the number of [3H]ouabain binding sites was lower in human platelets than in erythrocytes, both tissues exhibited a single class of high-affinity binding sites with an apparent KD similar to that found in human brain. Specific [3H]ouabain binding in basal ganglia tissue from patients with Huntington's disease was more than 50% lower than in tissue from age- and sex-matched controls. These results, along with previous findings in rat brain, suggest that high-affinity [3H]ouabain binding labels the neuronal form of Na, K-ATPase in human brain, and may prove useful in quantitating this enzyme in postmortem brain samples.

Dopaminergic receptor sites in human brain: positron emission tomography.

Positron emission tomography (PET) and 11C-labeled pimozide were used to study the dopaminergic (DA) receptor sites in the human striatum by comparing the latter with the cerebellum, which lacks DA receptors. Although 11C-pimozide concentration was not different in these two brain structures up to 53 minutes after IV injection (thus implying large nonspecific binding), a significant retention of radioactivity in striatum relative to cerebellum was found in controls but not in subjects pretreated with the unlabeled competitor haloperidol. This suggests that the striatal retention seen in controls was due to specific binding of 11C-pimozide to DA receptor sites, whereas prior occupation of the receptor sites by the unlabeled competitor was achieved in pretreated subjects.

Characterization and visualization of neurotensin binding to receptor sites in human brain

The binding of monoiodo [ 125I-Tyr 3 ]-neurotensin to human brain was characterized and visualized using radioreceptorassay and autoradiographic techniques. Specific binding to homogenates of human substantia nigra at 25°C was maximal at 20 min, reversible and saturable. Scatchard analysis of equilibrium data indicated the existence of two populations of binding sites with Kd values of 0.26 nM and 4.3 nM. Corresponding binding capacities were 26 and 89 fmol/mg of protein. Neurotensin analogs inhibited the binding of iodinated neurotensin with relative potencies that demonstrated the crucial role of the C-terminal hexapeptide portion of neurotensin for binding to its receptors. Autoradiography of human substantia nigra sections incubated with iodinated neurotensin revealed high levels of specific binding in the nucleus paranigralis and substantia nigra, pars compacta, and low levels in the substantia nigra, pars reticulata.

Chaeacterisation and distribution of vasoactive intestinal polypeptide binding sites in human brain

The binding of 125I-vasoactive intestinal polypeptide (VIP) to human brain membranes has been studied. The binding was saturable with a dissociation constant (KD) of 4.4 ± 2.0 nM and maximum binding value (Bmax) of 8.6 ± 6.2 fmoles mg tissue (mean ± SD, n = 4), and was displaced by unlabelled VIP with an IC 50 of 4 nM; secretin was much less potent with an IC 50 of approximately 8 μM. The regional distribution of 125I-VIP binding in human brain revealed highest binding values to be in frontal and temporal cortices with intermediate values in amygdala, caudate and cerebellum, with lowest values in hippocampus, substantia nigra and hypothalamus.

Calcium channel antagonist binding sites labeled by 3H-nimodipine in human brain.

In vitro binding of 3H-nimodipine to human brain membranes is demonstrated in this study. This binding was specific and saturable, and had an apparent affinity constant (KD) of 0.27 nM. The maximal number of binding sites for 3H-nimodipine was 5.8 pmoles/gm wet weight of human frontal cortex. The binding was shown to be dependent on calcium, with half-maximal stimulation obtained at 3 X 10(-5) M CaCl2. Other 1,4-dihyropyridine calcium antagonists were shown to be competitive antagonists of 3H-nimodipine binding. In contrast, the calcium antagonists, verapamil and diltiazem, had complex interactions with 3H-nimodipine binding. These results represent the first identification of 3H-calcium antagonist binding sites in human brain, and they confirm that various calcium antagonist drugs may differ with respect to both their potency and their molecular site of action.

Opiate receptor binding sites in human brain

Subclasses of opiate receptor binding sites in human brain membranes were investigated by means of competitive binding techniques. The experimental data were analyzed by use of computerized non-linear regression curve fitting program. μ-, δ- and χ-types of opiate binding were found in 5 different regions of the brain. A more extensive analysis of the regional distribution of subclasses of opiate binding sites was performed using a simple sequential inhibition technique. This method was shown to yield results which are comparable to those obtained by computer analysis of multiple tracer displacement curves. χ- and μ-sites represented the major component of binding in most brain areas whereas δ-sites were fewer in number. The 3 types of binding showed different distribution patterns, suggesting that they are independent from each other. The distribution pattern observed in human brain resembled the one observed in rat brain, although χ-sites appear to represent a more important, and δ-sites appear to represent a less important, fraction of binding in human as compared to rat brain.

