Extracellular metabolism of cyclic AMP.

Intravenously administered cyclic [8-3H]AMP to rats was quickly eliminated from the circulation. After 2 min 93% of the administered radioactivity disappeared from the plasues was recovered mainly in the form of nucleotides, ATP, ADP, AMP and IMP. In vitro contact of cyclic AMP with perfused liver, isolated liver cells and adipose tissue resulted in a rapid breakdown of the nucleotide, presumably on the outer surface of the cells. The degradation products have been identified mainly as adenosine and inosine. Incubation of adipose tissue and isolated liver cells with [3H] AMP also resulted in the breakdown of the nucleotide in themedium. The rate of AMP degradation by these tissues was faster than that for cyclic AMP degradation. The data suggest that cyclic AMP is readily metabolized on the outer surface of cells to products which may be converted within the cells to nucleotides. These findings seem of importance for the quantitative assessments of cellular cyclic AMP outflow during hormonal stimulation.     

Prolonged prostaglandin E1 stimulation of cyclic AMP production in transformed and normal WI-38 fibroblasts.

Long-term (48-hr) incubations of either the fibroblast strain WI-38 or its SV40-transformed counterpart, WI-38-VA13-2RA, in growth medium containing 1 micron prostaglandin E1 (PGE1) resulted in a sustained production and release of cyclic AMP from the cells into the medium. Despite the steady production, intracellular levels of the nucleotide decreased, reaching steady-state values within 4 hr of the initial exposure to PGE1. These values were maintained for the remainder of the 48-hr experimental period. The steady-state levels of intracellular cyclic AMP were higher than those observed in unstimulated cells, and cyclic AMP-dependent protein phosphokinase was in a highly activated state as compared to controls. Under these conditions little change in the growth or morphology of either the normal or transformed cells was observed. In contrast, inhibition of growth, apparent cell death, and unusual morphological changes were observed in both normal and transformed cells when high concentrations of either PGE1 (10 micron) or the phosphodiesterase inhibitor 1-methyl, 3-isobutylxanthine (0.5 mM to 2 mM) were used, which was indicative of toxic effects of the drugs. It was concluded that cyclic AMP-mediated activation of protein phosphokinase does not completely inhibit growth in WI-38 cells or restore normal growth and morphology to the SV40-transformed cells.     

Modulators of cyclic AMP metabolism induce syncytiotrophoblast formation in vitro

During placental development cytotrophoblast stem cells fuse to form the syncytiotrophoblast, a multinucleate cytoplasm with a brush border in contact with the maternal blood. Biochemical differentiation including the expression of placental-specific proteins and hormones accompanies this maturation. However, the biochemical mechanisms responsible for these events are unknown. We have defined a system in which single cytotrophoblast-like cells of the human choriocarcinoma (BeWo) cell line undergo fusion and extensive morphological differentiation following their treatment with effectors of cyclic AMP metabolism. Forskolin incubation caused a dose-dependent increase in intracellular and secreted cyclic AMP and a coordinate fusion of cells which yielded syncytia containing hundreds of nuclei per cytoplasm and a mature dense "placental-like" brush border. These fused cells also synthesized and secreted large amounts of both subunits of chorionic gonadotropin. However, they continued to synthesize several other placenta-specific proteins--placental-like alkaline phosphatase, placental lactogen, and SP1--at rates similar to those in control cells. Other reported effectors of cyclic AMP metabolism also induced cell fusion, although theophylline, an inhibitor of phosphodiesterase, induced fusion by a cyclic AMP-independent mechanism. Additionally, unlike the case with forskolin, treatment of BeWo cells with theophylline did not induce other morphological features of mature syncytiotrophoblasts. Thus, this system will allow one to examine the biochemical mechanism of placental cell fusion in the absence of other variables of cell differentiation.     

Cyclic AMP and cyclic GMP in rabbit blastocysts.

Concentrations of both nucleotides were significantly higher in Day-6 than in Day-5 blastocysts but the ratio of cAMP to cGMP changed from 0.5 to 1.5.     

Induction of cyclic AMP phosphodiesterase in Blastocladiella emersonii and its relation to cyclic AMP metabolism.

