Antibodies against actin were used to corroborate the presence ofactin as a major component protein ofisolated brain postsynaptic densities. The same antibodies also were used as an immunohistochemical stain to study the distribution of actin in sections of intact brain tissue. This showed two major sites where actin is concentrated: smooth muscle cells around blood vessels and postsynaptic sites. In the postsynaptic area the highest concentration of actin occurs in postsynaptic densities and there also is intense staining in the surrounding cytoplasm, especially within dendritic spines. Antiactin staining was much weaker in other parts of neurons and in glial cells. The high concentration of actin in dendritic spines may be related to shape changes that these structures have been found to undergo in response to prolonged afferent stimulation.
Preparations of isolated brain postsynaptic densities (PSDs) contain a characteristic set of proteins among which the most prominent has a molecular weight of 50,000. Following the suggestion that this major PSD protein might be related to a similarly sized component of neurofilaments (F . Blomberg et al ., 1977, J. Cell Biol ., 74 :214-225), we searched for evidence of neurofilament proteins among the PSD polypeptides . This was done with a novel technique for detecting protein antigens in SDS-polyacrylamide gels (immunoblotting) and an antiserum that was selective for neurofilaments in immunohistochemical tests . As a control, an antiserum against glial filament protein (GFAP) was used because antisera against GFAP stain only glial cells in immunohistochemical tests. They would, therefore, not be expected to react with PSDs that occur only in neurons . The results of these experiments suggested that PSDs contain both neuronal and also glial filament proteins at higher concentrations than either synaptic plasma membranes, myelin, or myelinated axons.However, immunoperoxidase staining of histological sections with the same two antisera gave contradictory results, indicating that PSDs in intact brain tissue contain neither neuronal or glial filament proteins . This suggested that the intermediate filament proteins present in isolated PSD preparations were contaminants . To test this possibility, the proteins of isolated brain intermediate filaments were labeled with ' 25 1 and added to brain tissue at the start of a subcellular fractionation schedule . The results of this experiment confirmed that both neuronal and glial filament proteins stick selectively to PSDs during the isolation procedure. The stickiness of PSDs for brain cytoplasmic proteins indicates that biochemical analysis of subcellular fractions is insufficient to establish a given protein as a synaptic junctional component. An immunohistochemical localization at PSDs in intact tissue, which has now been achieved for tubulin, phosphoprotein I, and calmodulin, appears to be an essential accessory item of evidence .Our findings also corroborate recent evidence which suggests that isolated preparations of brain intermediate filaments contain both neuronal and glial filaments.The postsynaptic density (PSD) is a disk-shaped proteinaceous structure that is attached to the postsynaptic junctional membrane at synapses in mammalian brain (17,(31)(32)(33) . In several recent studies, it has been shown that subcellular fractions highly enriched in PSDs can be made by treating isolated synaptosomal plasma membranes with detergents (7,8,28,29,42,49) .Analysis by SDS-gel electrophoresis has shown that isolated PSDs contain a characteristic set of polypeptides, among which is a major component of 50,000 mol wt. Initially, it was thought that this 50,000 PSD protein might be tubulin because the two comigrated in SDS gels and showed apparently similar 346
Fluid was collected by micropuncture from proximal and distal convolutions of anesthetized rats and analyzed for inulin, sodium, urea, and total osmotically active solute. The proximal fluid/plasma (F/P) sodium ratio was not significantly different from unity in antidiuretic animals, but was as low as 0.78 during mannitol diuresis. The distal F/P sodium ratio averaged 0.62 in antidiuresis, and 0.24 during osmotic diuresis. The data are interpreted to indicate active sodium transport by both proximal and distal convolutions. The F/P ratios for inulin, urea, and total osmotically active solute are in general agreement with previous studies.
Using micropuncture techniques, fluid was collected from loops of Henle at the tip of the renal papilla in anesthetized hamsters and Psammomys, and its composition compared with that of collecting duct urine. The osmolalities of the two fluids were essentially the same. Sodium and attendant anions constituted 64% and urea 19% of the osmotically active solute in loop fluid. In collecting duct urine, urea was the major solute present, and sodium was present in low concentration. Inulin ratios indicated that 9% of the filtered water reached the tip of these loops at a time when 0.9% was in the final urine. The data support the countercurrent hypothesis for urine formation.
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