Akihiko Himeno

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.

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.

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.

Involvement of glial endothelin/nitric oxide in delayed neuronal death of rat hippocampus after transient forebrain ischemia.

Abstract1. We examined time- and cell-type-dependent changes in endothelin (ET)-1-like immunoreactivity, ET receptors binding and nitric oxide (NO) synthase (NOS) activity in CA1 subfields of the hippocampus of stroke-prone spontaneously hypertensive rats subjected to a 10-min bilateral carotid occlusion and reperfusion.2. Microglia aggregated in accord with neuronal death and expressed a high density of ETB receptors and an intense NOS activity in the damaged CA1 pyramidal cell layer, 7 days after the induced transient forebrain ischemia. The increased NOS activity and ETB receptor in microglia disappeared 28 days after this transient ischemia.3. In contrast to microglia, astrocytes presented a moderate level of ET-1-like immunoreactivity, ETB receptors, and NOS activity in all areas of the damaged CA1 subfields, 7 days after the ischemia. These events were further enhanced 28 days after the ischemia.4. In light of these findings, the possibility that the microglial and the astrocytic ETB/NO system largely contributes to development of the neuronal death and to reconstitution of the damaged neuronal tissue, respectively, in the hippocampus subjected to a transient forebrain ischemia would have to be considered.

Characterization of nociceptin-stimulated in situ [35S]GTPγS binding in comparison with opioid agonist-stimulated ones in brain regions of the mice

We studied the characterization of receptor-mediated G protein activity by nociceptin throughout brain regions, using in situ GTPgammaS binding autoradiography. Nociceptin-stimulated GTPgammaS binding was markedly observed in amygdala, hippocampal pyramidal cell layers, temporal and entorhinal cortex, infralimbic organ, anterior olfactory nucleus, and rostral part of thalamus. These nociceptin-stimulated activities were not affected by naloxone, naltrindol nor norbinaltorphimine which completely blocked mu-, delta- or kappa-opioid agonist-stimulated GTPgammaS binding, respectively. In addition, the distribution of nociceptin-stimulated activities throughout brain regions was found to be different from such opioid receptor-mediated ones.

Specific binding sites for 125I-endothelin-1 in the porcine and human spinal cord.

Specific binding sites for 125I-endothelin-1 (125I-ET-1) in the spinal cord were investigated using quantitative receptor autoradiographic and chemical cross-linking methods. The binding sites were highly concentrated in porcine and human spinal cord areas corresponding anatomically to the dorsal horn (Rexeds laminae I-III), an area around the central canal (lamina X) and the principal part of the intermediolateral nucleus (IMLp). The localization of the binding sites differed from those of 125I-omega-conotoxin GVIA (125I-CgTx) and 125I-Bolton-Hunter substance P (125I-BH-SP), with the exception that the IMLp shared 125I-ET-1 with 125I-CgTx and 125I-BH-SP binding sites. Specific 125I-ET-1 binding sites in the areas examined were characteristically single and of high affinity. There were no differences between the potencies of unlabeled ET family peptides, ET-1, ET-2, ET-3 and sarafotoxin S6b at inhibiting 125I-ET-1 binding to the areas. Chemical cross-linking studies showed that 125I-ET-1 and 125I-ET-3 mainly bound to a protein with molecular mass of 43 kDa in the porcine and human thoracic spinal cord membranes. The present finding shows the neuronal significance of this newly discovered peptide in the spinal cord.

Quantitative receptor autoradiographic analysis for angiotensin II receptors in bovine retinal microvessels: quantitation with radioluminography.

Summary1.Specific125I-Sar1, Ile8-Angiotensin II (125I-Sar1, Ile8-AII) binding sites in bovine retinal microvessels were investigated using the quantitative receptor autoradiographic method with pellet sections.2.A quantitation was made with the computerized radioluminographic imaging plate system, a newly developed and highly sensitive method. Binding characteristics of the retinal microvessels were compared with those of the cerebral microvessels and the retinal macrovessels.3.We isolated microvessels from the bovine retina and bovine cerebral cortex using the method composed of two-size sievings and high-speed homogenization with a Polytron. The isolated microvessels were composed of capillaries, and the retinal macrovessels contained vessels with smooth muscle.4.There were specific binding sites for125I-Sar1, Ile8-AII which were single and of a high affinity, in both the cerebral and the retinal microvessels and the retinal macrovessels. There were no differences in affinity between the vessels, but the retinal microvessels did have a higher density of binding sites than the cerebral microvessels.5.The method we used is simple and sensitive for detecting and characterizing125I-Sar1, Ile8-AII binding sites in retinal capillaries. Knowledge of the existence of large numbers of specific binding sites, candidates of physiologically active angiotensin II receptors, aids with understanding the regulatory roles of angiotensin II in the blood-retinal barrier.

α-Atrial natriuretic peptide binding sites in the rat choroid plexus are increased in the presence of hydrocephalus

Specific alpha-rat atrial natriuretic peptide(1-28) [ANF-(99-126)] (rANP) binding sites in the choroid plexus of rats with kaolin-induced hydrocephalus were analyzed following incubation of related tissue sections with 125I-rANP, then using autoradiography and an image analysis coupled with computer-assisted micro-densitometry. The number of 125I-rANP binding sites in the choroid plexus of these rats was significantly higher, as compared to findings in the control rats, whereas no differences in the binding affinity were observed, 3 days and 3 weeks after the intracisternal injection of kaolin. The possibility that atrial natriuretic peptide may play a significant role in the function of cerebrospinal fluid production by interacting with specific, high affinity receptors in the rat choroid plexus has to be considered.

GLIAL ENDOTHELIN/NITRIC OXIDE SYSTEM PARTICIPATES IN HIPPOCAMPUS CA1 NEURONAL DEATH OF SHRSP FOLLOWING TRANSIENT FOREBRAIN ISCHAEMIA

1. When delayed neuronal death occurred in the hippocampus CA1 pyramidal cell layer of stroke‐prone spontaneously hypertensive rats (SHRSP) at 4 and 7 days after a 10 min bilateral carotid occlusion and reperfusion, intense endothelin‐1 (ET‐1)‐ and ET‐3‐like immunoreactivities became evident in astrocytes in the damaged hippocampus CA1 subfields.

The endothelin ETA receptor exists in the caudal solitary tract nucleus of the rat brain.

Abstract1. The receptor autoradiographic method done on the rat lower brain stem and cerebellum plus 125I-endothelin-1, BQ-123, an antagonist for the endothelin ETA receptor, and sarafotoxin S6c, an agonist for the ETB receptor, revealed minute amounts of the ETA receptor coexisting with the ETB receptor in the caudal solitary tract nucleus of the rat lower brain stem.2. The ETB receptor is present predominantly in other parts of the lower brain stem.3. Knowledge of the heterogeneous distribution of the central endothelin receptor subtypes aids in understanding the neurophysiology of endothelins.