Transthyretin localization in cultured and native human retinal pigment epithelium.

The aim of this study was to determine transthyretin subcellular localization in cultured and native human retinal pigment epithelium. Monoclonal and polyclonal antibodies directed against human plasma transthyretin were used to detect transthyretin-specific immunoreactivity in cultured human retinal pigment epithelium. At the light microscopic level, transthyretin-specific immunoreactivity was observed throughout the cytosol with intense perinuclear staining. Nuclear staining was faint, but detectable. Both monoclonal and polyclonal antibodies exhibited similar staining patterns. An electron microscopic immunogold labeling protocol detected transthyretin-specific immunoreactivity in cultured and native human retinal pigment epithelium. Transthyretin-specific immunogold labeling within mitochondrial, apical, basal, nuclear and cytosolic subcellular compartments was quantitated and statistically analyzed. The pattern of transthyretin labeling was similar for each antibody, and comparable between cultured and native human retinal pigment epithelium. Transthyretin labeling was observed in mitochondrial and nuclear compartments, and in close apposition to both apical and basal membranes. Transthyretin labeling density was highest in the mitochondrial compartment and was significantly greater than labeling in all other compartments. Detection of transthyretin labeling in mitochondrial and nuclear compartments suggests an intracellular role for transthyretin in human retinal pigment epithelium, possibly as a cytoplasmic carrier protein for thyroxine.

Kainic acid induces apoptosis in neurons.

Growing evidence suggests that non-N-methyl-D-aspartate receptor activation may contribute to neuronal death in both acute and chronic neurological diseases. The intracellular processes that mediate this form of neuronal death are poorly understood. We have previously characterized a model of kainic acid neurotoxicity using cerebellar granule cell neurons in vitro and we sought to determine the mechanism of kainic acid-induced neuronal degeneration. We found DNA laddering by agarose gel electrophoresis, cellular DNA fragmentation by in situ end labeling of DNA, and chromatin condensation using a fluorescent DNA intercalating dye, in cerebellar granule cells following exposure to kainic acid (100 microM). Aurintricarboxylic acid protected cerebellar granule cells from kainic acid-induced death. While the morphological and biochemical features of neuronal death induced by kainic acid resembled low K(+)-induced apoptosis in cerebellar granule cells, the time interval from the institution of the death promoting condition to neuronal death was shorter with kainic acid and did not require new protein or RNA synthesis. These results demonstrate that kainic acid receptor activation can induce transcription-independent apoptosis in neurons. This in vitro model should be useful in identifying the intracellular pathways that link kainic acid receptor activation with apoptosis.

Autoradiographic distribution of bombesin/gastrin-releasing peptide receptors in fetal cortex transplants.

Bombesin/gastrin-releasing peptide (BB/GRP) receptors were characterized in fetal cortex transplants in the rat. Using in vitro autoradiography techniques, the density of (125I-Tyr4)BB grains was low 2 weeks after transplantation but increased 4 weeks after cortex transplantation into the adult rat fourth ventricle. Densitometry analysis of the autoradiograms indicated that (125I-Tyr4)BB bound with high affinity (Kd = 5 nM) to a single class of sites in the transplant tissue. Specific (125I-Tyr4)BB binding was inhibited with high affinity by (Tyr4)BB but not by NMB (IC50 values of 3 and 100 nM, respectively). These data suggest that GRP may act as a novel growth factor and play a regulatory role in the development of fetal cortex transplants.

Kainate induces apoptosis in neurons.

