The present study investigates angiotensin (Ang) II effects on secretory protein synthesis in brain astrocytes cultured from neonatal and 21-day-old rats. Ang IIinduced changes in the de novo synthesis of [35S]methioninelabeled secretory proteins were visualized using two-dimensional NaDodSO4/PAGE. Astrocytes from 21-day-old rat brain possess specific high-affinity receptors for Ang II. These cells express two Ang 11-induced secretory proteins with M, 55,000 (AISP-55K) and Mr 30,000 (AISP-30K), which were time-and dose-dependent (EC50, 1 nM). [Sar', 11e8]Ang II (where Sar is sarcosine) inhibited Ang II-induced secretion of AISP-55K but not AISP-30K. N-terminal amino acid sequencing indicates that AISP-55K is identical to rat plasmiogen activator inhibitor 1, whereas AISP-30K exhibits 72-81% identity to three closely related proteins: human tissue inhibitor of metalloproteases, a rat phorbol ester-induced protein, and the murine growth-responsive protein 16C8. Immunofluorescent staining with rat plasminogen activator inhibitor 1 antibody was induced in the majority of cells in culture after Ang II treatment of astrocytes from 21-day-old rat brains. Absence of this response to Ang II in astrocytes from neonatal rat brain provides evidence that this action of Ang II on astrocytes is developmentally regulated.The octapeptide angiotensin (Ang) II has in recent years been implicated in the central control of blood pressure. Evidence in support of this view includes observations that centrally injected Ang II causes profound increases in blood pressure, that all components of the renin-Ang system are found in the brain (1-3), and that increased levels of Ang II and Ang II receptors are seen in brains of spontaneously hypertensive rats (4, 5). Cardiovascular effects of Ang II have been attributed to the modulation of sympathetic activity of neurons in relevant cardioregulatory centers of the brain (1, 2). In vitro studies with neurons in primary culture have further shown that occupation of neuronal Ang II receptors modulates both catecholamine synthesis and reuptake (6, 7). Increased levels of Ang II receptors have been found in neurons cultured from spontaneously hypertensive rats (8).In addition to a neuromodulatory role of Ang II, evidence exists for a distinct Ang II system in central nervous system (CNS) astrocytes. Receptors for Ang II (9), angiotensinogen mRNA (10,11), and Ang I and Ang 11 (12,13) have been demonstrated in cultures of astrocytes, and immunoreactive Ang I, Ang II, and angiotensinogen mRNA have been observed in astrocytes in vivo (14-16). Although the presence of an Ang II system in astrocytes is well documented, little is currently understood of its role in normal brain physiology.Recent studies have indicated that intercellular communication between astrocytes and other cells of the brain may be mediated by proteins secreted from astrocytes (17). Cells of astrocytic origin are thought to secrete several neurotrophic factors (18) and the neurotrophic properties of epidermal growth factor ...

Mid-to-late gestation rat placenta synthesizes a number of proteins related to prolactin, including rat placental lactogen II (rPL-II), rat prolactin-like protein A (rPLP-A) and rat prolactin-like protein B (rPLP-B). This study identifies a new family of proteins synthesized and secreted by gestation day 15 placental explants which exhibit amino acid homology to growth hormone precursors from several species. Placental explant medium was fractionated by ammonium sulfate precipitation and analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis to isolate four distinct proteins with Mr values of 28,000, 23,000, 25,000 and 30,000 and pI values of 5.7, 5.7, 5.4 and 5.3, respectively. These proteins represent a major fraction of secretory proteins with Mr in the 20,000 to 30,000 range. Immunoblot analysis showed that none of the four proteins crossreacted with antipeptide antisera against rPL-II, rPLP-A, or rPLP-B. These proteins were electrophoretically transferred from two-dimensional SDS-polyacrylamide gels onto an Immobilon PVDF membrane and N-terminal amino acid microsequencing carried out with a gas phase sequencer. N-terminal sequences of 45, 37, 37 and 32 amino acid residues were identified for proteins 1, 2, 3 and 4, respectively. The four proteins exhibit 76% to 97% homology. Computer analysis further revealed a 28% identity in a 32 amino acid overlap which begins at residue 14 of the 28,000 Mr protein (protein 1) and at residue 31 of growth hormone precursors of rat, mouse and human. The 32 amino acid overlap is 78% homologous if conservative amino acid replacements are included.

Studies characterized the structure and function of epidermal growth factor (EGF) receptors in astrocytic glial cells and neuronal cells in primary culture from neonatal rat brain. [125I]EGF binding to membranes prepared from glial and neuronal cultures was specific and dependent on protein concentration; however, glial preparations bound 5-fold more [125I]EGF per mg protein. Unlabeled EGF competed for binding to both glial and neuronal membranes with an IC50 of 5 nM, whereas insulin, insulin-like growth factor I, and nerve growth factor failed to compete. Scatchard plot analysis of binding data for glial cells yielded a curvilinear plot with dissociation constants of 7.12 nM for high affinity and 6.2 microM for low affinity sites. The higher level of binding in glial compared to neuronal membranes reflected a greater number of binding sites rather than differences in receptor affinity. In glial membranes, [125I]EGF covalently cross-linked to one major protein with a mol wt of 170,000, and EGF stimulated the phosphorylation of a 170,000 protein which was half-maximal at 20 nM. In contrast, neither covalent cross-linking nor receptor autophosphorylation could be detected in neuronal membranes. Culture of glial cells in the presence of EGF stimulated [35S]methionine incorporation into both cellular and secreted proteins, whereas no effect of EGF was observed in neuronal cultures. The addition of EGF to glial cultures produced a dose-dependent stimulation of [3H]thymidine incorporation as well as the multiplication of cells over a 6-day period. These observations show that functional EGF receptors in the neonatal brain are predominantly localized in glial cells.

3‐Chloro‐4‐methoxyaniline at concentrations of 10ppm or more in soil is converted to a mixture of 3,3′dichloro‐4,4′‐dimethoxyazobenzene, 3‐chlorobenzoquinone‐4‐(3‐chloro‐4‐methoxy)anil and its reduction product 2,3′‐dichloro‐4‐hydroxy‐4‐'methoxydiphenylamine, probably by a free radical mechanism. The herbicide metoxuron, N'‐(3‐chloro‐4‐methoxyphenyl)‐N,N‐dimethylurea and its demethylated metabolites probably break down to the amine too slowly in soil for coupling products to be detected. 3‐Chloro‐4‐methoxyacetanilide at 25 ppm rapidly gives rise to amine coupling products in soil slurries and ethyl N‐(3‐chloro‐4‐methoxyphenyl)carbamate does so after two months in the slurries.