The effects of theophylline and certain other purine derivatives on tyrosine aminotransferase activity in hepatoma cells in culture

The induction of tyrosine aminotransferase in HTC cells by derivatives of adenosine 3′,5′-monophosphate is not potentiated by theopylline, a commonly used inhibitor of cyclic nucleotide phosphodiesterase. In fact, the addition of theophylline to HTC cell cultures produces a rapid decrease in the level of tyrosine aminotransferase activity. The magnitude of this decrease is dependent upon the added concentration of theopylline in both the presence and absence of enzyme inducers. Among several other purines and pyrimidines tested, caffeine and adenine most strongly resemble theophylline in affecting tyrosine aminotransferase activity. Theophylline inhibits growth and both protein and RNA synthesis in HTC cells, but the inhibition of protein synthesis cannot account completely for the effect on tyrosine aminotransferase. Theophylline also seems to increse the rate of degradation of the enzyme without affecting the degradation rate for general cellular protein. The mechanism of this apparently specific increase in degradation rate differs from both the normal degradation process for the enzyme and the enhanced degradation produced by nutritional depletion of the medium.     

Possible involvement of cGMP in the control of tyrosine aminotransferase degradation in rat hepatocytes

Addition of theophylline to primary cultures of rat hepatocytes in which tyrosine aminotransferase had been preinduced with dexamethasone caused a further increase in specific activity of the enzyme. This increase was due in part to a reduction in the rate of tyrosine aminotransferase degradation that began about 2 hr after theophylline was added. The level of cGMP also increased with a similar time lag following the addition of theophylline. The concentration of theophylline which produced the above effects (1 mM) did not alter the rate of general protein degradation in hepatocytes. Addition of 8-bromo-cGMP (0.5 mM) resulted in an immediate reduction in the rate of tyrosine aminotransferase degradation and in an increase in the activity of the enzyme. Treating hepatocytes with MnCl2 (0.9 mM) caused an elevation of cGMP and a concomitant slowing of tyrosine aminotransferase degradation without changing the level of cAMP significantly. These results suggest an inverse relationship between the level of cGMP and the rate of tyrosine aminotransferase degradation in hepatocytes.     

Differential sensitivity of HTC and Fu5-5 cells for induction of tyrosine aminotransferase by 3',5'-cyclic adenosine monophosphate

The two independently derived hepatoma cell lines (HTC and Fu5-5) have previously been shown to display different sensitivities for the induction of tyrosine aminotransferase (TAT) enzyme activity and mRNA levels by glucocorticoids with the enzyme being half-maximally induced at approximately 7-fold higher concentrations of dexamethasone in HTC cells than in Fu5-5 cells. In the present study we investigated the induction of TAT activity by cAMP in order to see whether the difference is limited to the steroidal induction. Using the stable cAMP derivative (8-(4-chlorophenylthio)-cAMP) as an inducer, we found that a 6-fold higher cAMP concentration was needed in HTC cells to achieve the same extent of enzyme induction as in Fu5-5 cells. The induction of TAT enzyme activity could be accounted for by an increased amount of TAT mRNA. Further experiments involving sequential addition of both inducers in general showed a synergism of steroids and cAMP for TAT induction in HTC cells only at submaximal concentrations of steroid; in Fu5-5 cells, the occurrence of synergism depended on the order of addition of inducers. The maximal response in HTC cells was limited to the value that could be achieved by induction with steroid alone. In Fu5-5 cells, however, the steroid response could be augmented when cAMP was added to cells already maximally induced by steroid. This demonstrates that the effect of a combination of cAMP and steroids depends on their concentration, the sequence of their addition, and the rat hepatoma cell line used. Collectively the data suggest that a common pretranslational event determines the differential sensitivity of TAT induction by glucocorticoids and by cAMP in HTC and Fu5-5 cells. Furthermore a second, or possibly the same, common event also regulates the maximum level of TAT induction that is obtainable under most conditions with glucocorticoids and/or cAMP.     

