Studies on cellular adhesion in tissue culture. XIIA. Some effects of prostaglandins and cyclic nucleotides

The effects of cyclic nucleotides, and physiologically related substances, on the initial adhesion of three groups of cells to protein covered plastic surfaces in vitro, was studied over periods of up to 2 h. The rate of adhesion of Ehrlich ascites tumour (EAT) cells was decreased in the presence of prostaglandin F_2α and dibutyryl cyclic AMP; possibly decreased by PGE₁; increased in the presence of sodium fluoride, and unaffected by PGF_2β. Trypsinized EAT cells showed no adhesion-response to PGE₁ or PGF_2β, and generally a lesser degree of response to the other reagents than non-trypsinized EAT cells. L929 cells, following trypsin-treatment, showed a significant decrease in adhesion rate, only on culture in the presence of PGF_2α.The inhibition of initial adhesion in the responding cells, may be correlated with the assays of others and interpreted to indicate that increase in intracellular cyclic nucleotides is associated with decreased initial cell adhesion. The failure of the L929 to respond to at least some of the reagents is possibly partly due to their prior incubation with trypsin.     

Adrenal cells in tissue culture the effects of choleragen and ACTH on steroid and cyclic-AMP metabolism.

Primary cultures of mouse adrenocortical tumors provide a sensitive system for investigating the effects of the enterotoxin of the V. cholerae (choleragen) on cyclic-AMP metabolism in the intact cell. Like ACTH, the toxin stimulates the synthesis and release of steroids from these cells but its mode of action differs from that of ACTH. The steroidogenic response to ACTH is immediate and of limited duration. The initial rate of steroidogenesis is the highest. In contrast, the steroidogenic response to choleragen is preceded by a 30-240 minute lag period which is inversely related to the concentration of the toxin. Whereas prolongation of the response to a single dose of ACTH requires hormone concentrations above those producing maximal initial steroidogenic activity, persistent steroidogenesis is induced at all levels of the toxin. Steroidogenic responses are detectable with 10 pg/ml of choleragen or less. The respective effects of ACTH and choleragen on cyclic-AMP synthesis and release into the medium parallel those on steroidogenesis. Intracellular cyclic-AMP levels in ACTH-treated cells reach a peak within 20-30 minutes and decline to normal levels within 2-4 hours. In choleragen-treated cells, after the lage period, the levels of intracellular cyclic-AMP remain above control levels indefinitely. The effects of ACTH and choleragen on cyclic-AMP biosynthesis are additive at all levels of the two compounds. The effects of choleragen are blocked by prior treatment of the toxin with a five-fold molar excess of ganglioside GM1, a presumed constituent of the toxin-binding site.     

Effect of cyclic nucleotides on the mitotic activity of dental anlage and alveolar bone cells in tissue culture

A study was made of the effects of cAMP, db-cGMP and db-cAMP on the mitotic activity of the cells of the tooth anlage and alveolar bone in tissue culture of mouse embryo aged 15 days. The data indicate that db-cGMP and db-cAMP at concentrations 10(-6) M and 10(-8) M do not have any essential effect on the mitotic activity of the cells of the tooth anlage whereas cAMP inhibits the mitotic activity in these cells as compared with control. A definite relationship was established between the character of differentiation of the osseous and dental tissues in tissue culture and the concentration of cyclic nucleotides. db-cAMP, db-cGMP and cAMP raise the mitotic activity of the osteogenic cells, inhibit resorption of the alveolar bone, and stimulate formation of a new osseous tissue.     

Studies of cyclic AMP action using mutant tissue culture cells.

S49 mouse lymphoma cell mutants, each with a specific defect in its ability to generate or respond to cyclic AMP, have been isolated. Analysis of the properties of these cells has begun to provide information on complex and significant biologic problems related to the cyclic AMP system.     

Effects of purine cyclic nucleotides on the growth of neonatal rat hepatocytes in primary tissue culture

cGMP and db-cGMP administered for 20–24 h to neonatal rat hepatocytes in primary culture stimulated their DNA synthesis and proliferation only at concentrations higher than the physiological one, whereas at concentrations equal to or lower than the physiological concentration they were ineffective or inhibitory for both activities. Induction of DNA synthesis to be effected by cGMP required 15 h of treatment, preceded, however, by inhibition of the same process between the 6th and the 14th hour of exposure. In contrast, cAMP and db-cAMP stimulated the flow of cultivated hepatocytes into the S and M stages of their mitotic cycle when administered at very wide concentration range, including the physiological for cAMP and the sub-physiological for db-cAMP. cAMP was effective after 12–14 h of treatment. Equimolar mixtures of cGMP with cAMP and of db-cGMP with db-cAMP also stimulated the proliferative activity of primary hepatocytes, but only at very low doses, which induced a first peak of DNA synthesis between the 2nd and the 6th hour of treatment and a second peak at about the 18th hour. These actions of the cyclic compounds, employed singly or in equimolar combination, were shown to be specific, since they could not be reproduced by their main metabolites. The present results strengthen the view that cAMP plays a pre-eminent role in the positive regulation of hepatocyte proliferation. Contrary to the postulate of the dualistic doctrine, cGMP by itself is not proliferogenic in the physiological range; in fact, cGMP acts as an ancillary, possibly dispensable, compound whose physiological role may be to help, in cooperation with cAMP, liver cells to cross the G1/S boundary of their growth-division cycle.     