Extracts of vegetative cells of Blastocladiella emersonii contain 5% or less of the cyclic AMP phosphodiesterase activity in zoospore extracts. This difference in activity could be accounted for entirely by an increase in the differential rate of phosphodiesterase synthesis during sporulation, beginning after a lag period of about 60 min and extending for at least an additional 90 min into the 4-h sporulation process. To examine the relation between enzyme synthesis and cyclic nucleotide metabolicm, we determined the substrate specificity of phosphodiesterase synthesized during sporulation and partially purified from zoospores. Zoospore extracts contain two components, separable by gel filtration chromatography, with cyclic AMP phosphodiesterase activity. The larger component accounts for 20% of the total activity and the smaller component for 80%. Both components show essentially an absolute substrate specificity for cyclic AMP among several cyclic purine and cyclic pyrimidine nucleotides tested. Nevertheless, we found no change in the total cyclic AMP content of sporulating cells before, during, or after enzyme activity increased. We speculate that some other component of cyclic AMP metabolism or function limits the rate of cyclic AMP hydrolysis in sporulating cells.     

Morphology of the surface of normal and virus transformed cells in vitro]

The cell surface morphology of two cell lines--from the mink lung (Mv1Lu) and from the Kirsten sarcoma virus transformed derivate (Ki-Mv1Lu)--was studied by scanning electron microscopy. Marked differences are seen in cell morphology of these two lines at high cell densities. Mv1Lu cells at high densities had uniform flat polygonal shape with microvilli at their surface. A marked diversity in cell morphology was characteristic of Ki-Mv1Lu cells at comparable cell densities: variation in shape, in thickness degrees, and in the expression of cell surface ultrastructure (microvilli, blebs, filopodia). No dependence of Ki-Mv1Lu cell morphology from cell densities was observed. At low cell densities of Mv1Lu cells, cells with the morphology differing from the typical pattern of confluent Mv1Lu cells were seen. Morphological diversity of these cells was comparable with that of Ki-Mv1Lu cells. Nothing has been found in cell surface morphology that could be absolutely specific for transformed cells only.     

Prolonged prostaglandin E1 stimulation of cyclic AMP production in transformed and normal WI-38 fibroblasts

Summary Long-term (48-hr) incubations of either the fibroblast strain WI-48 or its SV40-transformed counterpart, WI-38-VA13-2RA, in growth medium containing 1 μm prostaglandin E₁ (PGE₁) resulted in a sustained production and release of cyclic AMP from the cells into the medium. Despite the steady production, intracellular levels of the nucleotide decreased, reaching steady-state values within 4 hr of the initial exposure to PGE₁. These values were maintained for the remainder of the 48-hr experimental period. The steadystate levels of intracellular cyclic AMP were higher than those observed in unstimulated cells, and cyclic AMP-dependent protein phosphokinase was in a highly activated state as compared to controls. Under these conditions little change in the growth or morphology of either the normal or transformed cells was observed. In contrast, inhibition of growth, apparent cell death, and unusual morphological changes were observed in both normal and transformed cells when high concentrations of either PGE₁ (10 μm) or the phosphodiesterase inhibitor 1-methyl, 3-isobutylxanthine (0.5mm to 2mm) were used, which was indicative of toxic effects of the drugs. It was concluded that cyclic AMP-mediated activation of protein phosphokinase does not completely inhibit growth in WI-38 cells or restore normal growth and morphology to the SV40-transformed cells. This work was supported by Grants AM 13904 and CA 21612 from the National Institutes of Health, Department of Health, Education and Welfare.     

Regulation of cyclic AMP metabolism in rabbit cortical collecting tubule cells by prostaglandins

