E. Kaukel

Permeation of dibutyryl cAMP into HeLa cells and its conversion to monobutyryl cAMP

Summary 3H-Dibutyryl cAMP (DBcAMP) when exposed to HeLa cultures proved rather resistant to extracellular degradation. It was taken up by the cells and led to an accumulation of monobutyryl cAMP (MBcAMP), which — in contrast to DBcAMP — showed a high affinity to Gilmans (1) cAMP-binding protein. cAMP and DBcAMP were not accumulated in the cells to a comparable degree under these conditions. Other than DBcAMP, cAMP was rapidly degraded extracellulary to various metabolites including adenosine which was taken up by the cells and converted to purine nucleotides. Only at high extracellular concentrations cAMP could permeate the cells and lead to a transient rise in intracellular cAMP levels. These levels, however, never reached the steadily increasing concentrations of protein kinase-binding substances (MBcAMP + cAMP) when similar concentrations of DBcAMP were added to cultures. These results indicate that the sustained hormone-like actions of DBcAMP come about mainly by a high resistance to extracellular and intracellular phosphodiesterase as well as by the enzymic conversion to MBcAMP accumulating in the cells.

Divergent action of cAMP and dibutyryl cAMP on macromolecular synthesis in HeLa S3 cultures

Abstract In HeLa S3 cultures, DBcAMP (1 × 10 −3 M) led to a decrease in glycogen content, cell proliferation and 3 H-thymidine incorporation into DNA. In contrast, cAMP increased glycogen levels, and incorporation of labeled thymidine and uridine into nucleic acids. Both nucleotides, however, inhibited net macromolecular synthesis and cell proliferation. The unusual actions of exogenous cAMP on nucleic acid synthesis may be explained by a depletion of the precursor pools, as UTP levels were drastically reduced under these conditions, leading to an elevated specific radioactivity of the remaining UTP on the addition of 3 H-uridine. Evidence is presented that the unusual effects of exogenous cAMP came about by enzymic degradation in the (calf serum-containing) medium to adenosine, while DBcAMP seemed to imitate intracellular cAMP.

Divergent action mechanism of cAMP and dibutyryl cAMP on cell proliferation and macromolecular synthesis in HeLa S3 cultures.

SummaryIn HeLa S3 cultures, exogenous cAMP and its dibutyryl derivative (DBcAMP) produced divergent effects with respect to glycogen levels as well as to incorporation of labeled precursors into nucleic acids, concentration of nucleotide triphosphates, and the extent of cell proliferation inhibition.The actions of exogenous cAMP were unspecific, as they could be imitated by various adenine nucleotides. Evidence is presented which shows that all effects seen with exogenous cAMP were brought about by adenosine set free continuously by the action of extracellular enzymes.Adenosine, when provided continuously by infusion or enzymically from exogenous adenine nucleotides, caused a depletion of pyrimidine nucleotides and a subsequent inhibition of net nucleic acid formation and of cell proliferation. The increased incorporation rates of (3H)uridine and (3H)thymidine into nucleic acids seen under these conditions were also consequences of the altered precursor pool sizes.Continuous provision of adenosine leading to effective cytostasis could also be achieved in tumor-bearing animals when high doses of adenine nucleotides like cAMP or NAD were injected.DBcAMP was able to imitate intracellular cAMP only after conversion to N6-monobutyryl cAMP which accumulated inside the cells and which had a high affinity to a muscle protein kinase. N6-MBcAMP also inhibited HeLa cAMP phosphodiesterases (low and high Km) competitively and to a similar degree as DBcAMP and theophylline. This inhibition, however, does not seem to contribute significantly to the effects of DBcAMP in HeLa cultures.The actions opposite to DBcAMP of exogenous cAMP disappeared when its extracellular degradation was prevented by theophylline or by heat-inactivation of the serum used in the culture medium. Its effects under these conditions, however, were still less pronounced than the alterations caused by DBcAMP.

cAMP dependent and cAMP independent alterations of lung hemodynamic in the pig

Abstract In vivo hypoxemia leads to an elevation of pulmonary vascular resistance (pvR) in pig lungs. Orciprenaline aminophylline and Ca 2+ are potent inhibitors of this effect, while propranolol augments the alveolo-vascular reflex. Evidence is presented that the effects of orciprenaline aminophylline and propranolol on pvR are mediated by intracellular alterations of cyclic AMP concentration. In contrast, the pvR-increasing effect of hypoxemia and its supression by Ca 2+ seem to be cyclic AMP independent mechanisms.

