Localization and characterization of insulin receptors in rat brain and pituitary gland using in vitro autoradiography and computerized densitometry.

In order to identify likely sites of action in insulin in rat brain we have used the technique of in vitro autoradiography and computerized densitometry to map, characterize, and quantify its receptors in coronal and sagittal sections. A discrete and characteristic distribution of insulin receptor binding was demonstrated, with specific binding representing 92% of total binding. Displacement and specificity competition curves in olfactory bulb are typical for authentic insulin receptors, and computer analysis indicates a single class of binding site with a dissociation constant (Kd) 0.48 nM for choroid plexus and 0.44 nM for olfactory bulb external plexiform layer. Insulin receptor density is maximum in the choroid plexus, and high in the external plexiform layer of olfactory bulb. Structures of the limbic system and hypothalamus reveal moderate to high insulin receptor density, particularly the lateral septum, amygdala, subiculum, hippocampal CA1 region, mammillary body, and arcuate nucleus. Moderate insulin receptor density occurs in regions of cerebral cortex and cerebellum, and moderate to low binding occurs in discrete brainstem and midbrain structures. Insulin binding in the pituitary gland is greatest in the anterior lobe, with clear distinction from intermediate and posterior lobes. The circumventricular organs and the thalamus show low insulin binding. We conclude that insulin receptors are widespread throughout rat brain, with concentration in regions concerned with olfaction, appetite, and autonomic functions. The distribution is distinct from other neuropeptides and not related to either vascularity or cell density. A common feature of regions rich in insulin receptors is that they contain dendritic fields receiving rich synaptic input. Whether insulin plays a specific neurotransmitter or metabolic role in these sites remains unclear, but these studies have provided detailed information on potential sites of action of insulin in the brain, and will allow further studies to examine insulin receptor function in specific brain regions.

Localization and Characterization of Insulin-Like Growth Factor-I Receptors in Rat Brain and Pituitary Gland Using in vitro Autoradiography and Computerized Densitometry* A Distinct Distribution from Insulin Receptors.

Abstract In order to identify likely sites of action of insulin-like growth factor-I (IGF-I) in rat brain and pituitary gland, we have used the technique of in vitro autoradiography and computerized densitometry to map, characterize and quantify its receptors in coronal and sagittal sections. A discrete and characteristic distribution of IGF-I receptor binding was demonstrated, with specific binding representing 85% of total binding. Displacement and specificity competition curves in the olfactory bulb were typical for authentic IGF-I receptors and computer analysis indicated a single class of binding site with a dissociation constant (K(d)) of 13 nM for the choroid plexus and 5.1 nM for the olfactory cortex. IGF-I receptor density was very high in the choroid plexus in ail ventricles, but the binding in other circumventricular organs was variable, with high levels in the median eminence and the sub-fornical organ, and low levels in the organum vasculosum of the lamina terminalis. Highest binding was seen in the glomerular layer of the olfactory bulb and its associated regions the taenia tecta and anteromedial olfactory nucleus. The preoptic and septal regions showed moderate binding, while the hypothalamus, with the exception of the median eminence, showed low IGF-I binding. The pituitary gland showed very high binding density in both anterior and posterior lobes, similar to the median eminence. The thalamus had high IGF-I binding density, while it was low in basal ganglia. In the limbic system the hippocampal CA2, CAS, CA4 layers showed high binding, with little in CA1, while binding was high also in the adjacent amygdala. Binding was low in the mid and hindbrain, with the exception of the geniculate bodies, and the sensory nucleus of the trigeminal nerve. Binding was high in the primary olfactory and endopyriform cortex and in specific superficial layers. Cerebellar binding was also high in the molecular layer. Fibre layers showed no binding. Comparison with insulin receptors revealed common distribution in the choroid plexus, paraventricular nucleus, cerebellum, entorhinal cortex and amygdala, with receptor density three- to five-fold higher for IGF-I than for insulin. In contrast, in the hippocampus, insulin binding was high in the CA1 field, and low in CA2, CA3, CA4 while for IGF-I binding the converse was seen. The arcuate nucleus showed prominent insulin labelling and minimal IGF-I binding, while the median eminence showed low insulin and high IGF-I binding. The hypothalamus was more widely labelled with insulin, while in the thalamus the converse was true. Olfactory bulb laminae were labelled with differing intensity by insulin and IGF-I. In common with insulin receptor distribution was the high density of IGF-I receptors over areas of extensive dendritic arborizations which receive rich synaptic inputs, in the cerebellum, hippocampus and olfactory bulb. We conclude that IGF-I receptors are widespread throughout rat brain and pituitary gland, with concentration in regions concerned with olfaction, autonomie and sensory processing, as well as in regulation of growth hormone release, via feedback at the median eminence and pituitary gland. Many of these regions have in common high rates of metabolic and synthetic activity, which may be mediated by IGF-I and its receptors.

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.

Overlapping distributions of receptors for atrial natriuretic peptide and angiotensin II visualized by in vitro autoradiography: morphological basis of physiological antagonism.

Atrial natriuretic peptides exert actions on many key organs involved in blood pressure and water and electrolyte balance. Many of these actions result in a physiological antagonism of angiotensin. To investigate the morphological basis of this interaction, we have mapped the distribution of receptors for atrial natriuretic peptide and angiotensin II in a number of target organs, using 125I-labelled rat atrial natriuretic peptide (99-126) and 125I-labelled [Sar1,Ile8]angiotensin II. In the kidney both atrial natriuretic peptide and angiotensin II receptors were observed overlying glomeruli, vasa recta bundles (high densities), and the outer cortex (moderate density). In the other tissues studied, atrial natriuretic peptide and angiotensin II receptors were codistributed in the adrenal zona glomerulosa, cerebral circumventricular organs including the subfornical organ, organum vasculosum of the lamina terminalis and area postrema, and the external plexiform layer of the olfactory bulb. The concurrent distribution of specific receptors for both peptides at these sites provides the basis for atrial natriuretic peptide to exert a functional antagonism of the actions of angiotensin II on blood pressure and water and electrolyte homeostasis at multiple sites.

In vitro autoradiographic localization of ANP receptors in rat kidney and adrenal gland.

The radioligand 125I-ANP-(99-126) was used to map receptors for atrial natriuretic peptide (ANP) in the rat kidney and adrenal gland using in vitro autoradiography and computerized densitometry. In the kidney a very high density of receptors was found overlying glomeruli; these sites had a binding affinity constant of 0.48 +/- 0.06 X 10(-9) M-1 and a site concentration 818 +/- 108 fmol X mg protein-1. A moderate density of receptors was seen in the inner renal medulla; these sites had a binding constant of 0.9 +/- 0.2 X 10(9) M-1 and a receptor concentration of 204 +/- 44 fmol X mg protein-1, and moderate receptor density was also seen in the inner stripe of the outer medulla overlying vasa recta bundles. Diffuse low-density binding was also detected in the outer cortex. In all cases these patterns were abolished by 1 micron ANP-(101-126) or 1 microM atriopeptin III but not by a range of unrelated peptides. In the adrenal, a high density of binding was found overlying the zona glomerulosa, whereas a moderate density occurred in the zona fasciculata. Binding was not detected in the adrenal medulla. These results provide evidence for several classes of ANP receptor distribution in kidney and adrenal and suggest multiple roles of the peptide in fluid and electrolyte homeostasis.