Human relaxin increases cyclic AMP levels in cultured anterior pituitary cells

Although relaxin acts at several abdominal sites and mammary tissue associated with pregnancy and parturition, the scope of target tissues and the signals conveying the relaxin message into the cell are poorly defined. We found that human relaxin rapidly elevates the cyclic AMP content of cultured rat anterior pituitary cells. This is a graded response (EC50 0.3 nM relaxin) that can be blocked by anti-relaxin antibodies or the hormones somatostatin and dopamine. Furthermore, other hormones with some sequence homology to relaxin, such as insulin and insulin-like growth factor-I, have no such action. We conclude that the anterior pituitary may be a target tissue for relaxin and that cyclic AMP may act as an intracellular messenger for relaxin in these cells.     

Effect of hormones on cyclic AMP levels in cultured human cells.

Cultured cells derived from human adipose tissue grew more slowly and had significantly higher basal levels of cyclic AMP than cultured fibroblasts. Cyclic AMP levels in cultured adipose tissue cells were unaffected by epinephrine and were elevated 15-fold by prostaglandin E₁ while fibroblast cyclic AMP levels were elevated 27-fold by epinephrine and 95-fold by prostaglandin E₁. These results support the postulate that the cultured adipose tissue cell is a distinct cell type which may represent an adipocyte or preadipocyte in culture.     

Regulation of cyclic AMP and cyclic GMP levels by adrenocorticotropic hormone in cultured neurons

The effect of adrenocorticotropic hormone (ACTH) on the intracellular concentration of cyclic nucleotides was studied in cultures of neurons from embryonic chick cerebral hemispheres. Incubation of neurons with ACTH(1-24) in the presence of phosphodiesterase inhibitor isobutylmethylxanthine resulted in a sustained increase in cyclic AMP while rise in cyclic GMP level was transient. The values obtained for half-maximal stimulation were 0.5 microM and 0.03 nM for cyclic AMP and cyclic GMP respectively. Concomitantly, ACTH(1-24) stimulated guanylate cyclase activity (half-maximal stimulation at 0.02 nM). These results suggest the existence of two distinct populations of ACTH receptors in neurons and provide the first evidence that cyclic GMP does mediate the action of ACTH in neurons.     

Tumor necrosis factor stimulates prostaglandin production and cyclic AMP levels in rat cultured mesangial cells

Human recombinant tumor necrosis factor-alpha (TNF) was found to stimulate the production of prostaglandins (PG) by cultured rat mesangial cells. This effect was demonstrable from 6 h, was dose dependent and affected the synthesis of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha. It required both RNA and protein synthesis but was not associated with a modification of cell proliferation. TNF also stimulated adenosine 3'-5' cyclic monophosphate (cAMP) levels in the mesangial cell culture medium. Indomethacin suppressed the effect of TNF on PGs but only reduced that on cAMP, indicating that PG production partly mediates the increase in cAMP. These findings demonstrate that mesangial cells can be a target for TNF and that the mechanism of TNF action includes stimulation of both PG production and cAMP levels.     

Thyrotropin and dibutyryl cyclic AMP increase levels of c-myc and c-fos mRNAs in cultured rat thyroid cells

