Kazuto Shigematsu

Localization and characterization of atrial natriuretic peptide binding sites in discrete areas of rat brain and pituitary gland by quantitative autoradiography

Atrial natriuretic peptide [rat (r) ANP6-33 or ANP99-126] binding sites were localized in discrete areas of rat brain and pituitary gland using quantitative autoradiographic techniques. High numbers of rANP6-33 binding sites were concentrated in the circumventricular organs (the organon vasculosum laminae terminalis, organon subfornicalis, and area postrema) and selected hypothalamic nuclei (the nucleus supraopticus, nucleus preopticus medianus and nucleus paraventricularis). High binding was also present in the choroid plexus and the bulbi olfactorii (laminae medullaris interna). A relatively low number of rANP6-33 binding sites was observed in other olfactory, limbic and brainstem areas (the nucleus tractus solitarii, nucleus motoris dorsalis vagii and nucleus hypoglossi), the eminentia mediana and the pituitary gland (anterior and posterior lobes). High-affinity rANP6-33 binding sites were demonstrated in the organon subfornicalis and the area postrema after incubation of consecutive sections from individual rat brains with 125I-rANP6-33 in concentrations from 20 to 400 pM. rANP6-33 binding sites were concentrated in areas associated with angiotensin II and/or vasopressin, suggesting an interaction among these peptides in the central nervous system.

Microglia with an Endothelin ETB Receptor Aggregate in Rat Hippocampus CA1 Subfields Following Transient Forebrain Ischemia

Abstract: We examined endothelin (ET) receptors in the hippocampus CA1 subfields of stroke‐prone spontaneously hypertensive rats subjected to a 10‐min bilateral carotid occlusion and reperfusion. When delayed neuronal death had occurred in the pyramidal cell layer at 7 days after transient forebrain ischemia, the quantitative receptor autoradiographic method we used revealed a dramatic increase in number of 125I‐ET‐1 binding sites in the hippocampus CA1 subfields. The highest number of de novo binding sites appeared in the area corresponding anatomically to the pyramidal cell layer with neuronal death. These binding sites were characteristically the ETB receptor. The de novo 125I‐ET‐1 binding was mainly present on microglia aggregating with a high density in the damaged pyramidal cell layer. As ET‐1‐ and ET‐3‐like immunoreactivities were highly expressed within astrocytes in damaged neural tissue, the possibility that microglia with the ETB receptor are activated to participate in the pathophysiology of ischemia‐related neural tissue damage by astrocytic ET‐1 and ET‐3 produced in response to transient forebrain ischemia would have to be considered.

Immunohistochemical expression of epidermal growth factor receptors in human adrenocortical carcinoma

Epidermal growth factor (EGF) receptors were examined immunohistochemically in 64 adrenocortical carcinomas obtained at autopsy, and in 23 adrenocortical adenomas and seven pheochromocytomas obtained during surgery. In the nonneoplastic adrenal gland, EGF receptors were scattered to the zona glomerulosa, zona fasciculata, and zona reticularis. Adrenocortical carcinomas (63 of 64), more than adrenocortical adenomas (10 of 23) or pheochromocytomas (four of seven), stained positively for EGF receptors (P less than .01). The immunoreactivity was limited to the cytoplasm, cell membrane, and chromatin. When the antibody was immunoabsorbed with an excess of immunogen peptide, there was no evidence of immunostaining. The adrenocortical carcinomas could be classified into 16 cases of the well-differentiated type, 33 cases of the moderately differentiated type, and 15 cases of the poorly differentiated type. There was no relationship between histologic grading and staining intensity of the EGF receptors. On the other hand, more than 80% of the cases of adrenocortical carcinomas revealed a moderate to high intensity for EGF receptors. In 62 of the 64 patients, there was already metastases to other organs. We conclude that the expression of EGF receptors is associated with tumor growth and/or metastatic potential in adrenocortical carcinoma.

Physiological Expression of the Gene for PrP-Like Protein, PrPLP/Dpl, by Brain Endothelial Cells and its Ectopic Expression in Neurons of PrP-Deficient Mice Ataxic Due to Purkinje Cell Degeneration