Stimulation of bovine endothelial cell angiotensin-I-converting enzyme activity by cyclic AMP-related agents

Bovine pulmonary artery endothelial cells in culture were used to assess the influence of cyclic nucleotides, isoproterenol (beta adrenergic agonist), and theophylline (phosphodiesterase inhibitor) on angiotensin-I-converting enzyme (ACE) activity of the cells and culture medium. Dibutyryl cAMP (10(-3) M) but not cAMP or dibutyryl cGMP stimulated angiotensin-I-converting enzyme (ACE) activity of cells in culture approximately 50-100% but had little influence on ACE activity of the medium. Theophylline at 10(-3) M concentration produced a three- to fourfold stimulation of both cellular and medium ACE activity. Isoproterenol by itself had no effect on cellular ACE activity but produced a stimulatory effect at 10(-7)-10(-5) M concentration after pretreatment of cells for 24 hr with 10(-4) M theophylline. The results support the concept that ACE activity of endothelial cells is influenced by the cyclic AMP system. ACE activity in cells and that released into medium may be under different regulatory controls.     

Antiglucocorticoid steroids have increased agonist activity in those hepatoma cell lines that are more sensitive to glucocorticoids

FU5-5 rat hepatoma (Reuber H35) cells are hypersensitive in that the same percentages of full induction of tyrosine aminotransferase (TAT) occur at much lower concentrations of glucocorticoids than in the related HTC rat hepatoma (Morris) cells. Unexpectedly, these hypersensitive FU5-5 cells also exhibited more agonist activity with the affinity labeling antiglucocorticoids cortisol 21-mesylate and dexamethasone 21-mesylate than did HTC cells (Mercier et al., Endocrinology 112, 601-609 [1983]). In the present study, several other antiglucocorticoids (11-desoxycortisone, progesterone, dexamethasone oxetanone, and RU 486 in addition to dexamethasone 21-mesylate) and the antiandrogen cyproterone acetate were examined to see if chemically unreactive, reversible antisteroids also would exhibit an altered activity (i.e. increased agonist activity) in FU5-5 cells. Each antiglucocorticoid examined did display a 2-fold increased amount of agonist activity in FU5-5 cells, as compared to HTC cells; only RU 486 was predominantly an antagonist in FU5-5 cells but the potency of RU 486 was about 9-fold less than in HTC cells. Dexamethasone, and especially progesterone, was metabolized in FU5-5 and HTC cells. However, differential metabolism in FU5-5 vs HTC cells cannot account for the increased induction of TAT in FU5-5 cells since the amount of agonist activity seen for dexamethasone mesylate (or its metabolites) depended not on the cell type used but rather on the glucocorticoid inducible enzyme monitored, i.e. TAT or glutamine synthetase. The combined data suggest that the hypersensitivity of FU5-5 cells towards glucocorticoid induction of TAT may be linked with the ability of both reversible and irreversible antiglucocorticoids to display increased TAT agonist activity in FU5-5 cells. This behavior was somewhat steroid specific since the antiandrogen cyproterone acetate did not display increased TAT agonist activity in FU5-5 cells compared to HTC cells and was only 2-fold less effective as an antiglucocorticoid in FU5-5.     

Acceleration of the degradation of tyrosine aminotransferase in rat hepatome (HTC) cells by inhibitors of cyclic nucleotide phosphodiesterase.

The following inhibitors of cyclic nucleotide phosphodiesterase reduce the specific activity of tyrosine aminotransferase when added to cultures of rat hepatoma (HTC) cells: theophylline, 1-methyl-3-isobutylxanthine, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone, and papaverine. Immunochemical measurements show that each inhibitor reduces the rate of tyrosine aminotransferase synthesis and increases the rate of degradation of the enzyme. The effect on synthesis also occurs with general proteins while that on degradation appears specific for tyrosine aminotransferase.     