Growing evidence suggests that non-N-methyl-D-aspartate receptor activation may contribute to neuronal death in both acute and chronic neurological diseases. The intracellular processes that mediate this form of neuronal death are poorly understood. We have previously characterized a model of kainate neurotoxicity using cerebellar granule cell neurons in vitro and we sought to determine the mechanism of kainate-induced neurons degeneration. We found DNA, and chromatin condensation using a fluorescent DNA intercalating dye, in cerebellar granule cells following exposure to kainate (100 microM). Aurintricarboxylic acid protected cerebellar granule cells from kainate-induced death. While the morphological and biochemical features of neuronal death induced by kainate resembled low-K(+)-induced apoptosis in cerebellar granule cells; the time interval from the institution of the death-promoting condition to neuronal death was briefer with kainate and did not require new protein or RNA synthesis. These results demonstrate that kainate receptor activation can induce transcription-independent apoptosis in neurons. This in vitro model should be useful in identifying the intracellular pathways that link kainate receptor activation with apoptosis.

Immunodetection of an arrestin-like protein in human retinal pigment epithelium.

The goal of this study was to determine if an arrestin/S-antigen-like protein is produced by human retinal pigment epithelial (HRPE) cells maintained in tissue culture. Arrestin immunoreactivity was examined in fixed, monolayer cultures of HRPE and on immunoblots of SDS-PAGE separations of whole cell lysates of HRPE using five monoclonal antibodies (mAbs A2G5, A9C6, 3C4.2, 3D1.2 and 5c6.47) that bind to different epitopes in bovine retinal S-antigen. Monolayers of HRPE cells showed immunoreactivity with four of the mAbs though the relative staining intensity varied among mAbs and donors. For example, mAb A2G5 which historically shows very limited crossreactivity among species, reacted strongly with cells from one donor, moderately with cells from a second donor and only weakly with other donor cultures examined. mAb 3D1.2 showed no reactivity with HRPE cells. Immunoblots of SDS-PAGE separations of whole cell lysates of HRPE established from ten different donors confirmed the presence of an arrestin-related polypeptide that comigrated with retinal arrestin. These results demonstrate the presence of an arrestin-like protein in HRPE cells which have been maintained in tissue culture. Though the function of this arrestin homologue in HRPE is not yet established, it could play a role in the downregulation of receptor and/or transport protein activity.

Kainic acid-induced lipid peroxidation: protection with butylated hydroxytoluene and U78517F in primary cultures of cerebellar granule cells.

The generation of free radicals in the progression of kainic acid (KA)-mediated neuronal death has been implicated in both in vitro and in vivo studies. In the present study, the association between KA-induced neurodegeneration and the appearance of lipid peroxidation products was investigated and compared to three well characterized free radical generating (FRG) systems: 200 microM ferrous ammonium sulfate (FAS), 20 microM copper (Cu2+), and 0.01 U/ml xanthine oxidase/2.3 mM purine/2.4 microM transferrin (XO). KA caused a dose-dependent increase in conjugated diene and lipid hydroperoxide formation as did the FRG systems. The antioxidant, butylated hydroxytoluene (BHT), decreased both FRG system- and KA-induced lipid peroxidation by approximately 60-70%. Unlike BHT, the potency of the lipid peroxidation inhibitor, U78517F, depended upon the system utilized to induce free radical generation. U78517F was most potent in attenuating FAS-induced lipid peroxidation (100 nM), followed by KA (1.5 microM), and then Cu2+ and XO (> 2 microM). Results were confirmed by measurement of cytolysis through the release of lactic dehydrogenase (LDH). These data provide further evidence that the generation of free radicals, subsequently leading to membrane disruption, is central to the mechanism of KA-elicited neuronal death in cultures of cerebellar granule cells.

Forebrain ischemia in the gerbil increases lambda opiate binding in hippocampal mossy fibers.

Transient forebrain ischemia was produced in gerbils by short-term occlusion of the common carotid arteries under halothane anesthesia. Histological analysis of brains 7 days post-ischemia demonstrated characteristic destruction of CA1 pyramidal cells. lambda Opiate binding (measured with [3H]naloxone in the presence of 300 nM diprenorphine) at 7 days post-ischemia was significantly increased in the stratum lucidum of the hippocampus (the mossy fiber layer), but not in any other region measured, including other hippocampal regions, cortex, amygdala, caudate putamen, thalamus, and hypothalamus. The increase in mossy fiber lambda binding was slow to develop (no increase detected up to 48 h post-ischemia), and long-lasting (binding remained elevated at 32 days post-ischemia). While MK-801 significantly inhibited CA1 pyramidal cell destruction when administered 20 min prior to ischemia, the increase in mossy fiber lambda binding was still evident. None of seven different opioid agonists and antagonists examined had an effect on either the pyramidal cell damage or increased mossy fiber lambda binding seen 7 days after ischemia.