Recently, a novel gene encoding a prion protein (PrP)-like glycoprotein, PrPLP/Dpl, was identified as being expressed ectopically by neurons of the ataxic PrP-deficient (PRNP(-/-)) mouse lines exhibiting Purkinje cell degeneration. In adult wild-type mice, PrPLP/Dpl mRNA was physiologically expressed at a high level by testis and heart, but was barely detectable in brain. However, transient expression of PrPLP/Dpl mRNA was detectable by Northern blotting in the brain of neonatal wild-type mice, showing maximal expression around 1 week after birth. In situ hybridization paired with immunohistochemistry using anti-factor VIII serum identified brain endothelial cells as expressing the transcripts. Moreover, in the neonatal wild-type mice, the PrPLP/Dpl mRNA colocalized with factor VIII immunoreactivities in spleen and was detectable on capillaries in lamina propria mucosa of gut. These findings suggested a role of PrPLP/Dpl in angiogenesis, in particular blood-brain barrier maturation in the central nervous system. Even in the ataxic Ngsk PRNP(-/-) mice, the physiological regulation of PrPLP/Dpl mRNA expression in brain endothelial cells was still preserved. This strongly supports the argument that the ectopic expression of PrPLP/Dpl in neurons, but not deregulation of its physiological expression in endothelial cells, is involved in the neuronal degeneration in ataxic PRNP(-/-) mice.

Endothelin-1 Binding to Endothelin Receptors in the Rat Anterior Pituitary Gland: Possible Formation of an ETA–ETB Receptor Heterodimer

Abstract1. Interaction in the recognition of endothelin-1 (ET-1), a typical bivalent ET receptor-ligand, between ETA and ETB receptors was investigated in the rat anterior pituitary gland, using our quantitative receptor autoradiographic method with tissue sections preserving the cell-membrane structure and ET receptor-related compounds.2. In saturation binding studies with increasing concentrations (0.77–200 pM) of 125I-ET-1 (nonselective bivalent radioligand), 125I-ET-1 binding to the rat anterior pituitary gland was saturable and single with a KD of 71 pM and a Bmax of 120 fmol mg−1. When 1.0 μM BQ-123 (ETA antagonist) was added to the incubation buffer, binding parameters were 8.3 pM of KD and 8.0 fmol mg−1 of Bmax, whereas 10 nM sarafotoxin S6c (ETB agonist) exerted little change in these binding parameters (KD, 72 pM; Bmax, 110 fmol mg−1).3. Competition binding studies with a fixed amount (3.8 pM) of 125I-ET-1 revealed that when 1.0 μM BQ-123 was present in the incubation buffer, ETB receptor-related compounds such as sarafotoxin S6c, ET-3, IRL1620 (ETB agonist), and BQ-788 (ETB antagonist) competitively inhibited 125I-ET-1 binding with Kis of 140, 18, 350 pM, and 14 nM, respectively, however, these compounds were not significant competitors for 125I-ET-1 binding in the case of absence of BQ-123.4. In cold-ligand saturation studies with a fixed amount (390 pM) of 125I-IRL 1620 (ETB radioligand), IRL1620 bound to a single population of the ETB receptor, and no change was observed in binding characteristics in the presence of 1.0 μM BQ-123. 125I-IRL1620 binding was competitively inhibited by ET-1 and ET-3 in the absence of BQ-123, with Kis of 20 and 29 pM, respectively, the affinities being much the same as those of 29 nM, in the presence of 1.0 μM BQ-123.5. Two nonbivalent ETA antagonists, BQ-123 and PD151242, were highly sensitive and full competitors for 125I-ET-1 binding (5.0 pM), in the presence of 10 nM sarafotoxin S6c.6. Taken together with the present finding that mRNAs encoding the rat ETA and the ETB receptors are expressed in the anterior pituitary gland, we tentatively conclude that although there are ETA and ETB receptors with a functional binding capability for ET receptor-ligands, the ETB receptor does not independently recognize ET-1 without the aid of the ETA receptor. If this thesis is tenable, then ET-1 can bridge between the two receptors to form an ETA–ETB receptor heterodimer.

Identification of a novel gene encoding a PrP-like protein expressed as chimeric transcripts fused to PrP exon 1/2 in ataxic mouse line with a disrupted PrP gene.

Abstract1. Mouse lines lacking prion protein (PrPC) have a puzzling phenotypic discrepancy. Some, but not all, developed late-onset ataxia due to Purkinje cell degeneration.2. Here, we identified aberrant mRNA species in the brain of Ngsk Prnp0/0 ataxic, but not in nonataxic Zrch Prnp0/0 mouse line. These mRNAs were chimeric between the noncoding exons 1 and 2 of the PrP gene (Prnp) and the novel sequence encoding PrP-like protein (PrPLP), a putative membrane glycoprotein with 23% identity to PrPC in the primary amino acid structure. The chimeric mRNAs were generated from the disrupted Prnp locus of Ngsk Prnp0/0 mice lacking a part of the Prnp intron 2 and its splice acceptor signal.3. In the brain of wild-type and Zrch Prnp0/0 mice, PrPLP mRNA was barely detectable. In contrast, in the brain of Ngsk Prnp0/0 mice, PrP/PrPLP chimeric mRNAs were expressed in neurons, at a particularly high level in hippocampus pyramidal cells and Purkinje cells under the control of the Prnp promoter.4. In addition to the functional loss of PrPC, ectopic PrPLP expression from the chimeric mRNAs could also be involved in the Purkinje cell degeneration in Ngsk Prnp0/0 mice.

