Modulation of adenylate cyclase/cyclic AMP response by thyrotropin and prostaglandin E2 in cultured thyroid cells. 2. Positive regulation.

Two different independent processes are operating in cultured thyroid cells to regulate adenylate cyclase/cyclic AMP responsiveness to thyroid stimulators (thyrotropin and prostaglandin E2): firstly, refractoriness or negative regulation [preceding paper], which is specific for each thyroid stimulator, is not mediated by cyclic AMP and is not accompanied by alteration of adenylate cyclase activity; secondly, positive regulation which is characterized by an augmentation of the cyclic AMP response stimulated by thyrotropin and prostaglandin E2. This process is not specific for each thyroid stimulator and is a state of increased susceptibility of cyclic AMP synthesis to stimulation, accompanied by increased activity of the catalytic subunit of adenylate cyclase. Positive regulation is apparently mediated by increased intracellular cyclic AMP levels. It is a time-dependent and dose-dependent process. Very low concentrations (5-50 micronU/ml) of thyrotropin augmented cyclic AMP synthesis stimulated by thyrotropin and prostaglandin E2 whereas higher concentrations (above 0.1 mU/ml) augmented prostaglandin E2 stimulation but induced refractoriness to thyrotropin. Prostaglandin E2 (0.1 to 10 micronM) augmented thyrotropin stimulation and dibutyryl adenosine 3':5'-monophosphate (0.3 to 2 mM) augmented thyrotropin and prostaglandin E2 stimulation. Positive regulation is a slow process which develops within days and increases up to day 5 in culture. Experiments using inhibitors suggested that protein synthesis is required for the full expression of the increase in adenylate cyclase activity induced by the studied thyroid stimulators.     

Cross-talk regulation between cyclic AMP production and phosphoinositide hydrolysis induced by prostaglandin E2 in osteoblast-like cells.

In cloned osteoblast-like MC3T3-E1 cells, PGE2 stimulated both cAMP accumulation and the formation of inositol trisphosphate (IP3) dose dependently. The cAMP accumulation showed the peak value at 5 min and decreased thereafter, whereas the IP3 formation reached a plateau almost within 10 min and sustained it up to 30 min. The effect of PGE2 on cAMP accumulation (EC50 was 80 nM) was more potent than that on IP3 formation (EC50 was 0.8 microM). 12-O-Tetradecanoyl-phorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, reduced the PGE2-induced cAMP accumulation, whereas 4 alpha-phorbol 12,13-didecanoate, a PKC-nonactivating phorbol ester, had little effect on the cAMP accumulation. 1-Oleoyl-2-acetyl-glycerol, a specific activator for PKC, inhibited PGE2-induced cAMP accumulation. TPA had little effect on cAMP accumulation induced by forskolin or NaF, a GTP-binding protein activator. So, the effect of TPA is presumed to be exerted at the point between the PGE2 receptor and Gs. On the other hand, forskolin and dibutyryl cAMP had little effect on the IP3 formation stimulated by PGE2. H-7, a PKC inhibitor, enhanced the PGE2-induced cAMP accumulation in comparison with HA1004, a control for H-7. Our data suggest that PGE2 regulates cAMP production through self-induced activation of PKC. These results strongly suggest that there is an autoregulatory mechanism in PGE2 signaling, and PGE2 modulates osteoblast functions through a cross-talk interaction between cAMP production and phosphoinositide hydrolysis in osteoblast-like cells.     

The stimulation of hepatic adenylate cyclase by prostaglandin E1

Adenylate cyclase activity associated with particulate preparations from rat, mouse, rabbit, and dog liver is stimulated 2-to 5-fold by prostaglandin E₁ (PGE₁). This stimulation is dependent upon the presence of guanosine-5′-triphosphate (GTP). Prostaglandins F_1a and F_2a do not alter the enzymatic activity under these same conditions. Optimal concentrations of PGE₁ + GTP stimulate rat liver adenylate cyclase more than glucagon alone, but less than glucagon + GTP. Activity measured with glucagon + GTP is not affected by addition of PGE₁. Stimulation from PGE₁ + GTP is increased by glucagon to the same level measured with glucagon + GTP.     

Modulation of thyroglobulin messenger RNA level by thyrotropin in cultured thyroid cells.

To examine the influence of thyrotropin (TSH) on the thyroglobulin (Tgb) mRNA content, the latter was evaluated in the cytoplasm of hog thyroid cells cultured in the absence (control cells) or presence of TSH. The Tgb mRNA levels were determined by, (i) kinetics of hybridization to sheep Tgb cDNA, (ii) capacity of coding for peptides immunologically related to Tgb in reticulocyte lysate. In cells cultured for 4 days in the absence of TSH, the content of Tgb mRNA sequences decreased to 30% of its initial value and the messenger activity to 15%. Conversely, TSH maintained the initial Tgb mRNA level in cells cultured in its presence, and TSH concentrations 50 micronU/ml or 5 mU/ml gave identical results. At each period tested poly (A) content was the same in TSH-treated and control cells. When TSH was added to media after 4 or 8 days culture without TSH, the Tgb mRNA level was partially restored. These results suggest that TSH exerts a positive control on Tgb gene expression through modulation of Tgb mRNA content of thyroid cells.     

