Linda L. Slakey

Angiotensin-converting enzyme: accumulation in medium from cultured endothelial cells.

Abstract The activity of angiotensin converting enzyme has been measured in endothelial cells cultured from hog aorta, and in serum-free culture medium taken from both endothelial cells and smooth muscle cells. Endothelial cells maintained in medium containing 20% fetal calf serum contained 43 pmol/min/106 cells of converting enzyme activity; freshly collected cells contained 518 pmol/min/106 cells. Endothelial cells held in serum-free medium release this activity into the medium in amounts up to 40 times that associated with the cells; at the same time the activity associated with the cells rises 2 fold. The rise in cell-associated activity and the appearance of activity in the medium are both blocked by cycloheximide. These observations provide direct evidence that endothelial cells can produce excess angiotensin-converting enzyme and release it in active form, and thus serve as the source of circulating converting enzyme activity.

Adenosine Receptors Activate Adenylate Cyclase and Enhance Secretion from Bovine Adrenal Chromaffin Cells in the Presence of Forskolin

Abstract: Cells of the adrenal medulla release not only catecholamines but also high concentrations of neuropeptides and nucleotides. Chromaffin cells, like many neuronal cells, have a diversity of receptors: adrenergic receptors, peptide receptors, histamine receptors, and dopamine receptors. We recently reported that these cells have nucleotide receptors that can mediate inhibition of the secretory response. The present studies show that adenosine, in the presence of enabling concentrations of forskolin, can potently enhance response to nicotinic stimulation. Neither adenosine nor forskolin alone produces a significant effect. A marked rise in intracellular cyclic AMP (cAMP) concentration is associated with the enhancement of secretion caused by forskolin plus adenosine. A phosphodiesterase inhibitor, Ro 20–1724, used together with forskolin produces significant increases in both cellular cAMP content and catecholamine secretion. However, the adenosine agonist 5′‐N‐ethylcarboxyadenosine elevates cellular cAMP content in the presence of forskolin without having any positive effect on secretion. This finding suggests that the rise in cAMP level may not be the sole cause of the increase in secretion by adenosine.

Synthesis of a collagenase inhibitor by smooth muscle cells in culture

Abstract Serum-free medium that was incubated for 24 hours with confluent cultures of smooth muscle cells contains a potent inhibitor of mammalian collagenase but not of bacterial collagenase. Synthesis of this inhibitor by these cultures is blocked by cycloheximide. The inhibitor is sensitive to trypsin but is stable to heating at 60° for 10 minutes and to acid treatment. Reduction with mercaptoethanol followed by alkylation destroys the inhibitory activity. This inhibitor may play a physiological role in the slow turnover of collagen that is observed in the vasculature.

Hydrolysis of diadenosine 5',5''-P',P''-triphosphate (Ap3A) by porcine aortic endothelial cells.

Diadenosine triphosphate is present in platelet-dense granules and released quantitatively on platelet aggregation. We have found that intact porcine aortic endothelial cells can efficiently hydrolyze extracellular diadenosine triphosphate. The products of diadenosine triphosphate hydrolysis are adenosine monophosphate and adenosine diphosphate. Adenosine diphosphate is a potent stimulus of platelet aggregation. Since platelet-dense granules contain high concentrations of adenosine triphosphate and adenosine diphosphate, we examined endothelial cell hydrolysis of a mixture of diadenosine triphosphate and adenosine triphosphate. We find that the presence of adenosine triphosphate severely inhibits the hydrolysis of diadenosine triphosphate. Thus, although endothelial cells can rapidly clear extracellular diadenosine triphosphate, during platelet aggregation the hydrolysis of diadenosine triphosphate may be slow due to the presence of high concentrations of other adenine nucleotides. This phenomenon may be important physiologically if, as current evidence implies, diadenosine triphosphate is involved in the maintenance of hemostasis.

Charge-coupled device imaging of rapid calcium transients in cultured arterial smooth muscle cells

Transient changes in the concentration of intracellular free calcium are associated with the transduction of primary signals and the subsequent employment of Ca2+ as a second messenger in a multitude of cell types. These transients, typically monitored with the calcium-sensitive fluorescent dye Fura-2, are known to occur with a time course in the order of seconds. In order to accurately monitor such rapid changes in intracellular free calcium concentration in both single cells and simultaneously in several cells in a single field, we have developed a digital fluorescence imaging system based on a charge-coupled device (CCD) camera. We report here on the detailed kinetics of calcium increases in cultured arterial swine smooth muscle cells in response to the agonist ATP.

