Kinetic studies of ATP uptake in amyloplasts from sycamore (Acer pseudoplatanus L.) have been performed with a newly developed method of centrifugation through a double layer of silicone oil; the results are compared with the frequently used method of centrifugation through a single layer. The present technique employs two separate silicone layers: the upper one prevents mixing of the organelle preparation with the incubation layer, which contains the metabolites, and the lower one prevents mixing of the incubation and pelleting layers. Incubation of the organelles takes place in the incubation layer after the upper layer of silicone inverts during centrifugation. Depending on the speed of centrifugation, incubation periods as short as 1 sec and as long as 2 min can be achieved. High reproducibility and accuracy, acquisition of multiple data in a single centrifugation, and maintenance of structural integrity and metabolic activity of the organelles make the technique advantageous for the analysis of metabolite transport in amyloplasts and other kinds of organelles.

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Cultured vascular endothelial cells were exposed to fluid shear stress by means of a rotary-disc shear-loading device, and the physiological effects of the conditioned medium (CM) and the homogenate (HM) of the cells on migration, adhesion and growth of endothelial cells (EC) or smooth muscle cells (SMC) were studied. Effects of shear stress on the production and secretion of collagen, one of the extracellular matrices of EC, were also studied. CM stimulated the adhesion and growth of SMC, but not of EC themselves. The ability to stimulate SMC adhesion and growth was similar in CM obtained from the static and shear-loaded cells. HM of the shear-loaded EC stimulated SMC migration. Further, HM of the shear-loaded EC contained increased amounts of collagen compared with the static EC. These results suggest that: 1) EC produce and secrete accelerators for the adhesion and growth of SMC, 2) EC react to the physical stimulus of fluid shear stress to produce stimulators of SMC migration, and 3) EC produce collagen, the production of which is enhanced by fluid shear stress.

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Embryonic stem (ES) cells are a pluripotent cell line derived from the inner cell mass of the mouse blastocyst. The mRNA levels of several growth-related genes were examined in ES cells by Northern blot analysis under several different growth conditions. In a coculture of ES cells with the mouse fibroblast cell line, Balb/c3T3 A31, the expression level of transforming growth factor β1 (TGF-β1) mRNA was elevated for 1 to 3 days. In a coculture of ES cells with primary embryonic fibroblasts, only slight accumulation of TGF-β1 mRNA was observed for 6 to 24 h and soon attenuated. In situ hybridization analysis revealed that TGF-β1 transcripts accumulated first in the masses of ES cells and subsequently in Balb/c3T3 A31 cells adjacent to ES cells' masses. The ability of ES cells to induce TGF-β1 in Balb/c3T3 A31 cells was not mediated by soluble factors and was lost upon differentiation. ES cells on primary embryonic fibroblasts grew in undifferentiated form, but those on Balb/c3T3 A31 cells stopped growing and formed embryoid cystic bodies. We suggest that TGF-βl mRNA induction in a coculture is triggered by an interaction between differentiating ES cells and Balb/c3T3 A31, and that this activity is limited to undifferentiated ES cells.

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Mutants of Dictyostelium discoideum were isolated and found to be defective in the epitope recognized by the monoclonal antibody 120 against the carbohydrate moieties of an integral membrane glycoprotein, contact site A, with the apparent molecular mass of 80×10³. One mutant, HG764, did not express any contact site A and had lost cell contact resistant to EDTA. The others, including HG794, expressed a 68-kDa form of contact site A. In comparison with the parental strain HG592, HG794showed weaker EDTA-resistant cell contact and the same degree of EDTA-sensitive cell contact. This suggested that the moieties which HG794 lacked were involved in EDTA-resistant cell contact. The 68-kDa contact site A in HG794could be labeled with wheat germ agglutinin and incorporated [³⁵S] sulfate. The modB mutant HL220also expresses 68-kDa contact site A, although it cannot be labeled with wheat germ agglutinin. Therefore, the mutants HG794and HL220 were compared by a complementation test. The diploid strain DG701expressed 80-kDa contact site A and showed the same degree of EDTA-resistant cell contact as strain HG592. In its EDTA-resistant cell contact, HG794was stronger than HL220.These results suggest that HG794is a new mutant, and that there might be at least two processes in the glycosylation of 68-kDa contact site A to the 80-kDa form. The carbohydrate moieties recognized by monoclonal antibody 120 and by wheat germ agglutinin might be involved in EDTA-resistant cell contact.

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Human umbilical vein endothelial cells grew well in dishes coated with collagen types I, II, III, or IV. However, the same cells tended to detach themselves from dishes coated with type V collagen, and cell proliferation in these dishes was inhibited. Such anti-adhesive activity was partially retained by heat-denatured type V collagen or by its α1 chain, but not by its α2 chain. Several other cell types did not adhere to the type V collagen substratum even in the presence of 10% serum. The cell types strongly inhibited from adhering by type V collagen included Swiss mouse 3T3 cells and their MSV-transformants, BALB/c3T3 cells and their methylcholanthrene-transformants, NIH 3T3 cells and their ras-transformants, BHK cells, CHO-9 cells, CHO-K1 cells, and mouse melanoma B16-F10 cells. Using Swiss mouse 3T3, we studied the effects of type V collagen on cell adhesion to fibronectin in serum-free medium. When the culture dishes were coated with a mixture of fibronectin with various concentrations of type V collagen, the adhesion of the cells was inhibited depending on the concentration of type V collagen. The inhibition of cell adhesion by type V collagen was competitively overcome by increased concentrations of fibronectin. The activity that interferes with the effects of fibronectin was retained mainly by the α1 chain of heat-denatured type V collagen.

