Frederick A. O. Mendelsohn

Localization of angiotensin converting enzyme by in vitro autoradiography in the rabbit brain

The distribution of angiotensin converting enzyme was examined in the rabbit brain by in vitro autoradiography with the specific radiolabelled inhibitor 125I-351A. In the rabbit, the highest concentrations of radioligand binding were found in the choroid plexus, blood vessels, subfornical organ, vascular organ of the lamina terminalis, area postrema and inferior olive. High levels of binding were found throughout the basal ganglia, consistent with the results in all other species studied. In the midbrain the central gray and the superior colliculus displayed high levels of binding. In the medulla oblongata high levels of binding were associated with the nucleus of the solitary tract and dorsal motor nucleus of vagus, consistent with the pattern in other species. There was moderate labelling throughout both the cerebral and cerebellar cortices, which contrasts to the rat but is consistent with the situation in primates. Angiotensin converting enzyme (ACE) is more widely distributed in rabbit brain that in rat, human and Macaca fascicularis, and the results suggest ACE has a very general role in the metabolism of neuropeptides. Inhibitors of converting enzyme are very widely used in the treatment of hypertension and heart disease, and the rabbit should provide a useful model for examining the effects of these drugs in the brain.

Catecholamine uptake sites in mouse brain: distribution determined by quantitative [3H]mazindol autoradiography

Because of the importance of the mouse brain catecholamine system in the study of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and because little information is available concerning the chemical neuroanatomy of the mouse, catecholamine uptake sites were mapped in C57 black mouse brain using [3H]mazindol autoradiography. Displacement studies with known dopamine (DA) and noradrenaline (NA) uptake blockers showed that binding in the striatum was entirely to DA uptake sites, while binding in the locus coeruleus was to NA uptake sites only. By using the selective noradrenergic uptake blocker desmethylimipramine (DMI), a complete map of both DA and NA uptake sites was generated. The mesostriatal DA system was the most clearly labelled and uptake sites were seen better in striatal terminals than the substantia nigra. Within the noradrenergic system, highest binding levels were seen over the locus coeruleus, although it was unclear whether these uptake sites were on cell bodies or terminals from the lateral tegmental noradrenergic system. These maps of the catecholamine uptake system in mouse brain provide a baseline for study of newly discovered neurotoxins and ageing processes.

Evidence for plasticity of the dopaminergic system in Parkinsonism

A series of compensatory mechanisms within the dopaminergic system have been shown to maintain clinical function in the presence of dopamine loss. Experimental evidence for increased presynaptic dopamine turnover owing to increased dopamine synthesis, release, and reduced reuptake exists. Direct evidence that these mechanisms maintain extracellular dopamine levels is provided by intracerebral microdialysis techniques. Postsynaptic denervation supersensitivity clearly occurs with D2 dopamine receptors, although this is less evident with D1 receptors.Similarly, mechanisms of plasticity have been shown to be relevant in human postmortem and Positron Emission Tomographic studies of patients with Parkinsons disease. However, although presynaptic increases in dopamine turnover are well documented, postsynaptic D1 and D2 receptor changes have been more difficult to establish, mainly because of methodological difficulties. D2 but not D1, receptor increases have been documented in drug naive Parkinsonian patients with PET techniques. In transplantation of adrenal gland to striatum in animal models and patients with Parkinsonism where clinical improvement occurs, plasticity of host response may be as important as plasticity of the graft.Although some elements of the compensatory mechanism of dopamine plasticity may be deleterious, such as dyskinesias owing to dopamine receptor supersensitivity, the overall effect of delay and minimization of the clinical expression of disease is advantageous. An even greater understanding of the mechanisms involved may assist in developing future therapeutic strategies.

Distribution of Angiotensin Converting Enzyme in Sheep Hypothalamus and Medulla Oblongata Visualized by in Vitro Autoradiography

In vitro autoradiographic mapping of angiotensin converting enzyme (ACE) in sheep brain using the specific ACE inhibitor, 125I-351A, revealed very high densities of binding in large blood vessels and choroid plexus. In the a very high density of labelling occurred in the organum vasculosum of the lamina terminalis and median eminence and a high density in the subfornical organ and moderate density in supraoptic, suprachiasmatic, arcuate and paraventricular nuclei. All fiber tracts were unlabelled. In the medulla oblongata, a very high density of binding was detected in the area postrema and a high density in the nucleus of the solitary tract and dorsal motor nucleus of the vagus; a moderate density was found in the substantia gelatinosa of the spinal tract and the inferior olivary nucleus.