Increased production of endothelins in the hippocampus of stroke-prone spontaneously hypertensive rats following transient forebrain ischemia: Histochemical evidence

Summary1.The effect of transient forebrain ischemia on endothelin-1 (ET-1) and endothelin-3 (ET-3) production in the hippocampus of stroke-prone spontaneously hypertensive rats (SHRSPs) was investigated using immunohistochemical techniques.2.In SHRSPs subjected to 10-min bilateral carotid occlusion, neuronal degeneration in the CA1 pyramidal cell layer of the hippocampus was detectable at 4 days and remarkable at 7 days after reperfusion.3.Coinciding with neuronal degeneration, ET-1- and ET-3-like immunoreactivities were intense in the CA1 pyramidal-cell layer, the stratum lacunosum moleculare, and the CA4 subfield of the hippocampus. Almost all of the immunostained cells had morphological characteristics of astrocytes.4.The possibility that ET has a role in the development of neuronal cell death following transient forebrain ischemia warrants further attention.

Upregulation of the Genes Encoding Lysosomal Hydrolases, a Perforin-Like Protein, and Peroxidases in the Brains of Mice Affected with an Experimental Prion Disease

ABSTRACT In an attempt to identify the molecules involved in the pathogenesis of prion diseases, we performed cDNA subtraction on the brain tissues of mice affected with an experimental prion disease and the unaffected control. The genes identified as being upregulated in the prion-affected brain tissue included those encoding a series of lysosomal hydrolases (lysozyme M and both isoforms of β-N-acetylhexosaminidase), a perforin-like protein (macrophage proliferation-specific gene-1 [MPS-1]), and an oxygen radical scavenger (peroxiredoxin). Dramatic increases in the expression level occurred at between 12 and 16 weeks after intracerebral inoculation of the prion, coinciding with the onset of spongiform degeneration. The proteinase K-resistant prion protein (PrPSc) became detectable by immunoblotting well before 12 weeks, suggesting a causal relationship between this and the gene activation. Immunohistochemistry paired with in situ hybridization on sections of the affected brain tissue revealed that expression of the peroxiredoxin gene was detectable only in astrocytes and was noted throughout the affected brain tissue. On the other hand, the genes for the lysosomal hydrolases and MPS-1 were overexpressed exclusively by microglia, which colocalized with the spongiform morphological changes. A crucial role for microglia in the spongiform degeneration by their production of neurotoxic substances, and possibly via the aberrant activation of the lysosomal system, would have to be considered.

Quantitative distribution of angiotensin II binding sites in rat brain by autoradiography

Angiotensin II binding sites were localized and quantified in individual brain nuclei from single rats by incubation of tissue sections with 1 nM 125I-[Sar1]-angiotensin II, [3H]-Ultrofilm autoradiography, computerized microdensitometry and comparison with 125I-standards. High angiotensin II binding was present in the circumventricular organs (organon vasculosum laminae terminalis, organon subfornicalis and area postrema), in selected hypothalamic nuclei (nuclei suprachiasmatis, periventricularis and paraventricularis) and in the nucleus tractus olfactorii lateralis, the nucleus preopticus medianus, the dorsal motor nucleus of the vagus and the nucleus tractus solitarii. High affinity (KA from 0.3 to 1.5 X 10(9) M-1) angiotensin II binding sites were demonstrated in the organon subfornicalis, the nucleus tractus solitarii and the area postrema after incubation of consecutive sections from single rat brains with 125I-[Sar1]-angiotensin II in concentrations from 100 pM to 5 nM. These results demonstrate and characterize brain binding sites for angiotensin II of variable high affinity binding both inside and outside the blood-brain barrier.

Prion Protein Is Necessary for Latent Learning and Long-Term Memory Retention

Abstract1. The cellular prion protein, designated PrPc, is a key molecule in the prion diseases but its physiological function remains unknown. To elucidate whether PrPc plays some role in the central nervous system, we established a line of mice in which the PrP gene had been disrupted and subsequently conducted long-term observations.2. Performance in latent learning and passive avoidance was evaluated using water-finding and step-through tests, respectively.3. PrP–/– mice showed impaired performance in the water-finding test, indicating a disturbance in latent learning, at 23 weeks of age. In the step-through test, although the PrP–/– mice showed normal learning ability and short-term memory retention, they evidenced a significant disturbance in long-term memory retention.4. These results indicate that PrPc is needed for certain types of learning and memory and that the loss of function of this protein may contribute to the pathogenesis of prion diseases.