Effects of traumatic brain injury in rats on binding to forebrain opiate receptor subtypes.

Sprague-Dawley rats were subjected to a moderate level (2.2 atm) of traumatic brain injury (TBI) using fluid percussion. Injured animals were allowed to survive posttrauma for periods of 5 min, 3 h, and 24 h. The effect of TBI on binding to forebrain opiate receptors was assessed using quantitative receptor autoradiography, and compared to a sham control group. Binding of [3H]DAGO to mu receptors in neocortex and the CA1 pyramidal layer of the hippocampus was significantly decreased in the 24-h group (p less than 0.05). [3H]Bremazocine binding to kappa receptors was unchanged at 5 min and 24 h, but showed large decreases 3 h after TBI in the CA1 pyramidal layer (65%, p less than 0.05) and dentate gyrus (43%, p less than 0.05). Levels of delta binding (measured with [3H]DSLET) and lambda binding (measured with [3H]naloxone) were unaffected by TBI. These data support previous suggestions of a role for endogenous opioids in TBI, and provide further evidence that mu and kappa opioid receptor subtypes in neocortex and hippocampus may have different functions in TBI.

Ontogeny of bombesin/gastrin-releasing peptide binding sites in rat brain.

The development of bombesin/gastrin-releasing peptide (BN/GRP) binding sites was determined in the rat brain. Using rat brain homogenate, the density of radio-labeled peptide binding sites increased dramatically around birth, remained constant during the first post-natal week, and then increased again between Postnatal (P) Days 7 and 10; the density of binding sites was similar in P10 and adult animals. Rat brain homogenates derived from P1, P7, or P10 animals bound ((125)I-Tyr(4))BN with high affinity (K(d) = 2-4 nM) to a single class of sites (B(max) = 11, 17, and 51 pmol/mg protein, respectively). BN, GRP, and GRP(18-27) inhibited specific radiolabeled peptide binding to rat brain homogenate with high affinity using P1, P3, P7, or P14 animals, whereas GRP(1-16) was inactive. These data indicate that the C-terminal of BN or GRP is essential for high-affinity binding activity in the rat brain during development. In vitro autoradiography revealed high ((125)I-Tyr(4))BN grain densities in the anterior olfactory nucleus, olfactory tubercle, nucleus accumbens, central medial thalamic nucleus, and hippocampus. Moderate grain densities were observed in the parietal cortex, periventricular hypothalamic nucleus, central gray, inferior colliculus, pontine reticular, and gigantocellular reticular nuclei in P1, P3, and P7 rat brains. In P14 and adult animals, relative to P1, P3, or P7 rat brains, the density of binding sites increased significantly in the parietal cortex. The ((125)I-Tyr(4))BN grain density was not significantly changed in the anterior olfactory nucleus, periventricular nucleus of the hypothalamus, olfactory tubercle, hippocampus, dentate gyrus, central medial thalamic nucleus, and nucleus accumbens but decreased significantly in the central grey, inferior colliculus, and pontine reticular nucleus. These data indicate that there is a redistribution of BN/GRP binding sites in the rat brain during development.

Bombesin-like, substance P and vasoactive intestinal polypeptide receptors in fetal cortical homografts to host cortex and spinal cord.

