Effects of adrenergic agents and phosphodiesterase inhibitors on outflow facility and cell shape of bovine trabecular meshwork.

Changes in the outflow facility of perfused calf eyes and in the shape of cells in cultured trabecular meshwork (TM) have been studied, following exposure to adrenergic agents and phosphodiesterase inhibitors (PDE). Dobutamine caused confluent TM cells to change their usual polygonal shape to a characteristic stellate shape. Salbutamol had no effect, but PDE inhibitors, isobutylmethylxanthine (IBMX), theophylline, and caffeine were very effective in producing this shape change. Epinephrine, isoproterenol, dobutamine, and salbutamol did not increase the outflow facility, either at 22 degrees C or 36 degrees C, while theophylline, caffeine, and IBMX did increase it in a dose-dependent manner. On the other hand, the high concentrations of beta-adrenergic agents required to produce even a small change in outflow facility and cell shape argue against the involvement of adrenergic-receptor mediation and may suggest another mechanism; on the other, the enhancement of epinephrine effects by PDE inhibitors and the similar effect produced by cyclic adenosine 3',5'-cyclic phosphate (cAMP) and purines suggest that changes in the cell shape are produced by beta-receptor activation. The beta-adrenergic agents were not effective in changing outflow facility, but the PDE inhibitors were remarkably effective both in changing the shape and in increasing facility.

Establishment of calf trabecular meshwork cell cultures.

Trabecular meshwork cell cultures have been grown from excised calf aqueous outflow pathway tissue using two different methods. In one method, tissue explants were placed directly in culture dishes and primary cultures were established by those cells migrating from the tissue. In the second method, a cell suspension was generated by incubating trabecular tissue strips with 0.1% pronase, and the washed cells were used to seed primary cultures. The cell cultures generated by each method appeared to progress through morphological and biochemical changes characteristic of the cell type, although at different respective rates. Comparisons of the cell morphologies, biosynthetic activities, and growth characteristics of the respective subcultures suggested that similar cell populations were obtained by each method. These studies further suggested that sufficient quantities of calf trabecular cells could be grown in culture to permit biochemical and pharmacological studies of trabecular-cell metabolism.

Factors influencing glycosaminoglycan synthesis by calf trabecular meshwork cell cultures.

When cell or explant cultures were established from excised calf aqueous outflow pathway tissue and subsequently labeled with radioactive precursors of glycosaminoglycans [( 3H]glucosamine and [35S]sulfate), hyaluronic acid was the predominant glycosaminoglycan produced initially. As the cultures reached confluence, sulfated glycosaminoglycans became more prominent products. Further investigation revealed that the cultured trabecular cells could synthesize all of the glycosaminoglycans found in isolated calf trabecular tissue once a critical cell density had been attained. Increasing concentrations of fetal bovine serum in the culture medium stimulated glycosaminoglycan accumulation without altering the quantitative distribution of the various glycosaminoglycan products. Treatment of confluent monolayers with enzymes capable of degrading extracellular matrix molecules caused marked changes in the distribution of glycosaminoglycans produced by calf trabecular-cell cultures. The results suggest that calf trabecular cell cultures can be used to study the dynamics of extracellular matrix production and turnover under controlled experimental conditions.

Isolation and characterization of an adenylyl-protein complex formed during the incubation of membranes from Dictyostelium discoideum with ATP.

When tritiated ATP is incubated with a membrane-enriched fraction prepared from the eukaryotic microorganism Dictyostelium discoideum significant levels of radioactivity can be precipitated with cold, 10% trichloroacetic acid. Reaction product was formed from ATP and dATP but not from GTP, CTP and UTP. Other studies showed that the maximum amount of the acid-insoluble product was formed about 1 min after the addition of the membranes and that, with further incubation, this reaction product was degraded. The rate of degradation of the reaction product was greatly reduced when the temperature was reduced to 4 degrees C, and when either NaF, Na2SO4 or dithiothreitol was added to the reaction mixture. These additions or conditions had no effect on the product-formation reaction. The rate of degradation was also reduced following the addition of adenosine to the reaction and this result did not occur following the addition of ADP, AMP or cyclic AMP. The acid-insoluble reaction product could be solubilized with SDS and analysis by gel-filtration chromatography on Sephadex G-75 revealed that the radioactivity was associated with a macromolecule that was not sensitive to RNAase or DNAase but was degraded by pronase. The nucleotide-protein complex was stable at room temperature but radioactivity was released in hot acid, which, after analysis by thin-layer chromatography, was found to co-migrate with authentic AMP, suggesting the formation of an adenylyl-protein complex as the reaction intermediate. The complex bond was stable at neutral and alkaline pH, suggesting a phosphoamide linkage between the protein and the adenylyl moiety.

Lysosomal abnormalities in hadacidin-treated Dictyostelium discoideum amoebae.

