Rat aortic smooth muscle cells were isolated and maintained in primary culture. After 2-3 days, cells recovered their contractile phenotype and could be induced to contract in response to vasopressin and angiotensin II. Vasopressin- and angiotensin-specific binding sites were detected on these cells, using tritiated Lys8-vasopressin, Asn1-Val5-angiotensin II, and Sarc1-Ile8-angiotensin II. Vasopressin binding sites had Kd values of 30 and 12 nM for Lys8-and Arg8-vasopressin, respectively, and a maximal binding capacity of 25,000 sites/cell. They displayed several of the expected characteristics of vasopressin receptors involved in the vasopressor response in vivo. A highly significant correlation was found between the relative agonistic or antagonistic vasopressor potencies of a series of vasopressin structural analogues and their relative abilities to inhibit [3H]vasopressin binding to aortic smooth muscle cells. Specific binding sites for Asn1-Val5-angiotensin II and Sarc1-Ile8-angiotensin II had the following characteristics: Kd = 2.3 and 1.3 nM, respectively; maximal capacity: 50,000 sites/cell. Vasopressin and angiotensin did not modify the intracellular cyclic AMP content of aortic smooth muscle cells.

No abstract

Neuroblastoma cells were synchronized by a combined isoleucine plus glutamine starvation. Adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] was measured under basal conditions and in the presence of dopamine, adenosine, and prostaglandin (PG) E1. A clear dissociation occurred between the respective evolution patterns of basal and agonistetimulated adenylate cyclase activities. The magnitudes of the enzyme response to PGE1, adenosine, and dopamine also exhibited different evolution patterns during the cell cycle. Evolution of adenylate cyclase responsiveness to PGE1 during the cell cycle exhibited striking similarities with the intracellular 3':5'-cyclic AMP changes observed elsewhere.Use of theophylline and fluphenazine as specific inhibitors of adenosine and dopamine, respectively, made it possible to demonstrate that adenosine, dopamine, and PGEI stimulated adenylate cyclase through independent receptor sites. Furthermore, whatever the stage of the cell cycle, responses to these three agonists were not additive, indicating that the receptors of adenosine, dopamine, and PGE1 control the same adenylate cyclase moieties. The data suggest that adenylate cyclase cell content and enzyme responsiveness to specific agonists can be independently controlled. In all synchronized cell types so far studied the intracellular 3':5'-cyclic AMP concentration varies at different stages of the cell cycle, being minimal at the time of mitosis and almost always reaching a maximal value in the early GI phase (1-4).More recently it was shown in fibroblasts that at the beginning of the G1 phase intracellular cyclic AMP drops abruptly while 3':5'-cyclic GMP rises. It was suggested that the changes of cyclic nucleotides regulate the cell cycle and more generally that cyclic nucleotides are involved in the "pleiotypic" control of cell growth (5,6 The aim of the present work was to analyze the evolution pattern of adenylate cyclase during the cell cycle in neuroblastoma cells and the evolution patterns of the responses to three different regulatory agents: adenosine, dopamine, and prostaglandin (PG) El. The latter seems to be a candidate for intracellular regulation because several cell lines, including neuroblastoma, are able to synthesize prostaglandins (7). MATERIAL AND METHODSMouse neuroblastoma cells were derived from the cholinergic clone NS20 (8). Current properties of these cells are: total protein content is 0.250 mg/106 cells; specific choline acetyltransferase activity is 60 pmol/mg protein per min. Present karyotype: 80% of the cells have 60 chromosomes and 20% have 120.Cell Culture. Cells were grown on Falcon petri dishes in Eagle's minimal essential medium containing Earle's salts solution supplemented with 10% fetal calf serum (Gibco), penicillin G at 50 international units/ml and streptomycin sulfate at 5 ttg/ml. Cells were incubated at 370 in 90% air/10% CO2.The doubling time of the cell population was close to 20 hr.Synchronization Procedure. Cells were collected during the exponentia...

No abstract