The influence of culture conditions on the induction of tyrosine aminotransferase by cyclic nucleotides in rat hepatoma cells.

The increase in tyrosine aminotransferase activity which occurs in rat hepatoma tissue culture (HTC) cells in response to cyclic AMP analogs has been shown to be an enzyme induction, similar to the larger response observed in certain other hepatoma cells and in liver. A specific antibody to tyrosine aminotransferase has been used to show that the number of enzyme molecules and the rate of enzyme synthesis are increased by N6,O2'-dibutyryl cyclic AMP in HTC cells. The effect on tyrosine aminotransferase is also produced by various 8-substituted derivatives of cyclic AMP and occurs whether or not the enzyme has been preinduced with a glucocorticoid. The response of the enzyme is greater when HTC cells are maintained in monolayer than in suspension cultures. Neither cell growth nor serum is required for the response.     

Adrenal cells in tissue culture. The effect of papaverine and amytal on steroidogenesis, respiration and replication

The smooth-muscle relaxant papaverine has been shown to be a potent inhibitor of cyclic AMP phosphodiesterase activity (Kukovetz, W. R., and Poch, G. (1970) Naunyn Schmiedebergs Arch. Pharmakol.267, 189). Because of this, papaverine was tested in monolayer cultures of functional mouse adrenal cortex tumor cells for possible stimulatory effects on Steroidogenesis. At 10^?5m, papaverine was found to inhibit ACTH-stimulated steroidogenesis 50% and at 10^?4m, > 95%. This was associated with a > 10-fold increase in [¹⁴C]lactate production from [¹⁴C]glucose and a 50% reduction in ³²P_i, incorporation into macromolecules. These findings were similar to those observed with the barbiturate amytal, an inhibitor of the mitochondrial electron-transport chain at the level of oxidation of NADH (Site I). Papaverine was 100 times more effective than amytal in inhibiting steroidogenesis and 1000 times more effective in initiating an increase in glycolysis. In intact tumor cells and mitochondria isolated from normal rat adrenals, papaverine (10^?4m) completely inhibits oxygen uptake supported by malate or α-ketoglutarate. Oxygen uptake is restored by the addition of succinate, suggesting that, like amytal, papaverine inhibits respiration at Site I.Papaverine does not inhibit NADPH-supported cholesterol side-chain cleavage in bovine adrenal acetone powders or 11β-hydroxylation in normal rat adrenal cortex mitochondria. By contrast, amytal inhibits both these activities at concentrations comparable to that effective in intact adrenal cells, suggesting a direct interaction of amytal with cytochrome P-450. Both papaverine and amytal inhibit incorporation of thymidine into nuclear DNA to an extent far greater than that observed with either maximally stimulating levels of cyclic AMP or high concentrations of ACTH. Succinate does not reverse the inhibitory effects of either papaverine or amytal on thymidine incorporation into DNA. Papaverine increases intracellular cyclic AMP in both resting and ACTH-treated cells. However, the effects of papaverine on steroidogenesis, glycolysis, ATP-P_i exchange, and DNA synthesis in adrenocortical cells are not directly attributable to this action.     

Separation of cyclic AMP and cyclic GMP from thymidine, electrolytes and polyvalent nucleotides in tissue samples.

Cyclic AMP and cyclic GMP can be separated from thymidine and its possible metabolites, electrolytes, and polyvalent nucleotides using columns of acidic alumina. Electrolytes and thymidine are not adsorbed on acidic alumina at pH 4.4 while cyclic nucleotides and polyvalent nucleotides are adsorbed at this pH. Cyclic AMP and cyclic GMP are eluted together from acidic alumina with 0.2 M ammonium formate (pH 6.0) and the polyvalent nucleotides remain adsorbed. The cyclic nucleotides are separated by chromatography on Dowex AG 1 X 8 resin. Recovery is 60--64% for cyclic AMP and cyclic GMP isolated from renal tissue samples. This methodology permits the separation of tritiated thymidine from cyclic nucleotides which are present in tissue preparations used in studies on the role of cyclic nucleotides in cellular growth.     

Hydrodynamic effects on cells in agitated tissue culture reactors

Tissue cells are known to be sensitive to mechanical stresses imposed on them by agitation in bioreactors. The amount of agitation provided in a microcarrier or suspension bioreactor should be only enough to provide an effective homogeneity. Three distinct flow regions can be identified in the reactor: bulk turbulent flow, bulk laminar flow, and boundary-layer flows. Possible mechanisms of cell damage are examined by analyzing the motion of microcarriers or free cells relative to the surrounding fluid, to each other, and to moving or stationary solid surfaces. The primary mechanisms of cell damage appear to result from (a) direct interaction between microcarriers and turbulent eddies, (b) collisions between microcarriers in turbulent flow, and (c) collisions against the impeller or other stationary surfaces. If the smallest eddies of turbulent flow are of the same size as the microcarrier beads, they may cause high shear stresses on the cells. Eddies the size of the average interbead spacing may cause bead-bead collisions which damage cells. The severity of the collisions increases when the eddies are also of the same size as the beads. Bead size and the interbead distance are virtually equal in typical microcarrier suspensions. Impeller collisions occur when the beads cannot avoid the impeller leading edge as it advances through the liquid. The implications of the results of this analysis on the design and operation of tissue culture bioreactors are also discussed.