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine on presynaptic dopamine uptake sites in the mouse striatum.

The effects of the specific dopaminergic neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), were studied on the kinetics of [3H]mazindol binding to striatal membranes of C57 black mice. This radioligand was used to label dopamine uptake sites and when administered in vivo, MPTP caused an irreversible, non-competitive inhibition of mazindol binding, consistent with damage to dopaminergic terminals. This effect was abolished by pretreatment with pargyline, a MAOB inhibitor, suggesting that oxidation of MPTP to the pyridinium moiety, MPP+, is a necessary step for toxicity when mazindol binding is used as an end point. In keeping with these findings, pretreatment of mice with mazindol protected against the dopamine-depleting effects of MPTP in vivo. This data suggests that MPTP exerts its toxic effects via MPP+ which is concentrated intraneuronally via the dopamine uptake system. During this process the neurotoxin irreversibly inactivates the dopamine uptake sites.

Inhibition of angiotensin converting enzyme by N-terminal fragments of substance P

A range of N-terminal fragments of substance P (SP) were evaluated for inhibitory activity against angiotensin converting enzyme (ACE) from rat lung and brain (striatum). SP inhibited the enzyme from both sources in a concentration dependent manner (IC50 30 microM). The N-terminal fragments SP[1-7], SP[1-6], SP[1-4] and SP[3-4] were equipotent with SP for both sources of the enzyme. However, SP[1-3] showed a difference in its activity, being more active than SP (IC50 10 microM) in inhibiting the brain enzyme, but inactive against lung ACE. These results suggest that the inhibitory action of SP on ACE resides in the N-terminus of the peptide. The difference in reactivity towards SP[1-3] lends support to the idea that lung and brain ACE are different isozymes.

Identification and Localization of Insulin-Like Growth Factor Receptors in Human Infant Pituitary Gland—Similar Distribution to Somatotrophs

Insulin‐like growth factors(IGF) are involved in feedback regulation of growth hormone(GH) secretion from the pituitary. Though receptors for IGF‐I and IGF‐II have been identified on particulate preparations of rat pituitary, their localization and relationship to GH‐secreting acidophils has not been determined. We used quantitative in vitro autoradiography and immunocytochemistry to simultaneously determine the distribution of IGF receptors and GH‐secreting cells in human infant pituitary gland. Frozen or fixed post‐mortem human infant pituitary glands were sectioned for binding studies, or immunocytochemistry. Binding for IGF‐I and IGF‐II showed characteristic specificity for respectively Type I and Type II receptors. Binding sites were visualized throughout the pituitary gland, with similar density and distribution for IGF‐I and IGF‐II receptor sites. Receptor density was two‐fold higher in anterior than posterior pituitary, with highest density in the lateral horns of the anterior pituitary. The distribution of GH‐containing cells (acidophils) was similar to IGF receptor distribution. Increased density of IGF receptors in regions of GH‐secreting cells may point to the mechanism whereby IGF uniquely inhibits synthesis of human GH in contrast to its promotion of synthetic processes in other cells.

The Distribution of Catecholamine Uptake Sites in Mouse Brain

Uptake sites rapidly remove catecholamines from the synaptic cleft and terminate neurotransmitter action prior to recycling into storage vesicles ready for further release. These uptake sites are sodium dependent and are probably present over much of the neuron since both uptake and release of dopamine (DA) has been demonstrated in dendrites, at least in the substantia nigra. Mazindol is a potent inhibitor of catecholamine uptake and binds with a high affinity to single binding sites. Affinity for noradrenaline (NA) uptake sites is greatest (IC50 for [3H] amine uptake into synaptosomes 0.65 µM), while affinity for DA uptake sites is also high (IC50 18 µM) (Hyttel 1982). Because of the high affinity of desipramine (DMI) for noradrenergic uptake sites (IC50 0.97), but low affinity for DA uptake sites (IC50 9100) the addition of this compound may be used to distinguish between NA and DA uptake sites when using [3H] Mazindol binding. Since we and others have used [3H] Mazindol binding as a useful technique to study the effects of the neurotoxin 1-methyl-4-phenyl, 1,2,3,6 tetrahydropyridine (MPTP) on DA uptake sites in mouse brain (Donnan et al 1987, Javitch et al 1986), we have documented the regional distribution of binding to DA and NA uptake sites as a basis for further study.