Studies of cyclic AMP action using mutant tissue culture cells

Summary 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. Presented in the Opening Symposium on Nutritional Factors and Differentiation at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, June 6–9, 1977. The work was supported in part by National Science Foundation Grant BMS 75-06764 and National Institutes of Health Grants GM 16496 and GM 00001. P.C. is the recipient of National Institutes of Health Research Career Development Award GM 00308. P. A. I. is an Established Investigator of the American Heart Association.     

Endogenous cyclic AMP in thyroid cell culture. Effect of thyroid-stimulating hormone and dibutyryl cyclic AMP.

We have studied the variations of endogenous cyclic AMP levels in thyroid cells cultured over a period of 7 days in several conditions: in the presence of thyroid-stimulating hormone or dibutyryl cyclic AMP which both promote the aggregation of isolated cells into follicles, and in their absence when cells develop as a typical monolayer. In follicle-forming cells, the cyclic AMP level was found to rise during the first day of culture, then to fall rapidly. In monolayer-forming cells, the cyclic AMP content slightly increases attaining the same level as found in other cells at the fourth day, which remains stable till the seventh day. We have investigated the response of these cells cultured in the presence of dibutyryl cyclic AMP retain the capability of increasing their cyclic AMP concentration whereas monolayer-forming cells do not preserve this quality of thyroid cells.     

Endogenous cyclic AMP in thyroid cell culture. Effect of thyroid-stimulating hormone and dibutyryl cyclic AMP

We have studied the variations of endogenous cyclic AMP levels in thyroid cells cultured over a period of 7 days in several conditions: in the presence of thyroid-stimulating hormone or dibutyryl cyclin AMP which both promote the aggregation of isolated cell into follicles, and in their absence when cells develop as a typical monolayer. In follicle-forming cells, the cyclic AMP level was found to rise during the first day of culture, then to fall rapidly. In monolayer-forming cells, the cyclic AMP content slightly increases attaining the same level as found in other cells at the fourth day, which remains stable till the seventh day. We have investigated the response of these cells to the acute effect of thyroid-stimulating hormone: only cells cultured in the presence of dibutyryl cyclic AMP retain the capability of increasing their cycli AMP concentration whereas monolayer-forming cells do not preserve this quality of thyroid cells.     

Gonadotropin stimulation of cyclic AMP levels in Chinese hamster ovary cells in culture.

When Chinese Hamster Ovary (CHO) cells, incubated in serum-free medium, are exposed to gonadotropins a transient increase in the intracellular concentration of cyclic AMP is observed. Maximum accumulation of cyclic AMP is noted 30 minutes after addition of either human chorionic gonadotropin (hCG) or follicle stimulating hormone (FSH). Within one to two hours after hormone addition, the intracellular concentrations of cyclic AMP have returned to basal levels. The enhancement of intracellular cyclic AMP levels by hCG is hormone concentration dependent, with maximal stimulation observed at 10 micrograms/ml hCG. The exogenous addition of gonadotropins also slows the growth rate of CHO cells. This effect on growth seems to be mediated through cyclic AMP since the growth rate of a mutant of CHO cells defective in the catalytic subunit of cyclic AMP dependent protein kinase is only slightly decreased.     

Modulation of cyclic AMP in purified rat mast cells. II. Studies on the relationship between intracellular cyclic AMP concentrations and histamine release.