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_L-Gliitamate, a putative photoreceptor cell neurotransmitter, causes thinning of the inner layers of the retina and has been used for preparing biologically fractionated photoreceptor cells. However, it is possible that absence of the inner retinal layers may affect the remaining retina, and/or glutamate may directly affect photoreceptor cells. We evaluated quantitatively the effects of _L-glutamate on the developing photoreceptor cells by measuring the rod photoreceptor cell-specific protein, opsin. Wepurified rat rhodopsin and used it as the standard for measuring opsin content of rat retinas with competitive enzyme-linked immunosorbent assay. Various concentrations of glutamate were injected into 7-day-old rats, and the effects of the amino acid concentration on opsin expression were determined on postnatal day 14. Inner layers of the retina degenerated when10 μl or 15 μl of 2.4 M glutamate/gram body weight was administered subcutaneously. Opsin content of these glutamate- treated retinas decreased significantly compared with control retinas. We administered glutamate to rats at various stages of development and determined the effects by light microscopy on postnatal day 14. The administration of glutamate resulted in no degeneration of the inner retina if injected on postnatal day 1 or 2, degeneration of the inner retina between day 3 to 7, and again, no degeneration after postnatal day 13. Opsin content decreased significantly when glutamate was administered between postnatal day 1 to 7, but not after day 13, the day the blood-retinal barrier seems to reach maturity. Our findings indicate that systemic administration of _L-glutamate affects the expression of opsin in the developing rod photoreceptor cells.

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The roles of potassium and chloride ions in CAMP-mediatedamylase exocytosis were studied using intact and saponin-permeabilized parotid acini. Cyclic AMP-evoked amylase release from saponin-permeabilized parotid acini decreased markedly when KCl in the incubation medium was isoosmotically replaced by K-glutamate, NaCl, Na-isothionate, or mannitol. Quinidine and barium, K⁺ channel blockers, clearly inhibited amylase release from the permeabilized acini, but not from intact ones. The chloride channel blocker DPC (diphenylamine-2-carboxylate) also inhibited amylase release, while DIDS (4, 4'-diisothiocyanostilben-2, 2'-disulfonate) or bumetanide had little effect, if any, on the exocytosis. Hyperosmolarity with mannitol markedly reduced amylase release from permeabilized acini. These results suggest that potassium and chloride ions play important roles in cAMP-mediated amylase exocytosis, and that these ions act on secretory granules inside the acinar cells.

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Spatiotemporal change of the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in response to a variety of secretagogues was examined in rat pancreatoma AR-42J and AR-IP cells by microspectroflurometry and digital imaging microscopy after loading with fura-2. In the presence of external Ca²⁺, carbachol, CCK-OP (cholecystokinin-octapeptide), gastrin, norepinephrine or high K⁺ evoked a large transient increase in [Ca²⁺]_i in AR-42J cells which declined to a sustained level before slowly declining towards the resting level. In the absence of external Ca²⁺, a transient increase in [Ca²⁺]_i were evoked by all the ligands except for high K⁺ stimulation, which declined rapidly towards the resting level. The [Ca²⁺]_i increase caused by carbachol and high K⁺ treatment was inhibited by muscarinic receptor antagonist, atropin, and by L-type Ca²⁺ channel blocker, nifedipin, respectively. The transient [Ca²⁺]_i increase induced by gastrin stimulation was not blocked by Ca²⁺ channel blocker, lanthanum. In the AR-IP cells, which are non-differentiated pancreatoma cell line, all stimulations including high K⁺ treatment have failed to evoke [Ca²⁺]_i response. These intracelluar Ca²⁺ mobilizations in response to ligands in AR-42J cells were displayed by digital imaging microscopy. From these results we conclude that AR-42Jcells has an α-adrenergic receptor, in addition to muscarnic acetylcholine receptor, CCK-OP receptor, gastrin receptor and voltage dependent Ca²⁺ channel. In marked contrast, AR-IP cells have neither any hormonereceptor for the above ligands nor voltage dependent Ca²⁺channel.

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By immunogoldelectron microscopy we have shown that in mouse cerebellar Purkinje cells fixed by per fusion with formaldehyde-glutaraldehyde solution, the InsP₃ receptor are numeously detected on the stacks of flattened cisterns (OTSU et al, (1990) Cell Struct. Funct., 15: 163-173). In the present experiment we investigated distribution, structure and properties of the stacks by conventional electronmicroscopy, lectin cytochemistry and immunoelectron microscopy. The size and number of stacks were variable depending on their intracellular localization; short stacks with 2-4 parallel cisterns predominate in the perikaryon, long stacks with 4-15 cisterns in the proximal dendrite, and long stacks with 3-4 cisterns in the distal dendrites. The flattened cisterns bind with concanavalin A but not with wheat-germ agglutinin and may contain KDEL proteins loaded with Lys-Asp-Glu-Leu at their C-terminin in their lumens, indicating that the cisterns are derived from ER membranes. The electron dense materials sandwiched between the cisternal membranesare composed of small particles, short cylindrical in shape and 20 nm in diameter, and markedly labeled with anti InsP₃R antibody. We suggest that they correspond to the tetramer of the InsP₃R or their related molecules. It is not clear whether the stacks of flattened cisterns exist per se in the Purkinje cells or smooth ER existing in singlet in vivo in the Purkinje cells forms stacks during fixation. It is strongly suggested, however, that the smooth ER membranes covered by the InsP₃R or their related molecules can easily interact and stack each other in the Purkinje cells.

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