Prostaglandin E1 (PGE1) at 1 nM inhibits arginine-vasopressin (AVP)-induced water reabsorption in the rabbit cortical collecting tubule (RCCT), while 100 nM PGE1, by itself, stimulates water reabsorption (Grantham, J. J., and Orloff, J. (1968) J. Clin. Invest. 47, 1154-1161). To investigate the basis for these two responses, we measured the effects of prostaglandins on cAMP metabolism in purified RCCT cells. In freshly isolated cells, PGE2, PGE1, and 16,16-dimethyl-PGE2 acting at high concentrations (0.1-10 microM) stimulated cAMP accumulation; however, one PGE2 analog, sulprostone (16-phenoxy-17,18,19,20-tetranor-PGE2 methylsulfonilamide), failed to stimulate cAMP accumulation or to antagonize PGE2-induced cAMP formation; PGD2, PGF2 alpha, and a PGI2 analog, carbacyclin (6-carbaprostaglandin I2), also failed to stimulate cAMP synthesis. These results suggest that there is a PGE-specific stimulatory receptor in RCCT cells which mediates activation of adenylate cyclase. Occupancy of this receptor would be anticipated to cause water reabsorption by the collecting tubule. At lower concentrations (0.1-100 nM) PGE2, PGE1, 16,16-dimethyl-PGE2, and, in addition, sulprostone inhibited AVP-induced cAMP accumulation by fresh RCCT cells in the presence of cAMP phosphodiesterase inhibitors. Pertussis toxin pretreatment of RCCT cells blocked the ability of both PGE2 and sulprostone to inhibit AVP-induced cAMP accumulation. In membranes prepared from RCCT cells, sulprostone prevented stimulation of adenylate cyclase by AVP. These results suggest that E-series prostaglandins (including sulprostone) can act through an inhibitory PGE receptor(s) coupled to the inhibitory guanine nucleotide regulatory protein, Gi, to block AVP-induced cAMP synthesis by RCCT cells. Occupancy of this receptor would be expected to cause inhibition of AVP-induced water reabsorption in the intact tubule. Curiously, after RCCT cells were cultured for 5-7 days, PGE2 no longer inhibited AVP-induced cAMP accumulation, but PGE2 by itself could still stimulate cAMP accumulation. In contrast to PGE2, epinephrine acting via an alpha 2-adrenergic, Gi-linked mechanism did block AVP-induced cAMP formation by cultured RCCT cells. This implies that some component of the inhibitory PGE response other than Gi is lost when RCCT cells are cultured.     

Extracellular metabolism of cyclic AMP

Intravenously administered cyclic [8-³H]AMP to rats was quickly eliminated from the circulation. After 2 min 93% of the administered radioactivity disappeared from the plasma, and most of it was recovered in the kidney, liver and muscles. The label in the tissues was recovered mainly in the form of nucleotides, ATP, ADP, AMP and IMP.In vitro contact of cyclic AMP with perfused liver, isolated liver cells and adipose tissue resulted in a rapid breakdown of the nucleotide, presumably on the outer surface of the cells. The degradation products have been identified mainly as adenosine and inosine.Incubation of adipose tissue and isolated liver cells with [³H]AMP also resulted in the breakdown of the nucleotide in the medium. The rate of AMP degradation by these tissues was faster than that for cyclic AMP degradation.The data suggest that cyclic AMP is readily metabolized on the outer surface of cells to products which may be converted within the cells to nucleotides. These findings seem of importance for the quantitative assessments of cellular cyclic AMP outflow during hormonal stimulation.     

Amylase secretion by rabbit parotid gland. Role of cyclic AMP and cyclic GMP.

Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10(-4) M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by alpha-adrenergic blockade with phenoxybenzamine. Epinephrine (4 - 10(-5) M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the beta-blocking agent, propranolol. Pure alpha-adrenergic stimulation with methoxamine (4 - 10(-4) M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 - 10(-6) M, isoproterenol (a beta-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 - 10(-5) M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cyclic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 - 10(-6) M). These data strongly suggest that cholinergic muscarinic agonists and alpha-adrenergic agonists stimulate amylase output in rabit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by alpha-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this tissue to the effects of cholinergic stimuli.     

Insulin receptors and functions in normal and spontaneously transformed cloned rat hepatocytes.

Spontaneous transformation of late passages of a cloned line of normal, differentiated, diploid rat hepatocytes (RL-PR-C) leads, not only to a loss of contact-inhibition of growth and ultimately to oncogenicity, but also to a variety of characteristics suggestive of a loss of involvement with insulin: lack of a serum requirement for growth; reductions in numbers and affinity of insulin receptors; increased rate of dissociation of insulin; loss of insulin stimulation of protein synthesis; and, a reduction in insulin's ability to activate glycogen synthase and to stimulate glycogen synthesis from glucose.     

Different effects of selenium on cyclic AMP metabolism in hepatoma cells and normal liver cells

Sodium selenite administered to normal and hepatoma (HA)-bearing mice sc (2 mg/kg) or ip (1 mg/kg) led to a significant increase in cyclic AMP (cAMP) level and a decrease in cAMP phosphodiesterase (PDE) activity in HA cells. In contrast, in tumor host liver and normal liver the cAMP level was reduced, and the PDE activity was slightly elevated, whereas the cAMP adenylate cyclase (AC) was little affected by Na₂SeO₃, if at all. These results imply that selenite-induced changes in PDE activity play a decisive role in regulating intracellular cAMP level. Kinetic studies revealed that there were different forms of PDE. TheK _m value of PDE isozymes in normal liver and host liver were identical, but differed from those of HA. It seems likely that the selective responsiveness of PDE to selenite may be related to the difference in PDE isozyme patterns. Addition of DBcAMP to the culture medium resulted in an inhibition of³H-thymidine incorporation into hepatoma cells, indicating that the inhibition of HA cell proliferation was cAMP-mediated. Thus, selenium has been shown to exert a selective effect on cAMP metabolism of hepatoma cells, which may account for its inhibitory effects on cancer cells.     