The significance of cyclic 3′5′-AMP for the euler-liljestrand mechanism

Changes in the pulmonary vascular resistance and the cAMP levels in the lung parenchyma of four pigs were recorded during normoxia and hypoxia and following the administration of orciprenaline under hypoxia.Additional measurements recorded were pulmonary artery pressure, left-ventricular pressure, dp/dtmax, mean arterial pressure, arterial oxygen saturation, blood gas values. The rise of the pulmonary vascular resistance during hypoxia is not preceded by a decrease in intracellular cAMP levels; extreme cAMP concentrations, however, block the Euler-Liljestrand mechanism.The findings suggest that alveolar hypoxia and/or intracellular metabolic acidosis suppresses the inhibiting action of cAMP on the Ca++ influx in the smooth vascular muscles or affect the Ca++-binding process of the sarcoplasmic reticulum of the vascular musculature, resulting in vascoconstriction.

Basal cAMP in HeLa cells is protected from phosphodiesterase and does not turn over

Abstract In HeLa S3 cultures, theophylline even after 24 hours scarcely increased basal cAMP levels although it is an effective inhibitor of HeLa phosphodiesterases in vitro as well as in intact cells. The persistance of cAMP in concentrated homogenates incubated at 0° or 25°, and its resistance to charcoal adsorption suggest that it is protected from PDE by being strongly bound to specific proteins. In contrast to HeLa cultures, Ehrlich ascites carcinoma (EAC) cells do respond to theophylline by a rapid increase in cAMP. On incubation of EAC homogenates, free cAMP is degraded while the bound cAMP fraction again is protected. The existence of two cAMP pools - free cAMP and protein-bound cAMP - in tissues is postulated. They are not in (rapid) equilibrium with each other, and they differ in turnover rates.

Chronic Hypoxia: Evidence for Parasympathetic Stimulation and cGMP/cAMP-Mediated DNA Synthesis

Female Wistar rats exposed for 1–9 days to chronic hypobaric hypoxia (440 Torr or 4,250 m altitude) showed a significant increase of 3H-thymidine incorporation into the DNA of hypoxic lungs that was p

Effects of hypoxia on pulmonary vascular reactions and on lung cGMP and cAMP in pigs.

Abstract Hypoxia leads to an elevation of the pulmonary vascular resistance (pvR) in pigs. This was accompanied by an increase of intraparenchymatous concentrations of cyclic GMP (cGMP), while cyclic AMP (cAMP) levels were not different from values measured during normoxia. The inhibition of the hypoxia-induced vasoconstriction by a β 2 -receptor stimulating agent was associated with a remarkable increase in intraparenchymatous cAMP-concentrations and a decrease in cGMP levels. The correlation analysis between pvR versus the ratio cGMP/cAMP revealed a significant linearity. These data may suggest that cAMP and cGMP are involved in hypoxia induced pulmonary vasoconstriction and its pharmacological inhibition.

Histamine-an important mediator for the Euler-Liljestrand mechanism?

During normoxia and acute alveolar hypoxia the pulmonary vascular-resistance, histamine concentrations in the lung parenchyma and partially in the arterial blood, and cAMP levels in the lung tissue of young pigs were measured. A significant correlation between the pulmonary vascular resistance and the lung or blood histamine concentrations was not found during either normoxia or hypoxia. The influence of terbutaline and meclastine on the pulmonary vascular resistance and biochemical parameters was also studied. Terbutaline significantly reduced the hypoxic pulmonary vascular response. An inverse relation between the pulmonary vascular resistance and the cAMP levels during the drug induced inhibition of the Euler-Liljestrand mechanism was found. Meclastine had no influence on the hypoxic pressor response.

Partial beta-adrenergic receptor blockade in experimental bronchial asthma

The dose-response curves of Terbutaline on isolated tracheae of actively sensitized guinea pigs was significantly (p < 0,001) shifted to the right in comparison with control tracheae (ED 50 11,8 × 10−8 M resp. 5.2 × 10−8 M). Furthermore, these isolated, sensitized organs dilated significantly (p < 0.0005) slower than did controls (0.186 ± 0.072 resp. 0.350 ± 0.121 cm H2O/sec), whereas the dilation velocity after addition of theophylline did not differ. Finally, the stimulation with Fenoterol led to significantly (p < 0.0005) lower amounts of cAMP in sensitized than in control tracheae (24.3 ± 5.3 resp. 33.2 ± 7.5 pmole/mg prot.). The Km1 and Km2 of cAMP-phosphodiesterases of tracheal homogenates did not differ in both groups. These results indicate, that sensitization led to a partial blockade of the β-adrenergic-receptor-adenylatecyclase-system.