Interactions of [ 3H]LSD with serotonin receptors in human brain

The binding of [ 3H]LSD to serotonergic sites in human brain was studied. The pharmacological profile of [ 3H]LSD binding in frontal cortex differed to that in hippocampus. Analysis of the inhibition of [ 3H]LSD binding by serotonin and spiperone was consistent with the presence of two binding sites, which differed in pharmacological specificty. The results are discussed in relation to previously published findings in experimental animals.

Depressed patients have decreased binding of tritiated imipramine to platelet serotonin "transporter".

The high-affinity tritiated (3H) imipramine binding sites are functionally (and perhaps structurally) associated with the presynaptic neuronal and platelet uptake sites for serotonin. Since there is an excellent correlation between the relative potencies of a series of antidepressants in displacing 3H-imipramine from binding sites in human brain and platelet, we have examined the binding of 3H-imipramine to platelets from 14 depressed patients and 28 age- and sex-matched controls. A highly significant decrease in the number of 3H-imipramine binding sites, with no significant change in the apparent affinity constants, was observed in platelets from the depressed patients compared with the controls. These results, coupled with previous studies showing a significant decrease in the maximal uptake of serotonin in platelets from depressed patients, suggest that an inherited or acquired deficiency of the serotonin transport protein or proteins may be involved in the pathogenesis of depression.

Binding of adrenergic ligands ([ 3H]clonidine and [ 3H]WB-4101) to multiple sites in human brain

In order to identify alpha-adrenoceptors in post-mortem human brain and to detect the possible existence of multiple types of binding sites for adrenergic [ 3H]ligands, we studied the binding of [ 3H]clonidine and [ 3H]WB-4101 to human brain cerebral cortex, hippocampus, hypothalamus and striatum. Frontal cortex revealed two binding sites for [ 3H]clonidine (with K D values of approximately 1 and 8 nM), as indicated by the biphasic Scatchard plot, the biphasic pattern of dissociation kinetics, and the biphasic inhibition by phentolamine on the binding of [ 3H]clonidine; the high-affinity site was heat-labile. Two high-affinity binding sites for [ 3H]WB-4101 were also detected in the human frontal cortex (with K D values of about 0.09 and 1.5 nM), as revealed by a biphasic pattern of dissociation. A third site with low affinity binding for [ 3H]WB-4101 was detected by the biphasic inhibition by phentolamine (as well as by WB-4101 and prazosin) on the binding of [ 3H]WB-4101. The three other brain regions revealed very similar patterns exhibited by the frontal cortex, except that the density of the [ 3H]clonidine sites (of either high or low affinity) was highest in the hypothalamus, whereas the density of [ 3H]WB-4101 sites was highest in the hippocampus.

Multiple binding sites in human brain for [ 3H]-clonidine and [ 3H]-WB-4101

Multiple binding sites in human brain for [ 3H]-clonidine and [ 3H]-WB-4101

Regional concentration of putative nicotinic-cholinergic receptor sites in human brain

Various anatomically distinct regions from postmortem human brain were analyzed for α-bungarotoxin (α-BuTX) binding and choline acetyltransferase activity. There was a heterogeneous distribution of both α-BuTX and enzyme activity in the regions studied. Although levels of α-BuTX binding were lower than those reported in other mammalian brain studies, the relative regional concentration was, in general, similar to that of other mammals. High binding activity was noted in the mammillary body, uncus, colliculi, and cortex. Lowest activity was observed in the caudate nucleus and cerebellum. Choline acetyltransferase activities in parallel assays confirmed prior data in human brain.

Benzodiazepine binding in human brain: Characterization using [ 3H] flunitrazepam

[ 3H]Flunitrazepam was used to characterize benzodiazepine binding sites in human brain. The benzodiazepine binding sites exhibited high affinity, pharmacological specificity and saturability in their binding of [ 3H]flunitrazepam. The dissociation constant (K D) of [ 3H]flunitrazepam binding was determined by three different methods and found to be in the range of 2–3 nM. The potency of several benzodiazepine analogs to inhibit specific [ 3H]-flunitrazepam binding in vitro correlated well with their potency in several in vivo human and animal tests. The density of [ 3H]-flunitrazepam binding sites was highest in the cerebrocortical and rhinencephalic areas, intermediate in the cerebellum, and lowest in the brain stem and commissural tracts.

Biochemical identification of the site of action of benzodiazepines in human brain by 3H-diazepam binding

Benzodiazepines, a group of minor tranquillizers, bind in a selective, stereo-specific fashion to a receptor site in human brain. Diazepam, their main representative, is bound with an apparent dissociation constant of 7 nM. The potency of various benzodiazepines in displacing diazepam parallels closely their therapeutic and pharmacological potencies. The density of the receptor site varies 24 fold in human brain, with the highest level in cerebral cortex and cerebellum. The apparent affinity of the receptor site, however, is remarkably similar in twenty different brain regions. The characteristics of the benzodiazepine receptor site suggest that it represents the site of therapeutic action of the benzodiazepines in human brain.