Modulation of the human sperm acrosome reaction by effectors of the adenylate cyclase/cyclic AMP second-messenger pathway.

The acrosome reaction of spermatozoa appears to be analogous to various somatic cell exocytotic events which involve cascade reactions, i.e., transmission of an external signal across the cell membrane resulting in activation of an "amplifier" enzyme and the generation of a second messenger. Using a synchronous acrosome reaction system (De Jonge et al., J. Androl., 10:232-239, '89a), it was found that analogues of the second-messenger cAMP, dibutyryl cAMP (dbcAMP) and 8-bromo cAMP, stimulated the acrosome reaction of capacitated spermatozoa. Additionally, treatment of spermatozoa with either xanthine or non-xanthine phosphodiesterase inhibitors induced a significant (P less than 0.05) increase in the percent acrosome reaction after a period of capacitation in comparison to untreated controls. These results indicate that analogues of cAMP or inhibitors which prevent cAMP hydrolysis can induce the human sperm acrosome reaction. Subsequent experiments were conducted to test whether the amplifier enzyme in the cascade reaction, adenylate cyclase, has a role in the acrosome reaction. Forskolin, an adenylate cyclase stimulator, caused a significant (P less than 0.01) increase in the percent acrosome reaction in comparison to controls. Modulators of adenylate cyclase--adenosine, 2'-0-methyladenosine, and 2',3'-dideoxyadenosine--significantly (P less than 0.01) inhibited the forskolin-induced acrosome reaction. dbcAMP was able to overcome the inhibition by adenosine. Two inhibitors of protein kinase A, the Walsh inhibitor and H-8, caused a significant (P less than 0.01) inhibition of the dbcAMP-induced acrosome reaction. Finally, in the absence of extracellular calcium, dbcAMP induced a significant (P less than 0.01) increase in the acrosome reaction in contrast to A23187. These results suggest that: 1) a molecular mechanism for the human sperm acrosome reaction involves the cAMP second-messenger system; i.e., activation of adenylate cyclase, the amplifier enzyme that produces cAMP, production of cAMP as a second messenger, and activation of cAMP-dependent kinase A; and that 2) activation of adenylate cyclase occurs after calcium influx.     

Cyclic AMP formation and release by cultured bone cells stimulated with prostaglandin E2.

PGE2 produced a marked and dose-related increase in cAMP content of cultured bone cells and in the release of cAMP into the incubation medium. The amount of cAMP released from the cells by PGE2 was proportional to the cellular concentration, and was dependent upon the time of incubation with PGE2. The cAMP levels released into the media increased slowly at a linear rate during a 60 min treatment with PGE2. This release was blocked by theophylline, probenecid, ouabain and dinitrophenol, suggesting that the release of cAMP was not a simple diffusive process and required energy. SC-19220 reduced the formation of cAMP more than the release, suggesting that the formation and the release may arise from separate events. Inability of D600 to inhibit PGE2-induced release of cAMP indicates that the release does not require calcium.     

Inhibition of a parathyroid hormone-stimulated renal adenylate cyclase by cyclic AMP.

Plasma membranes were isolated from bovine renal cortex. This particulate, adenylate cyclase-containing fraction was stimulated to produce cyclic AMP by parathyroid hormone and fluoride. When the time-course of adenylate cyclase activity was investigated, it was found that while PTH-stimulated cyclic AMP production comes to a halt in about 15 minutes after the initiation of the reaction, fluoride-stimulated activity continues unabated for at least an hour. Experiments to determine the cause of this showed that the cyclase enzyme is not degraded under our experimental conditions, but is inhibited by a soluble, unbound product of the reaction which requires ATP for its synthesis. In our experiments degradation of parathyroid hormone was relatively slow and could not account for the rapid inhibition of PTH-stimulated cyclase activity. Of the various agents tested, cyclic AMP was found capable of inhibiting PTH-stimulated cyclic AMP production by our purified membrane preparation. Half-maximal inhibition was observed at around 10(-6) M concentrations of the nucleotide. Pyrophosphate, adenosine, 5'-AMP and ADP had no effects. The significance of these results in relation to the regulation of adenylate cyclase activity is discussed.     

Bacterial adenylate cyclase increases cyclic AMP and hormone release in pituitary tumor cells

Calmodulin-activated, adenylate cyclase toxin, a virulence factor produced by the human respiratory pathogen Bordetella pertussis, elicits marked accumulation of cyclic AMP in cell lines from rat pituitary tumors. This effect is associated with and apparently responsible for an enhanced release of prolactin and/or growth hormone from GH3, GH4C1 and 235-1 cells. The utility of this novel toxin in probing cyclic AMP-mediated responses is supported by these observations and studies with pertussis and cholera toxins.     