We have studied the effects of thyrotropin (TSH) on the growth and on the levels of the mRNAs of the cellular proto-oncogenes, c-myc, and c-fos, in the specific target of TSH action, the thyroid follicular cell. FRTL5 cells, a cloned line from normal rat thyroid gland that depends upon TSH for its replication, were maintained in a quiescent state for 5 days by keeping them in a medium devoid of serum or TSH. The addition of bovine TSH (bTSH, 1 nM) increased DNA synthesis and stimulated cell proliferation after a lag period of 24 h. This growth response was anteceded by prompt, but transient, increases in the levels of c-myc and c-fos mRNAs, with peak responses at 60 and 30 min, respectively. The minimally and maximally effective concentrations of bTSH were 0.01 mM and 1.0 nM, respectively. Dibutyryl cAMP (Bt2cAMP) stimulated cell growth and increased the level of c-myc mRNA in a concentration-dependent manner, with maximum effects at a Bt2cAMP concentration of 1 mM. At the single concentration tested (1 mM), Bt2cAMP also increased the level of c-fos mRNA. Hence, bTSH-stimulated mitogenesis in quiescent FRTL5 cells is associated with rapid, but short-lived, increases in the levels of the mRNAs of the proto-oncogenes, c-myc and c-fos. Since bTSH is known to stimulate adenylate cyclase in these cells, and since the effect of TSH on c-myc and c-fos mRNAs is mimicked by Bt2cAMP, it is possible that these responses to bTSH are mediated, at least in part, by cAMP.     

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.     

Role of phosphodiesterase in cyclic AMP signaling in cultured rat granulosa cells

Inactivation of the cyclic nucleotide signal in granulosa cells depends on a complex array of cyclic nucleotide phosphodiesterases (PDE). In order to examine the role of PDE in cyclic AMP (cAMP) signaling in granulosa cells, the present study examined the expression of PDE4D proteins and regulation of cAMP-PDE activities in cultured rat granulosa cells. The results of immunoblot analyses showed that two predominant PDE4D subtypes of approximately 80 and 70 kDa appeared when immature rat granulosa cells were treated with FSH. However, these two new subtypes presumed to be PDE4D proteins were not influenced by treatments of DETA/NO, cGMP and PKB inhibitor, LY294002. Immature rat granulosa cells treated with medium alone displayed low cAMP-PDE activity throughout 48 h of culture while those treated with FSH (2 ng.mL-1) showed a marked increase in cAMP-PDE activity between 6 and 12 h of culture, followed by a decline. The findings from the present study indicate that the increased cAMP-PDE activity by FSH is mainly related to the changes of PDE4D protein levels. However, the inhibitory effects of NO on cAMP accumulation in rat granulosa cells are not via the increased cAMP-PDE activity.     

The action of pulsed magnetic fields on cyclic AMP levels in cultured fibroblasts

Pulsed magnetic fields, similar to those used clinically to promote bone repair, have been applied to cultured fibroblasts obtained from chick embryo tendons and rabbit bone marrow. Cyclic adenosine monophosphate (cAMP) levels were found to be lower in field-treated cultures in response to hormones such as prostaglandin E2 and isoproterenol, and the fibroblasts appear less sensitive to environmental perturbation prior to hormone incubation. We propose that the adenylate cyclase complex is temporarily inactivated by prolonged exposure to pulsed magnetic fields, and that this effect might be analogous to desensitisation phenomena.     

Regulation of the proliferation rate of cultured,androgen-responsive cells does not involve changes in cyclic AMP levels

The proliferation rate of cultured cells from the mouse mammary carcinoma Shionogi 115 is regulated both by local cell population density and by androgens. Measurement of intracellular levels of cyclic AMP has shown that these levels are constant over a wide range of proliferation rates (mean doubling times varied from 23 hr to more than 200 hr). Addition of dibutyryl cyclic AMP or theophylline to the culture medium resulted in inhibition of growth—even in the presence of androgen. This inhibition of growth and the relationship between cyclic AMP levels and cell proliferation is discussed.     

Regulation of mRNA levels for cellular retinol binding protein in rat Sertoli cells by cyclic AMP and retinol

The levels of mRNA for cellular retinol binding protein (CRBP) were studied in primary rat Sertoli cell cultures treated with cAMP analogues and retinol. In the presence of cyclic AMP analogues a dose- and time-dependent reduction (70-90%) of the levels of mRNA for CRBP was observed. Retinol concentrations above 10 nM induced a dose- and time-dependent increase (2-3 fold) in mRNA levels for CRBP. Assuming that CRBP is important for vitamin A action, our data indicate that both cAMP and retinol itself modulate the sensitivity of the Sertoli cells for retinol.     