Changes in intracellular and extracellular rat mast cell adenosine 3':5' monophosphate (cAMP) concentrations during stimulation of histamine release by 48/80 were studied. There was a rapid and progressive fall in intracellular cAMP beginning within 10 sec after the addition of 48/80. The lowest cAMP values were obtained at 10 min, with return to control levels by 30 min. The fall in cAMP was dose-related with progressive decreases in 10-min cAMP measurements as the 48/80 concentration was increased from 0.25 to 1.00 mug/ml. There was a graded increase in histamine release over the same concentration range. Attempts to demonstrate significant amounts of cAMP in the medium during 48/80 stimulation were unsuccessful, indicating that the changes in cAMP intracellularly are not due to altered cellular permeability. There was a general correlation between the ability of pharmacologic agents to sustain high intracellular levels of cAMP in the presence of 48/80, and inhibition of histamine release. Theophylline (20 mM) which increased cAMP levels 2- 3-fold prevented a detectable decrease in cAMP after 1 mug/ml 48/80 (measured at 10 min) and almost completely inhibited histamine release. Prostaglandin E1 (27 muM) also raised cAMP levels, decreased the 48/80-induced fall in cAMP (by 42%). Epinephrine increased mast cell cAMP levels, but did not prevent the subsequent 48/80-induced decrease in cAMP and did not inhibit histamine release. Carbamylcholine (1 nM), adenine (1 muM), and diazoxide (10 muM) lowered mast cell cAMP and potentiated 48/80 induced release. In view of previous studies from this laboratory indicating that 48/80 stimulates mast cell phosphodiesterase, it seems likely that the 48/80-induced fall in cAMP is due, at least in part, to increased cAMP destruction. Since agents which prevent the fall in cAMP inhibit histamine release, it is apparent that cAMP is an important part of the control mechanism of histamine secretion. On the other hand, it cannot be concluded that a decrease in cAMP alone is sufficient to produce a response since carbamylcholine, diazoxide, and adenine which lower cAMP do not alter histamine release unless 48/80 is also present.     

Effect of prostaglandin E2 on cyclic AMP levels in limb cells of mouse mutant brachypodism.

Mouse embryo limb cells carrying either the brachypodism (bpH/bpH) mutation or its wild-type (+/+) allele were tested for their ability to accumulate cyclic AMP in response to prostaglandin E2 (PGE2) between Embryonic Days E12 and E14. Mutant cells exhibited a precocious increase in cyclic AMP. In the absence of PGE2 but in the presence of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX), the brachypodism cells accumulated a significantly lower amount of cyclic AMP by Day E14. Limb cells carrying the bpH mutation may provide a useful experimental system to study the PGE2-cyclic AMP-cartilage differentiation interrelationship.     

Coexpression of mutant and wild type protein kinase in lymphoma cells resistant to dibutyryl cyclic AMP.

A mutant clone resistant to dibutyryl cyclic AMP was isolated from S49 mouse lymphoma cells. The mutant expressed a form of cyclic AMP-dependent protein kinase distinguishable from wild type kinase by its decreased sensitivity to activation by cyclic AMP and its increased thermal lability. Hybrids formed between mutant and wild type cells were resistant to dibutyryl cyclic AMP and expressed both mutant and wild type activities in about equal amount. The parent mutant cells also appeared to express wild type kinase activity, but at a lower level. We conclude that wild type S49 cells have and express two identical alleles for the regulatory subunit of protein kinase, one of which has undergone mutation in the mutant cells.     

New signaling pathway for parathyroid hormone and cyclic AMP action on extracellular-regulated kinase and cell proliferation in bone cells. Checkpoint of modulation by cyclic AMP

cAMP signaling, activated by extracellular stimuli such as parathyroid hormone, has cell type-specific effects important for cellular proliferation and differentiation in bone cells. Recent evidence of a second enzyme target for cAMP suggests divergent effects on extracellular-regulated kinase (ERK) activity depending on Epac/Rap1/B-Raf signaling. We investigated the molecular mechanism of the dual functionality of cAMP on cell proliferation in clonal bone cell types. MC3T3-E1 and ATDC5, but not MG63, express a 95-kDa isoform of B-Raf. cAMP stimulated Ras-independent and Rap1-dependent ERK phosphorylation and cell proliferation in B-Raf-expressing cells, but inhibited growth in B-Raf-lacking cells. The mitogenic action of cAMP was blocked by the ERK pathway inhibitor PD98059. In B-Raf-transduced MG63 cells, cAMP stimulated ERK activation and cell proliferation. Thus, B-Raf is the dominant molecular switch that permits differential cAMP-dependent regulation of ERK with important implications for cell proliferation in bone cells. These findings might explain the dual functionality of parathyroid hormone on osteoblastic cell proliferation.     

A mutant strain of Polysphondylium pallidum deficient in production of cyclic AMP.

A mutant strain (PN507) of the cellular slime mold Polysphondylium pallidum is described which: (a) is morphogenetically abnormal in stalk formation; (b) secretes unusually low quantities of cyclic AMP; (c) responds to exogenous cyclic AMP in the same manner as wild type, by differentiating stalk cells and synthesizing several specific proteins; (d) complements with other morphogenetic mutants secreting normal amounts of cyclic AMP to produce fruiting structures resembling wild type. The tentative conclusion is that the critical defect of PN507 is low production of cyclic AMP.     

