Joseph S. Makarski

Stimulation of cyclic AMP production by vasoactive agents in cultured bovine aortic and pulmonary artery endothelial cells.

SummaryThe ability of selected vasoactive agents to influence cyclic AMP levels of confluent, early-passaged bovine calf aortic and pulmonary artery endothelial cells was investigated. Among the agents tested, only the catecholamines (isoproterenol, epinephrine, nonrepinephrine) and prostaglandins (PGE1, PGE2, PGF2a) resulted consistently in increased cyclic AMP production in both cell populations. The degree of cyclic AMP stimulation obtained with other vasoactive compounds (angiotensins I and II, bradykinin, and serotonin) tended to be either very small or difficult to reproduce. Isoproterenol stimulation was blocked completely by propanolol, a β-blocking agent, but not by phentolamine, an α-blocking agent. These results reveal that bovine calf aortic and pulmonary artery endothelial cells are responsive to catecholamines and prostaglandins, and therefore presumably possess both sensitive adenylate cyclases and plasma membrane receptors for these compounds.

Effect of protein-hydroxyethylmethacrylate hydrogels on cultured endothelial cells.

The use of protein hydroxy ethylmethacrylate (HEMA) hydrogels to control cell morphology and growth, as well as the synthesis of extracellular matrix components, is described in this communication. HEMA hydrogels prepared with collagen support growth of embryonic lung fibroblasts (IMR-90), as well as bovine aortic and pulmonary artery endothelial cells at a level comparable to the respective cells grown on tissue culture surfaces. On the other hand, HEMA hydrogels prepared with solubilized elastin inhibit the fibroblast growth and prevent both types of endothelial cell cultures from achieving their normal morphology. These morphologically altered endothelial cells resume a normal cobblestone-like appearance when subcultivated from the elastin-HEMA hydrogels to tissue culture plastic. When pulsed with [14C]proline, the procollagens synthesized by the endothelial cells on the different surfaces vary, as shown by immunoprecipitation and polyacrylamide gel electrophoresis. On the standard tissue culture plastic, the confluent cells produce mainly type III procollagen in the medium, whereas those endothelial cells grown on collagen and elastin-HEMA hydrogels synthesize primarily type I procollagen (much like sprouting cells on tissue culture plastic), regardless of their morphology.

Studies on the Mechanism of Decreased Angiotensin I Conversion in Rat Lungs Injured with Alpha-Naphthylthiourea

Lung endothelial cell injury may be an important early event in the pathogenesis of increased permeability pulmonary edema. Since angiotensin converting enzyme (ACE) is located on the luminal surface of the endothelial cell membrane, we sought to determine whether the conversion of angiotensin I (AI) to angiotensin II is decreased after acute lung injury to rats, induced by alpha-naphthylthiourea (ANTU), and we investigated the mechanism of the decrease. We found that lungs isolated from rats treated 4 h earlier with ANTU at a dose of 15 mg/kg body weight (BW) had decreased AI conversion when perfused with Krebs-Henseleit at a constant flow rate of 30 ml/min/kg BW. When perfusate flow rate was increased from 30 to 50 ml/min/kg BW, lungs isolated from rats treated with 10 mg/kg BW ANTU also had decreased AI conversion when compared to controls treated with a vehicle, Tween 80. Investigating the mechanism of decreased AI conversion, there were no differences among experimental groups in pulmonary arterial pressures or effluent perfusate pH or pO2. There was no correlation between lung wet/dry weight ratios and the extent of AI conversion among control rat lungs. Lung homogenate and serum ACE activity did not differ among control rats and rats pretreated with the two doses of ANTU. Ultrastructural studies revealed an increased percentage of capillaries with blebbing of endothelial cells in lungs injured with ANTU, as compared to controls, but no evidence of increased endothelial cell denudation in injured lungs. We conclude that angiotensin I conversion is decreased after ANTU lung injury and that the extent of decrease is related to the dose of ANTU and to perfusate flow rate. Although we cannot exclude decreased vascular surface area perfused as a cause of decreased conversion, we speculate that subtle changes in the luminal endothelial cell membrane may have caused decreased AI conversion after ANTU lung injury.

Loss of adenylate cyclase hormonal sensitivity in chemically transformed epithelial cells.

The hormonal sensitivity of adenylate cyclase from a normal rat liver epithelial cell line (K16) and its chemically transformed derivative (W8) were compared. Intact normal rat liver cells had markedly increased cAMP levels after brief exposure to epinephrine, isoproterenol, norepinephrine or prostaglandin E1. In contrast, the cAMP levels of chemically transformed cells were relatively unaffected by these same compounds even after prolonged incubation. A comparison of broken cell adenylate cyclase activities revealed a decreased basal activity in the chemically transformed cells; the response to NaF was similar in the two cell lines, while the response to catecholamines and prostaglandins paralleled the intact cell studies. These data suggest that one reason for loss of adenylate cyclase hormonal responsiveness in chemically transformed rat liver epithelial cells may be a dysfunction or loss of hormone binding sites.

Inhibition of human prostatic epithelial cell replication by cAMP and selected analogs.

Abstract The inhibition of human prostatic epithelial cell (MA-160) replication by cAMP and certain analogs was explored in tissue cultures. When untreated fetal bovine serum was used to supplement the culture medium, cyclic AMP (cAMP) markedly inhibited cell growth. The inhibition was reversed by equimolar concentrations of uridine. Inhibition by 8-methyl-thio-cAMP (MES) was somewhat less effective and was not reversed by uridine. After heat treatment of the fetal bovine serum, which inactivated the cAMP phosphodiesterases, cAMP became less effective in cell growth inhibition, whereas the activity of MES remained unaltered. Dibutyryl cAMP (db-cAMP) had no effect on cell growth, however, when combined with the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX), significant retardation of cell replication was observed. Cells treated for 24 h with 0.5 mM MES took up and incorporated significantly less [3H]TdR and [3H]uridine than control cells. Treatment of cells with 0.5 mM cAMP for 24 h, on the other hand, resulted in both substantially increased [3H]TdR uptake and increased [3H]uridine incorporation into RNA. The effects of similar treatment with db-cAMP plus MIX closely paralleled those of MES with marked inhibition of the uptake and incorporation of both thymidine and uridine.

Adenylate cyclase activity in cultured epithelial cells

SummaryThe cyclic AMP metabolism of cultured epithelial cells was investigated. Epinephrine or 1-methyl, 3-isobutylxanthine (MIX) alone had no effect on cyclic AMP levels in intact cells, whereas the combination of the two agents yielded a 6- to 10-fold increase in cyclic AMP levels. Both basal and stimulated cyclic AMP levels decreased with increasing cell density. Cell-free adenylate cyclase preparations were stimulated markedly by epinephrine or isoproterenol in the absence of MIX. Since the epithelial cells were found to have a relatively small amount of cyclic nucleotide phosphodiesterase (PDE) activity, the requirement for MIX to visualize intact cell responsiveness to epinephrine could be explained only partially by its PDE inhibitory properties.