Iron accumulation by bovine aortic endothelial cells.

Bovine aortic endothelial cells in monolayers were used to study iron and transferrin binding and transport mechanisms. Diferric bovine transferrin labeled with 59Fe was used as an iron donor. We have shown the presence of saturable iron uptake when cells were incubated with varying concentrations of diferric transferrin. This uptake decreased when the cells were treated with trypsin, ammonium chloride and methylamine. The effects of the latter two could be reversed by the addition of 2.0 mM Ca2+. Energy dependence was shown by using various electron transport/oxidative phosphorylation inhibitors. The presence of transferrin receptors on the cell surface was confirmed by their isolation, SDS-PAGE and autoradiography. There were approximately 1.5 x 10(6) transferrin receptors per cell with a Kd of 9.1 x 10(-7) M in the physiological iron range. Iron was also taken up when the cells were incubated with radioactive ferrous iron without transferrin. Uptake was not affected by receptor-mediated endocytosis inhibitors. Calcium increased ferrous iron uptake and overcame the effects of metabolic inhibitors on iron uptake from transferrin. A ferrireductase was detected in cell membranes. It is proposed that iron is transported by bovine endothelial cells by two mechanisms: one is receptor-mediated endocytosis from transferrin, and the other involves a non-endocytic mechanism from transferrin and Fe2+, which is possibly promoted by Ca2+.

Arterial receptors for adrenal steroids and transport of electrolytes in vascular smooth muscle.

The role of arterial receptors to mineralocorticoids (MC) and glucocorticoids (GC) in the induction by MC and GC of changes in transmembrane transport of sodium (Na+) and water was investigated. Implantation of Silastic rubber strips impregnated with 11-desoxycorticosterone acetate (DOCA) in rabbits was followed by a marked increase in vascular smooth muscle cell membrane permeability to Na+ and hypertension. Both of these effects were preventable with progesterone, an anti-MC at the steroid-receptor level, implanted in relative excess simultaneously with DOCA, in approximately 50% of the implanted animals. The other 50% were hydroxylating in vivo progesterone to 11-desoxycorticosterone (DOC) efficiently enough not to yield the necessary ratio of progesterone to DOC for the sufficient MC receptor blockage. In vascular smooth muscle cell culture, grown in the presence of steroids, GC but not MC increased intracellular water space. This increase was preventable by a potent synthetic anti-GC,RU 38486, a steroid with high affinity for GC receptors, added to culture medium along with GC. These results provide evidence that both the in vivo effect of MC on Na+ permeability and the induction of hypertension, and the in vitro effect of GC on water transport in cultured vascular smooth muscle cells are elicited through the receptor-mediated molecular mechanism(s) for action of these steroids in the arterial wall.

Regulation of intracellular arachidonate in normal and stressed endothelial cells.

The uptake of arachidonate and stearate from serum-free media by endothelial cells was investigated over a 48 h period. Arachidonate was rapidly incorporated into both the phospholipids and triacylglycerols. Triacylglycerol incorporation reached a maximum at 2 h and then rapidly declined with a concomitant increase in phospholipid incorporation. High initial arachidonate incorporation into phosphatidylcholine was followed by a partial transfer of that arachidonate to phosphatidylethanolamine. In contrast, stearate was slowly incorporated into all of the phospholipids and was not incorporated into the triacylglycerols. Cells stimulated with A23187 for 24 h cleaved stearate from all the phospholipids equally, whereas more arachidonate was cleaved from phosphatidylethanolamine than from the other phospholipids. Released arachidonate was both metabolized and reacylated into the triacylglycerols. Our results suggest that triacylglycerols serve as a modulator of intracellular arachidonate concentrations in endothelial cells.

Comparison of two types of endothelial cells in long term culture.

Bovine aortic endothelial cells have been serially subcultured and maintained for ten months (24 passages) without any apparent loss of endothelial-like properties. There appear to be two endothelial cell types; one type is mononucleated, 40-60 mu in diameter, and the other (giant) cell type, multinucleate and 300-600 mu in diameter. Both cell types were identified as endothelial cells by established criteria; 1. ultrastructurally, by the presence of Weibel-Palade bodies, microfilaments and pinocytotic vesicles and 2. immunologically, by the presence of factor VIII antigen and thrombosthenin.

Adenosine cyclic 3',5'-monophosphate-dependent protein kinase from human platelets.

A single cyclic AMP-dependent protein kinase (EC 2.7.1.37) has been isolated from human platelets by using DEAE-cellulose ion-exchange chromatography and Sephadex G-150 gel filtration. The molecular weight of the protein kinase was estimated to be 86 490. In the presence of cyclic AMP, the protein kinase could be dissociated into a catalytic subunit of molecular weight 50 000, and either one regulatory subunit of molecular weight 110 000 or two regulatory subunits of molecular weights 110 000 and 38 100, depending on the pH used. Recombination of either of the regulatory subunits with the catalytic subunit restored cyclic AMP-dependency in the catalytic subunit. The apparent Km for ATP in the presence of 10 muM Mg2+ was 4 muM (plus cyclic AMP) and 4.3 muM (minus cyclic AMP). The concentration of cyclic AMP needed for half-maximal stimulation of the protein kinase was 0.172 muM and apparent dissociation constants of 3.7 nM (absence of MgATP) and 0.18 muM (presence of MgATP) were exhibited by the "protein kinase-cyclic AMP complex". The enzyme required Mg2+ for maximum activity and showed a pH optimum of 6.2 with histone as substrate. In addition to four major endogenous platelet protein acceptors of apparent molecular weights 45 000, 28000, 18 500, and 11 100, the platelet protein kinase also phosphorylated the exogenous acceptor proteins thrombin, collagen and histone, all capable of inducing platelet aggregation. Prothrombin, a nonaggregating agent, was not phosphorylated.

Culture of arterial endothelial cells: characterization and growth of bovine aortic cells.

Arterial endothelial cells were obtained from bovine aortae by mild treatment with collagenase and medium perfusion. These cells were cultured in RPMI-1640 medium containing 15mM Hepes buffer and 35% fetal calf serum at pH 7.35. Essentially all (90-95%) the effluent cells were viable and 80% of these cells attached to the substratum within 1 hour. Small patches of attached cells coalesced to form confluent monolayers in 3-5 days. Confluent monolayers of endothelial cells consisted of a homogeneous population of tightly packed, polygonal cells. Selected cultures were serially subcultured (trypsin-EDTA) for 12-14 months (30-35 passages) without any apparent change in morphology or loss of growth characteristics. Primary and three-month old (15 passages) cultures had population doubling times of 32-34 hours and 29-31 hours, respectively. These cells (primary and subcultures) did not require a minimum cell number to become established in culture. Bovine endothelial cells (primary, first, fifth and thirteenth passages) were characterized ultrastructurally by the presence of Weibel-Palade bodies, pinocytotic vesicles and microfilaments and immunologically by the presence of thrombosthenin-like contractile proteins and Factor VIII antigen. The intercellular junctions of post-confluenct cultures stained specifically with silver nitrate. From these data, we concluded that identifiable endothelial cells could be obtained from bovine aortae and cultured and maintained for prolonged periods of time.