We have explored the cellular loci of endothelin (ET) actions and formation in the brain, using cerebellar mutant mice as well as primary and continuous cell cultures. A glial role is favored by several observations: (i) mutant mice lacking neuronal Purkinje cells display normal ET receptor binding and enhanced stimulation by ET of inositolphospholipid turnover; (ii) in weaver mice lacking neuronal granule cells, ET stimulation of inositolphospholipid turnover is not significantly diminished; (iii) C6 glioma cells and primary cultures of cerebellar astroglia exhibit substantial ET receptor binding and ET-induced stimulation of inositolphospholipid turnover; (iv) ET promotes mitogenesis of C6 glioma cells and primary cerebellar astroglia; and (v) primary cultures of cerebellar astroglia contain ET mRNA. ET also appears to have a neuronal role, since it stimulates inositolphospholipid turnover in primary cultures of cerebellar granule cells, and ET binding declines in granule cell-deficient mice. Thus, ET can be produced by glia and act upon both glia and neurons in a paracrine fashion.Endothelin (ET) is a potent vasoconstricting peptide with multiple isoforms that originally was isolated from vascular endothelial cells (1, 2). In many parts of the body, ET acts on smooth muscle or other cells close to sites of synthesis (3, 4). In peripheral tissues and the brain, ET is a potent stimulator ofinositolphospholipid turnover (5). It also stimulates growth of smooth muscle cells and fibroblasts (6, 7).The sites of synthesis and action of ET in the brain are unclear, though one report describes the in situ hybridization and immunohistochemical localization of ET to motor neurons in human spinal cord (8). ET receptor binding sites mapped by autoradiography occur heterogeneously in the brain independently of the distribution of blood vessels, but the cellular elements containing these receptors have not been established (9, 10). In the present study, we use mouse cerebellar mutants and enriched cell populations to demonstrate glial as well as neuronal actions of ET. ET enhances inositolphospholipid turnover within glia, stimulates glial cell growth, and is synthesized within astroglia. MATERIALS AND METHODSMaterials. ET-1 and ET-3 were purchased from Peptides International (Louisville, KY). 125I-labeled ET-1 (125I-ET-1) was obtained from NEN/DuPont. Male mutant mice with Purkinje cell degeneration (PCD; pcd/pcd, C57BL/6J-pcd strain), mice with the nervous mutation (nr/nr, C3HeB/FeJ-nr strain), mice with the weaver mutation (wv/wv, B6CBA/ Ca-AWJ/A-wv strain), and their phenotypically normal littermates were purchased from The Jackson Laboratory. The mice were sacrificed by decapitation 1-3 mo after birth.Cerebellar primary cell cultures were prepared from 8-to 12-day-old rats as described (11) ET Binding Sites. For binding experiments, '25I-ET-1 (10,000-20,000 cpm; 2200 Ci/mmol; 1 Ci = 37 GBq) was added to tissue or cellular homogenates or to 12-/,m freshfrozen cryostat-cut sections in 50 mM Tris maleate, pH 6.5/1...

Endothelin (ET) is a recently identified vasoactive peptide with three isoforms for which three genes have been cloned. The cellular sites of synthesis of this peptide have not yet been identified in vivo. Using Northern blot analysis, we have detected two forms of ET mRNA in rat tissues: a 3.7-kilobase form in the kidney, eye, and brain, a 2.5-kilobase form in the intestine, and both forms in the lung. We have localized these forms of ET mRNA in several rat tissues using in situ hybridization. In the 19-day rat fetus, ET mRNA is highest in the lung, intestine, and meninges. At high resolution, ET mRNA is localized in the lung to respiratory epithelial cells of bronchioles and apparently in blood vessels. In adult tissues, ET mRNA is present throughout the lung, in the renal medulla vasa recta, and in the iris of the eye. ET mRNA is synthesized in close proximity to ET binding sites in many organs (e.g., lung, kidney, intestine, and eye), suggesting a local action of this peptide. However, in other areas (e.g., heart and renal cortex), ET binding sites are present in the absence of ET mRNA, suggesting an action of ET from the bloodstream or from neurons. Northern blot analysis of ET mRNA in microvascular endothelial cells in culture indicates that ET is synthesized in small blood vessels and regulated similarly to its regulation in large vessels. Our results provide evidence that ET, like other regulatory peptides, may serve in several tissues as a neuromodulator or local hormone.Endothelin (ET) is a 21-amino acid peptide isolated from aortic endothelial cells in culture and shown to be an extremely potent vasoconstrictor (1). Genes for three forms of ET have been identified in the human and rat and have been cloned from a human genomic library (2). However, mRNA for only one of these forms has been identified and only in endothelial cells from aorta or umbilical vein (1, 11). In aortic endothelial cells, levels of mRNA for ET-1 are regulated by several humoral substances or analogues (1). In peripheral tissues, ET receptor binding sites have been identified primarily in smooth muscle, especially of blood vessels and airways (3)(4)(5). Because ET is relatively resistant to peptidases, it has been suggested that ET functions primarily as a circulating hormone, while an alternative model is for ET to be elaborated by endothelial cells adjacent to the muscle cells on which it acts. However, the cellular sites for biosynthesis of ET have not yet been identified in any tissue in vivo. In the present study we have localized sites of ET formation by in situ hybridization techniques. We provide evidence that in most parts of the body ET is formed near the site of its action and also demonstrate dynamic regulation of ET mRNA in microvessel endothelial cells. METHODSTwo oligonucleotide probes (39 bases long) were synthesized based on the published rat ET-3 sequence (6) and purified by HPLC. Sequences were chosen to avoid internal repeats, regions with known homology to other sequences, and regions with greater th...

Key Points Question What are the incidence rates and risk factors for intraocular inflammation (IOI) and/or retinal vascular occlusion (RO) after brolucizumab treatment for neovascular age-related macular degeneration (AMD) in clinical practice? Findings In this cohort study of patient eyes with neovascular AMD treated with brolucizumab, the incidence rate for any form of IOI and/or RO was approximately 2.4%. A history of IOI and/or RO was a key risk factor for IOI and/or RO after brolucizumab treatment initiation. Meaning These early findings explore potential risk factors for inflammation-associated adverse events that may occur following real-world treatment with brolucizumab.