Dissociation of the regulation of fatty acid synthesis and cyclic AMP levels in cultured glial and neuronal cells.

Abstract— Cultured C-6 glia and neuroblastoma were utilized to investigate the relation of rates of fatty acid synthesis (from ³H₂O) to levels of cyclic AMP under conditions of short-term and long-term regulation. The data demonstrate a consistent dissociation of alterations in rates of fatty acid synthesis and levels of cyclic AMP. Thus, marked alterations in the rate of fatty acid synthesis occurred when serum or albumin-bound palmitic acid was present in the culture medium, but there were no accompanying alterations in levels of cyclic AMP. Similarly, when high intracellular and/or extracellular levels of cyclic AMP were induced by exposure of the cells to dibutyryl cyclic AMP or isoproterenol, no change in the rate of fatty acid synthesis occurred. Although the data raise serious doubt about an important role for cyclic AMP in the regulation of fatty acid synthesis, they do not rule out such a role. The findings do indicate that any such role must involve alterations in compartmentalization, metabolism or binding of the mononucleotide within the cell.     

The turnover of cyclic AMP in cultured fibroblasts

The determination of the turnover of cAMP in WI-38 and VA13 cultured fibroblasts stimulated by prostaglandin E1 is reported. The method made use of data obtained from a process of continuously labeling the cellular adenine nucleotide pools by incubation with [3H]-adenine. The turnover of the cAMP was estimated from the delay in appearance of tritium label in the cAMP pool was compared to the cellular ATP. For WI-38 cells the half-life of cAMP when accumulation had reached a steady-state was 1.46 minutes; in the presence of 0.5 mM 1-methyl-3-isobutylxanthine (IBMX) the half-life was increased to 9.24 minutes. For VA13 transformed fibroblasts the half-life of cAMP determined by this method was 6.30 minutes. cAMP in these latter cells in the absence of hormone had a half-life of 3.01 minutes. This decrease supports the contention that the hormone has profound effects on phosphodiesterase as well as adenylate cyclase activities in these cells.     

Coordinated modulation of albumin synthesis and mRNA levels in cultured hepatoma cells by hydrocortisone and cyclic AMP analogs.

The treatment of Hepa-2 cells, a permanent mouse hepatoma cell line, for 72 h with hydrocortisone (10(-6) M), N6,O2-dibutyryl cyclic AMP (10(-3) M), or 8-bromocyclic AMP (10(-3) M) results in a 2-,3- or 4-fold increase, respectively, in rates of synthesis and secretion of mouse serum albumin. Simultaneous treatment with hydrocortisone and N6,O2-dibutyryl cyclic AMP results in a 10-fold stimulation in these parameters, an effect that is significantly more than additive for the two compounds tested. The number of albumin mRNA sequences, determined by hybridization of total cell RNA to albumin complementary DNA, was increased in direct proportion to the increases in albumin synthesis in all experiments. The relative rate of albumin synthesis approaches in vivo levels in cells treated simultaneously with hydrocortisone and N6,O2-dibutyryl cyclic AMP. We propose that these factors may be necessary to maintain the maximal level of differentiated function in the continuous culture of Hepa-2 cells.     

Thyrotropin and cyclic AMP regulation of ras proto-oncogene expression in cultured thyroid cells

We examined the effect of thyrotropin (TSH) on intracellular levels of c-ras mRNA in a line of differentiated rat thyroid cells obtained from normal Fischer rat thyroids. These cells are totally dependent on TSH for growth. TSH stimulation of quiescent cells increased c-ras mRNA content, with a maximal response (730% of basal) after 6 h, and a decline towards basal levels after 24 h. Dibutyryl cAMP and forskolin mimicked this stimulatory effect of TSH on c-ras, but did not enhance beta-actin mRNA content. This study demonstrates hormonal and cyclic nucleotide control of c-ras expression in a well-differentiated, non-tumorogenic mammalian cell.