Effects of probenecid on transport and metabolism of cyclic AMP by isolated rabbit renal tubules

The effects of probenecid on the transport and metabolism of cyclic [14C]-AMP were studied in isolated rabbit kidney cortex tubules. Incubation in a medium with 10-400 microM probenecid for 30 min caused a 30-70% decrease in the tubular uptake of labeled material from a medium containing 0.1 mM cyclic [14C]AMP. The radioactivity in the tubules, after 30 min incubation, with or without probenecid, was mostly in the form of inosine and hypoxanthine. The disappearance of external cyclic [14C]AMP was retarded by probenecid and the concentration ratio of cyclic AMP to inosine + hypoxanthine was increased. Cyclic AMP phosphodiesterase activities, from both the soluble and particulate fractions of the kidney, were inhibited by probenecid. These findings indicate that the changes caused by probenecid on the renal disposal of extracellular cyclic AMP can be accounted for by a decrease in the accumulation of the products of cyclic AMP metabolism secondary to inhibition of extracellular cyclic AMP phosphodiesterase activity.     

The growth and differentiation of transitional epithelium in vitro

The development of rat transitional epithelial cells grown on conventional non-permeable surfaces was compared with development on permeable collagen supports. On glass or plastic surfaces, cells grew as expanding nomolayer sheets. Once confluent, growth continued with a bilayer being formed in most areas and apical cells being continuously sloughed off. Although most cells were interconnected by desmosomes, and junctional complexes were formed, no other indications of differentiation were observed. After 2-3 wk of growth, division stopped and cel death ensued. In contrast, single-cell suspensions plated on collagen-coated nylon disks reassociated into multicellular islands and commenced growth. Mitoses were confined to the basal cells in contact with the permeable substrate. The islands developed into epithelial trilayers, tapering to monolayers along spreading edges. Once the islands were confluent, stratification was completed and appeared similar to that observed in vivo. Germinal cells formed a basal lamina, and the upper layer was composed of large, flattened cells with an unusually thick asymmetrical plasma membrane on the apical surface. Electron microscopic and radioactive tracers demonstrated "leaky" zonulae occludentes with a restricted permeability to small molecules. The movement of urea was retarded in comparison to water. Unlike the slow turnover of adult epithelium in vivo, maturation and sloughing of apical cells were measurable. Transfer of cells could be effected and growth maintained for up to 4 mo. These results may indicate the necessity of a nutrient-permeable growth surface for the polarized differentiation of adult transitional epithelium.     

Synthesis of mucous glycoproteins by rabbit tracheal cells in vitro. Modulation by substratum, retinoids and cyclic AMP.

One function of airway epithelium is the secretion of mucins, which comprise an important component of the mucous lining layer. We demonstrate that rabbit tracheal epithelial cells grown in primary culture incorporate [3H]glucosamine into material released into the medium which is characterized as mucin by the following criteria: high Mr, monosaccharide composition, ion-exchange behaviour different from that of glycosaminoglycans and oligosaccharides attached via N-acetylgalactosamine. The production of mucin by the cells requires growth on a substratum of collagen gel and is enhanced by retinoids in the extracellular medium. In the presence of retinoids, 8-bromo cyclic AMP and factors present in medium from 3T3 fibroblasts each further stimulate mucin production. These results indicate that an isolated epithelial-cell culture system, in the absence of nervous, mesenchymal or other tissue types, can be used to answer questions about the regulation of mucin production at the cellular level.     

Effect of LH on the release of cyclic AMP by the rabbit ovary perfused in vivo and in vitro.

After perfusion of 10 rabbit ovaries in vitro with a modified Krebs bicarbonate buffer containing dextran and glucose, the concentration of cAMP in the perfusion medium was significantly increased 2-5 min after stimulation with 10 mug LH/ml medium and was higher at 15 and 30 min. Intravenous injection of 100 mug LH/rabbit caused a significant increase of cAMP concentrations in the ovarian venous blood from 8 ovaries 10 min after the injection and the cAMP concentrations were higher after 15 and 30 min. The ovarian blood flow was not changed after the LH injection. It is concluded that perfusion techniques can be useful in analysis of the mechanisms and physiological significance of release of cAMP from the ovary after hormonal stimulation.