Platelet prostaglandin E1 hyposensitivity in schizophrenia: reduction of prostaglandin E1- or forskolin-stimulated cyclic AMP response in platelets

Cyclic 3',5'-adenosine monophosphate (cAMP) formation via prostaglandin E1 (PGE1)-or forskolin-stimulation were determined in washed intact platelets from 32 schizophrenic patients and 30 normal controls. Regarding basal cAMP levels in the platelets, there were no differences between schizophrenic patients and normal controls. Both PGE1-and forskolin-stimulated cAMP response reduced in platelets from schizophrenics compared with normal controls. These results suggested that platelets in schizophrenics were impaired not only in the adenylate cyclase unit per se but also extensively in the cAMP generating system coupled to a PGE1 receptor.     

Stimulation by thyrotropin and cyclic AMP of the proliferation of quiescent canine thyroid cells cultured in a defined medium containing insulin

We have developed serum-free primary cultures of differentiated follicular dog thyroid cells which allow the study of the hormonal control of cell proliferation. The cooperation of insulin and increasing cellular cyclic AMP by thyrotropin triggers the DNA synthesis and the proliferation. Dog thyroid cells are an example of a system in which cyclic AMP is a sufficient signal to stimulate the proliferation in quiescent cells.     

Effect of gelatin on the cyclic AMP response of primocultured hog thyroid cells to acute thyrotropin stimulation.

The cyclic AMP response of cultured hog thyroid cells to acute thyrotropin stimulation was shown to be under a dual regulatory control by thyrotropin: both positive and negative regulation have been described. When added to the culture medium, gelatin (0.25%) promoted the reorganization of the cells into folicle-like structures, as does thyrotropin. Unlike thyrotropin, gelatin did not induce an increase in intracellular cyclic AMP but enhanced the acute cyclic AMP response to thyrotropin in cells cultured in gelatin-containing medium. When both gelatin and thyrotropin were present, the positive effect of low concentrations of hormone (less than 50 microU/ml) was increased whereas the refractory process observed in the presence of higher concentrations of hormone (greater than 50 microU/ml) was unchanged. These effects of gelatin might be mediated by interaction of the denatured collagen molecules with external proteins of the plasma membrane of thyroid cells.     

Noninactivation of thyrotropin by cultured porcine thyroid cells.

Freshly isolated porcine thyroid cells were cultured in the presence of highly purified porcine thyrotropin. Cells associate into follicles between the second and tenth day of culture and later form a monolayer. The biological and immunological activity of thyrotropin was measured daily in the media. Thyrotropin concentration and biological activity remained unchanged from the onset of the culture up to day 14. Limiting factors influencing thyroglobulin biosynthesis do not appear before day 13. The loss of follicular organization at day 10 cannot be explained by thyrotropin degradation in the medium. Considering the number of receptors per cell and the half life of the thyrotropin . receptor complex in the two dissociation compartments previously demonstrated, it appears in terms of both biological activity and affinity for the receptors that the thyrotropin molecules released from the first compartment do not differ from native molecules. It can be calculated that at least 31% of the molecules released from the second compartment are not inactivated. Thus, it is probable that the catabolism of thyrotropin on the receptor, or near the receptor site, does not play an important role in the regulation of thyroid cell function in vitro.     

Specific refractoriness of adenylate cyclase in skin to epinephrine,prostaglandin E,histamine and AMP

The cyclic AMP level in pig skin (epidermis) increases markedly after incubation with epinephrine, prostaglandin E, histamine or adenosine 5′-monophosphate. This increase is transient and “spiking” is the consistent response to these four stimulators. The “spiking” is due to a non-responsiveness or refractoriness which develops within minutes and is specific to any one stimulating hormone but not to the others. The addition of inhibitors of protein syntheses did not prevent the development of the refractoriness. Adenylate cyclase and phosphodiesterase activities measured in skin homogenates prepared from skin samples taken before, during and after the “spiking” did not change significantly. The hormone-induced refractoriness in this skin system appears to be due to a specific, localized loss of function of the adenylate cyclase system.     

On the regulation of cyclic AMP level in bacteria. II. In vitro regulation of adenylate cyclase activity. Solubilization and reconstitution of a functional membrane-bound adenylate cyclase system responsive to regulation by glucose.

1. The in vitro regulation of the membrane bound adenylate cyclase of Escherichia coli B/r by a variety of carbohydrates and one mammalian hormone was examined. 2. The membrane bound adenylate cyclase was found responsive to regulation by the various growth substrates and to glucagon. 3. Solubilization of the bacterial membrane preparation by a procedure specific for the solubilization of the phosphotransferase enzyme E1 1 to its E1 1 A and E1 1 B subunits was found to be accompanied by the loss of the adenylate cyclase regulation by glucose. 4. Reconstitution of the membrane was found to result in a recovery of the regulative response of the adenylate cyclase to glucose. 5. A model for the intermediate steps in the interaction between glucose and phosphotransferase E1 1 and the adenylate cyclase is discussed.