Plasma-derived serum as a selective agent to obtain endothelial cultures from swine aorta

SummaryEndothelial cell and smooth muscle cell cultures from artery wall provide a potential model system for studying cellular processes involved in atherogenesis. To prepare serial subcultures of swine arterial endothelial cells that are free of smooth muscle cells without either selecting a small population or subjecting the cells to cytotoxic conditions, we used swine plasma-derived serum (SPDS) to establish conditions in which endothelial cells have a growth advantage. Endothelial cells were collected by collagenase digestion and smooth muscle cell cultures were prepared by outgrowth from explants of arterial medial segments. Growth rates were compared when each cell type was maintained on SPDS, or fetal bovine serum (FBS), or swine whole serum (SWS). When 20% FBS or SWS were used the doubling times were <30 h for both endothelial cells and smooth muscle cells. On 20% SPDS the doubling time for endothelial cells was 32 h, but for smooth muscle cells it was at least 168 h. Using SPDS, we prepare endothelial subcultures from swine aorta that express principally polygonal morphology at confluence. Endothelial cell cultures grown on SPDS have higher angiotensin-converting enzyme than those grown on FBS.

Angiotensin converting enzyme in cultured endothelial cells and growth medium: Relationships to enzyme from kidney and plasma

We have previously reported that cultured cells from swine aorta possess angiotensin converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) and release it into serum-free culture medium. The present work compares enzyme from these two sources, and from swine kidney and serum, with respect to antibody and lectin binding. Purified enzyme from swine kidney, and the activity in swine serum, cultured endothelial cells and culture medium bind similarly to rabbit antibodies prepared against the kidney converting enzyme. Enzyme from each of these sources was allowed to bind to an immobilized lectin (Ricinus communis), which binds to terminal galactose residues of glycoproteins. Increasing concentrations of galactose were used to remove enzyme from the lectin column and the distribution of enzyme activity in the galactose eluates was determined. The elution pattern was similar for kidney and endothelial cell enzyme, and different from the pattern found for both serum and medium enzymes. Neuraminidase treatment of either serum or medium enzyme altered the distribution of activity eluted to that found for endothelial cell or kidney enzymes. The effects of neuraminidase suggest that the difference in lectin binding between cell and medium enzyme reflects differences in the number of terminal sialic acid residues that cover galactose residues.

Accessibility of lysolecithin in catecholamine secretory vesicles to acyl coa: Lysolecithin acyl transferase

Abstract Lysolecithin (monoacylglycerophosphorylcholine) accounts for 13 to 20% of the lipid phosphorous of the bovine adrenal catecholamine secretory vesicles (chromaffin granules). We have incubated purified vesicles with [1-14C] oleyl coenzyme A and rat liver microsomes containing acyl coenzyme A: monoacylglycerophosphorylcholine acyl transferase to determine the accessibility of the granule membrane lysolecithin to another membrane. No acylation of lysolecithin occurs when the chromaffin granules are intact. The accessibility of the granule membrane lysolecithin increases markedly when the vesicles are broken.

Properties of a collagenase inhibitor partially purified from cultures of smooth muscle cells.

An inhibitor of mammalian collagenase has been partially purified from the spent medium of smooth muscle cells grown in culture. The inhibitor is a glycoprotein with an apparent molecular weight of 25,000. It is stable to heat, acid, and mercurials, but is destroyed by trypsin treatment and by reductive alkylation. The inhibitor interacts with active mammalian collagenase and this interaction results in the loss of enzymatic activity. This presumptive collagenase-inhibitor complex is stable to the treatment with mercurials and to trypsin. These latter observations suggest that this inhibitor is different from other collagenase inhibitors that are thought to be responsible for the latency of the enzyme.

Modulation of response to adenosine in vascular smooth muscle cells cultured in defined medium.

SummaryCultured pig aortic smooth muscle cells maintain a viable, quiescent state in a chemically defined medium that contains 10−6M insulin, 5µg/ml transferrin, and 0.2 mM ascorbate. DNA synthesis and DNA content were determined by measuring tritiated thymidine incorporation and DNA-binding to the fluorescent probe 4′,6-diamidino-2-phenylindole, respectively. The majority of the population of cells in defined medium cultures were diploid. Tritiated thymidine uptake in cells in defined medium was one-tenth that observed in cells in fetal bovine serum-containing medium. The study of cellular cyclic AMP level in response to extracellular adenosine stimulation in dividing cells and quiescent cells showed that cells in defined medium had a lower extent of response to adenosine compared to cells cultured in serum-containing medium. Both the cell growth index and the response to adenosine of cells cultured in defined medium were reversible after replacing the medium with 10% fetal bovine serum-containing medium, which suggests that the cells in defined medium were healthy and were capable of modulating cellular metabolism depending on culture conditions.