Adenosine, AMP, cyclic AMP, theophylline and the action and production of erythropoietin.

Erythropoiesis, as measured by the uptake of 59Fe into plethoric mice, is stimulated by adenosine, AMP, cyclic AMP, and dibutyryl cyclic AMP, but not by cytidine, its nucleotides or cyclic GMP. This stimulation is erythropoietin dependent, because it is prevented by anti-erythropoietin. Theophylline neither stimulates erythropoiesis nor potentiates the action of erythropoietin on bone marrow cells in plethoric mice. Theophylline does potentiate the production of erythropoietin in rats following a frief hypoxic exposure but does not cause a similar increase in mice.     

Modulation of cyclic AMP in purified rat mast cells. III. Studies on the effects of concanavalin A and anti-IgE on cyclic AMP concentrations during histamine release.

Changes in rat mast cell cyclic adenosine 3',5' monophosphate (cAMP) concentrations during stimulation of histamine release by concanavalin A (con A) and anti-IgE were studied. Con A caused an increase in cAMP with a mean peak level at 20 sec of 232% of control (range 164% to 365%). Con A-stimulated cells demonstrated falls toward control levels after 20 sec, but generally remained above control for at least 5 min. By 10 min cAMP had returned to control values. The con A effect on cAMP occurred in the absence of phosphatidyl serine but was markedly inhibited by 5 mM alpha-methyl-D-mannose. Anti-IgE induced a less marked increase in cAMP (157% of control, range 110% to 540% of control) which reached a peak at 20 sec. Two monospecific goat anti-rat myeloma IgE antisera induced similar changes in cAMP whereas normal goat IgG had no effect. These peak values were followed by a rapid decrease in cAMP. Within 2 min the cAMP content of anti-IgE stimulated cells had fallen to levels well below control and remained below control levels from 45 sec to over 15 min. Histamine release in both systems began after the peak cAMP levels, during the period of rapid destruction of cAMP.     

Quantitative aspects of regulation of cellular cyclic AMP levels. II. Kinectics of drug action in a model cyclic AMP generating system.

The cyclic AMP generating system introduced in paper I is considered as a simple well-defined pharmacological-endocrine system. Its behaviour, as anticipated by equations derived from a model of the system, is compared with that predicted by the expressions presented by Clark, Ariens and Stephenson in order to quantitate drug actions in general. Consideration is given to the anticipated effect on steady state levels of cyclic AMP of (1) modifying the structure of the hormone which regulates adenylate cyclase activity, and of (2) introducing simultaneously two hormones which compete to bind at the same receptor site on this enzyme. The coverage is extended further (3) to situations where two hormones interact simultaneously with adjacent sites on the adenylate cyclase molecule and (4) to circumstances where inhibitors of phosphodiesterase operate both alone and in combination with hormones which influence adenylate cyclase activity. Finally, the value of the approach both in elucidating the regulation of cellular cyclic AMP levels and in quantitating the actions of hormones and drugs is briefly discussed.     

Regulation of cyclic AMP levels in Arthrobacter crystallopoietes and a morphogenetic mutant.

The extracellular levels of cyclic AMP (cAMP), cAMP phosphodiesterase activity, and adenylate cyclase activity were measured at various intervals during growth and morphogenesis of Arthrobacter crystallopoietes. There was a significant rise in the extracellular cAMP level at the onset of stationary phase, and this rise coincided with a decrease in intracellular cAMP. The phosphodiesterase activity measured in vitro increased in the early exponential phase of growth as intracellular cAMP decreased, and, conversely, prior to the onset of stationary phase the phosphodiesterase activity decreased as the intracellular cAMP levels increased. Adenylate cyclase activity was greater in cell extracts prepared from cells grown in a medium where morphogenesis was observed. Pyruvate stimulated adenylate cyclase activity in vitro. A morphogenetic mutant, able to grow only as spheres in all media tested, was shown to have altered adenylated cyclase activity, whereas no significant difference compared to the parent strain was detectable in either the phosphodiesterase activity or the levels of extracellular cAMP. The roles of the two enzymes, adenylate cyclase and phosphodiesterase, and excretion of cAMP are discussed with regard to regulation of intracellular cAMP levels and morphogenesis.