When Dictyostelium discoideum amoebae were harvested from nutrient medium and suspended in a starvation buffer to initiate development, approximately 30% of the total cellular beta-N-acetylglucosaminidase activity was secreted into the extracellular fluid within 4 h. During this same period, only 10% of the total cellular acid phosphatase and acid protease activities were secreted. When the cells were pretreated overnight with 5 mg sodium hadacidin ml-1 and then suspended in starvation buffer, 60% of the glucosaminidase and 30% of the acid phosphatase activities were secreted, while the level of acid protease secretin remained at 10%. The secretory behaviour of hadacidin-treated cells was, however, identical to that of untreated cells when 0.1 M-sucrose was added to the starvation buffer to enhance lysosomal enzyme secretion. Treatment with hadacidin also affected the intracellular content of these enzyme activities. After 16 h exposure to 5 mg hadacidin ml-1, the cellular levels of glucosaminidase and acid protease activity were decreased by 50% and 30% respectively, while acid phosphatase activity remained unchanged. All of the changes observed upon hadacidin treatment were time dependent and were not evident if the cells were exposed to the drug for only 4 h. These results suggest that hadacidin treatment affects the lysosomal system of D. discoideum.

Products of endogenous N-acetylglucosaminyltransferase activity of Dictyostelium discoideum during growth and early development.

In the presence of Mn2+ and uridine diphosphate-N-acetyl-D-[14C]glucosamine, a total particulate fraction prepared from Dictyostelium discoideum amoebae catalyzed the transfer of N-acetyl[14C]glucosamine to endogenous membrane protein acceptors. No transfer to lipid acceptors was evident. The 14C products obtained from growth-phase and aggregation-competent amoebae were converted to glycopeptides by pronase digestion. The respective glycopeptides appeared identical in their chemical and chromatographic properties, suggesting that the same activity was functioning in both growing and differentiating cells. The results provided no evidence for developmental regulation of this activity in D. discoideum.

Effect of hadacidin on growth and adenylosuccinate synthetase activity of Dictyostelium discoideum.

The growth of the eucaryotic microorganism Dictyostelium discoideum in liquid culture was completely inhibited by the aspartic acid analog hadacidin (N-formylhydroxyamino-acetic acid). Growth arrest occurred both in chemically defined medium and in complex growth medium containing aspartic acid and AMP precursors such as adenine and adenosine. Although these compounds could not overcome the effect of hadacidin, growth was restored if cells were washed and resuspended in fresh growth medium. Additional experiments showed that D. discoideum contains adenylosuccinate synthetase, the enzyme which catalyzes the synthesis of adenylosuccinate from IMP, aspartic acid, and GTP in the de novo biosynthesis of purines. A partially purified preparation of this enzyme was obtained, and the effect of hadacidin on its activity was studied. We found that maximum inhibition of the D. discoideum activity occurs at a ratio of aspartic acid to hadacidin of 5:1, suggesting that the affinity of the drug for this enzyme is less than for the enzyme from rabbit muscle and plants but greater than for that from Escherichia coli. The effect of the drug can be overcome by a 10-fold excess of aspartic acid, suggesting that the drug acts as a competitive inhibitor. A comparison of the adenylosuccinate synthetase activity levels at various stages of growth showed that its specific activity decreases about 60% as cells enter the stationary growth phase, and decreases about 75% after starvation for 2 h. Further studies showed that in cells treated with hadacidin the rate of uptake of exogenous nutrients is reduced about 75% and that these cells are more resistant to rupture by osmotic shock. While the results of this study are consistent with the proposal that growth arrest is contingent upon inhibition of adenylosuccinate synthetase activity, they also suggest that, as a consequence of this inhibition, some physiological properties of the cell have been altered.

Protease secretion during onset of development in Dictyostelium discoideum.

At the onset of development, the single cells of the eukaryotic micro-organism Dictyostelium discoideum secrete proteolytic activity which can be assayed using the insoluble substrate remazolbrilliant blue hide. The activity is not secreted by exponentially growing cells, but does appear extracellularly at the onset of the stationary growth phase. When growth phase cells are resuspended in non-nutrient buffer, proteolytic activity begins to appear outside the cells. It accumulates in the buffer at a rate similar to that observed for 2 glycosidases of lysosomal origin and reaches a maximum after about 2 h of incubation. After 3--4 h incubation, centrifugation of the non-nutrient buffer removes the cells, producing a supernatant which we refer to as conditioned medium. Subsequent experiments with conditioned medium showed: (a) its incubation with purified plasma membranes results in the release of polypeptides which can be recovered and, when displayed on polyacrylamide gels, can be shown to be stage specific; and (b) that conditioned medium can decrease the rate of detachment of cells from a collagen substratum. Both effects can be prevented by the addition of remazolbrilliant blue hide suggesting that they are due to proteolytic activity present in the conditioned medium. Finally, we were able to show that conditioned medium contains components which, when spread over the bottom of plastic Petri dishes, enhance the rate of multicellular structure formation. Additional studies showed that this effect of conditioned medium could also be brought about by components which remained behind on uncoated plastic dishes after the removal of a D. discoideum cell layer. These data may be accommodated to a model in which the protease secreted during the onset of development acts on the cell membrane releasing components which coat the substratum and facilitate migration and multicellular structure formation.

Synthesis of a mannosyl phosphoryl polyprenol by the cellular slime mold Dictyostelium discoideum.

A membrane fraction isolated from the cellular slime mold Dictyostelium discoideum was incubated with GDP-[14C]mannose and found to catalyze the incorporation of [14C]mannose into an endogenous acceptor to yield a product with the chemical and chromatographic properties of a polyprenol phosphate sugar derivative. These results suggest that D. discoideum can synthesize a mannosyl phosphoryl polyprenol.