Neuropeptide receptors were visualized in homografts of fetal cortex (E14) into adult rat cortex (immediate or 7 day delay) and spinal cord using in vitro autoradiographic techniques to explore the expression of peptide receptors in the same graft tissue in different central nervous system implantation sites. Receptors for bombesin (BN)-like peptides developed in the grafts by 3 weeks postimplantation regardless of location or age of implantation pocket in host. After 4 weeks, the density of BN receptors was confined to the graft. In grafts to spinal cord, however, high densities of BN-like receptors were not confined to the graft but were distributed throughout the spinal cord. In contrast, the density of vasoactive intestinal polypeptide (VIP) and substance P (SP) receptors was moderate and low to undetectable in the fetal grafts. The development of the peptide receptors studied was graft donor tissue specific since they were not altered by central nervous system implantation site.

Localization of receptors for bombesin-like peptides in the rat brain.

BN-like peptides and receptors are present in discrete areas of the mammalian brain. By radioimmunoassay, endogenous BN/GRP, neuromedin B, and ranatensin-like peptides are present in the rat brain. High-to-moderate concentrations of BN/GRP are present in the rat hypothalamus and thalamus, whereas moderate-to-high densities of neuromedin B and ranatensin-like peptides are present in the olfactory bulb and hippocampus, as well as in the hypothalamus and thalamus. While the distribution of neuromedin B and ranatensin-like peptides appears similar, it is distinct from that of BN/GRP. When released from CNS neurons, these peptides may interact with receptors for BN-like peptides. BN, GRP, ranatensin, and neuromedin B inhibit specific [125I-Tyr4]BN binding with high affinity. By use of in vitro autoradiographic techniques to detect binding of [125I-Tyr4]BN to receptors for BN-like peptides, high grain densities were found in the olfactory bulb and tubercle, the nucleus accumbens, the suprachiasmatic and paraventricular nucleus of the hypothalamus, the central medial and paraventricular thalamic nuclei, the hippocampus, the dentate gyrus, and the amygdala of the rat brain. Some of these receptors may be biologically active and mediate the biological effects of BN-like peptides. For example, when BN is directly injected into the nucleus accumbens, pronounced grooming results and the effects caused by BN are reversed by spantide and [D-Phe12]BN. Thus, the putative BN receptor antagonists may serve as useful agents to investigate the biological significance of BN-like peptides in the CNS.

Neuropeptide receptors are present in fetal neocortical transplants.

Transplants of fetal neocortex (E15-19) within the minimally traumatized fourth ventricle were examined for the presence of receptors for the peptides bombesin (BN) and vasoactive intestinal peptide (VIP). These peptides are involved in cellular growth or regulation of cerebral blood flow. Although fetal and adult cortex in situ have low and moderate receptor densities for BN-like peptides respectively, neocortical transplants developed high densities between 4 and 16 weeks postoperative. VIP receptors, which have moderate density in adult cortex, were initially high but later showed a variable distribution in the transplants. The results suggest that neuropeptides, particularly BN and VIP may have a role in the growth and vascularization of central nervous system transplants.

Astrocytes secrete basal lamina after hemisection of rat spinal cord.

Basal lamina is reconstructed over the lesioned surface of the spinal cord. The following experiment (90 rats) studies the ultrastructure of the formation of this membrane and the immunohistochemistry of laminin production (a major secreted component of basal lamina). After hemisection of the spinal cord at T6 animals were prepared for electron microscopy or antilaminin-biotin-avidin-peroxidase incubation. Three-5 days posthemisection, antilaminin reaction product was observed in astrocytes and their processes which faced the lesion, endothelia of blood vessels or pia. Ultrastructurally (3 days), basal lamina was polymerizing as small projections on the surface of astrocytic membranes facing the lesion, endothelia or pia. By 5 days the basal lamina was a single membrane, folded multiple sheets or in swirls. At 6-10 days the antilaminin reaction and the basal lamina (except for duplications) did not differ from normal. Reactive astrocytes secrete laminin for at least 3-5 days after hemisection and form basal lamina on the lesioned surface of the spinal cord after spinal cord hemisection.