Capillaries were isolated from epididymal fat, and a catecholamine-sensitive adenylate cyclase found in these capillaries was characterized. The effect of various hormones on the accumulation of adenosine 3': 5'-cyclic monophosphate in capillary endothelial cells was determined and the cyclase was found to exhibit mixed alpha and beta characteristics. Cyclase was cytochemically localized in these endothelial cells with 5'-adenylyl-imidodiphosphate as a specific cyclase substrate and alloxan as a specific cyclase inhibitor. Lead imidodiphosphate was precipitated at or near the site of cyclase activity upon hydrolysis of 5'-adenylyl-imidodiphosphate by cyclase. This reaction product was observed primarily on the luminal surface of intact capillaries, in micropinocytic invaginations, in free vesicles within the cytoplasm, and in the intracellular junctions.Digestion of epididymal fat with collagenase has been used by several investigators to obtain pure fractions of fat cells (1, 2). Upon centrifugation of the dissociated adipose tissue, adipocytes collect at the top and the vascular and stromal cells are pelleted. This study describes a method for partial purification of the vascular-stromal pellet to obtain a relatively homogeneous preparation of intact capillaries and reports the biochemical characterization and cytochemical localization of a catecholamine-sensitive adenylate cyclase (cyclase) associated with capillary endothelial cells.A modification of the method of Reik et al. (3) was used in the cytochemical localization of capillary cyclase activity. The substrate, 5'-adenylyl-imidodiphosphate, which is specifically hydrolyzed by cyclase but not by other ATPases (4, 5), was used in comparison with ATP; the specificity of localization was enhanced by a reaction in which lead imidodiphosphate is precipitated at or near the site of cyclase activity. Further specificity was achieved by the use of alloxan, which has been shown to selectively inhibit cyclase while sparing other membrane ATPase (6), and thus would be expected to inhibit product formation when 5'-adenylyl-imidodiphosphate is used as substrate. METHODS AND MATERIALSIsolation of Capillaries. The distal portion of epididymal fat pads from Sprague-Dawley rats (250-300 g) were removed and placed in Tyrode's basal salt solution (pH 7.2) at 00, thoroughly minced, and placed in siliconized vials (three fat pads per vial) each containing 40 mg of crude collagenase (Worthington Biochemicals), 6 mg of bovine-serum albumin, and 6 ml of Tyrode's balanced salt solution. The mixture was incubated at 370 for 45 min on a wrist action shaker and centrifuged in siliconized conical tubes (30 X g for 2 min). The resulting pellet contained larger blood vessels, stromal cells, and attached fat cells, and was discarded. The supernatant containing the fat cake was decanted and centrifuged (800 X g for 2 min). The vascular pellets from the second centrifugation were twice pooled and washed in Tyrode's solution, and collected by centrifugation at 800 X g for 2 min. T...

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A vascular corrosion cast of an entire mouse kidney was scanned with a modular multiresolution X-ray nanotomography system. Using an isotropic voxel pitch of 0.5 mm, capillary systems such as the vasa recta, peritubular capillaries and glomeruli were clearly resolved. This represents a considerable improvement over corrosion casts scanned with microcomputed tomography systems. The resolving power of this system was clearly demonstrated by the unique observation of a dense, subcapsular mat of capillaries enveloping the entire outer surface of the cortical region. Resolution of glomerular capillaries was comparable to similar models derived from laser scanning confocal microscopy. The high-resolution, large field of view and the threedimensional nature of the resulting data opens new possibilities for the use of corrosion casting in research.

The addition of 0 .08 M sucrose to a culture medium containing Chang-strain human liver cells causes intense cytoplasmic vacuolation . Electron microscopy of these cells grown inferritin, time-lapse cinematography, and radioautography reveal that the vacuoles arise by endocytosis and that the sucrose is taken into the cell and localized in the vacuoles . Tracer studies demonstrate that sucrose-'H provides a marker for quantitation of endocytosis and that it neither induces nor stimulates endocytosis . Electron micrographs of vacuolated liver cells show microfilaments in close proximity to the inside of the plasma membrane, in the pseudopodia, and to the cytoplasmic side of the membrane surrounding endocytosis vacuoles . Cytochalasin B (CB), a mold metabolite that inhibits various types of cell motility, has a dose-dependent inhibitory effect on the uptake of sucrose-3H by these cells . This inhibition is accompanied by a cessation of the movement of ruffles and pseudopodia on the surface of the cells and the formation of blebs which arise from the cell's surface . These morphological changes are quickly reversible upon removal of CB. Alterations in the appearance and location of microfilaments are also observed in CB-treated cells .

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These results indicate that pericytes are capable of two types of contractile responses in culture, depending on the specific vasoactive stimulus.