Calcium requirement for the inhibition by theophylline of histamine release from mast cells

When added to Ca2+-free Hanks' solution, Ca2+ (0.1-2.5 mM) had no significant effect on antigen-induced histamine release from rat mast cells, but Sr2+ (1.0-3.0 mM) dose-dependently increased the release. Ba2+ (1.0 and 2.0 mM) also enhanced the release. Ca2+ and Ba2+ inhibited compound 40/80-induced histamine release, in a dose-dependent manner. In ordinary Hanks' medium, theophylline and 3-isobutyl-1-methylxanthine (IBMX) dose-dependently inhibited the antigen-induced histamine release but these drugs were ineffective in Ca2+-free medium. Theophylline (1.0 mM) also inhibited compound 48/80-induced histamine release in the presence but not absence of Ca2+. There was an optimal Ca2+ concentration for the theophylline effect. Sr2+ but not Ba2+ could substitute for Ca2+ in supporting the theophylline effect. Theophylline (1.0 mM) and IBMX (1.0 mM) increased mast cell cyclic AMP levels both in the presence and absence of Ca2+. These results suggest that Ca2+ is required in the interaction of theophylline and specific sites on mast cells or in the mast cell response to theophylline which probably does not involve the cyclic AMP increase and is linked to the inhibition of histamine release.     

Increasing intracellular cAMP and cGMP inhibits cadmium-induced oxidative stress in rat submandibular saliva

The effect of cadmium on induction of oxidative stress in rat submandibular saliva and protective role of increasing intracellular cAMP and cGMP by use of specific phosphodiesterase inhibitors, theophylline and sildenafil were investigated. Pure submandibular saliva was collected intraorally by micro polyethylene cannula from anaesthetized rats using pilocarpine as secretagogue. Acute administration of cadmium (10 mg/kg) caused significant oxidative stress by increasing lipid peroxidation by-products (thiobarbituric reactive substances, TBARS) and decreasing total thiols and total antioxidant power of the saliva. Concurrent therapy of rats by theophylline (25 mg/kg) and sildenafil (5 mg/kg) prevented cadmium-induced oxidative stress in saliva. Theophylline and sildenafil inhibited cadmium-induced increase in lipid peroxidation and decrease in total thiols and antioxidant power. It is concluded that cadmium administration results in oxidative stress in rat submandibular saliva, which can be protected by concurrent administration of specific cyclic nucleotide phosphodiesterase inhibitors.     

Inhibition of uridine transport in cultured mammalian cells by theophylline

Theophylline (theobromine, caffeine) reversibly inhibits the incorporation of labeled RNA precursors both in confluent 37 RC and in exponentially growing HeLa cells. As measured in 37 RC after 2 h labeling, 20 mM theophylline reduces the incorporation of [3H]UTP and [14C]uridine into acid-precipitable material to 5% and 9% of the control, respectively. This reduction is paralleled by a comparably lowered incorporation of the same precursors into the acid-soluble pool. The initial rate of incorporation into total cell material is similarly affected by theophylline, the inhibition being of a simple competitive type. Theophylline does not alter the turnover rate of pulse labeled RNA during actinomycin D chase nor does it preclude the utilization of the endogenous pool of nucleoside phosphates. Upto a concentration of 10 mM, it does not inhibit uridine kinase neither in 37 RC nor in HeLa cells. The mentioned inhibitory effects of theophylline cannot be mimicked by exogenously added cyclic AMP. All the data support the conclusion that theophylline inhibits the transport of uridine into the cell.     

The stimulus-secretion coupling of glucose-induced insulin release. Insulin release due to glycogenolysis in glucose-deprived islets.

1. When pancreatic islets are preincubated for 20h in the presence of glucose (83.3mM) and thereafter transferred to a glucose-free medium, theophylline (1.4mM) provokes a dramatic stimulation of insulin release. This phenomenon does not occur when the islets are preincubated for either 20h at low glucose concentration (5.6mM) or only 30 min at the high glucose concentration (83.3mM). 2. The insulinotropic action of theophylline cannot be attributed to contamination of the islets with exogenous glucose and is not suppressed by mannoheptulose. 3. The secretory response to theophylline is an immediate phenomenon, but disappears after 60min of exposure to the drug. 4. The release of insulin evoked by theophylline is abolished in calcium-depleted media containing EGTA. Theophylline enhances the net uptake of 45Ca by the islets. 5. Glycogen accumulates in the islets during the preincubation period, as judged by both ultrastructural and biochemical criteria. Theophylline significantly increases the rate of glycogenolysis during the final incubation in the glucose-free medium. 6. The theophylline-induced increase in glycogenolysis coincides with a higher rate of both lactate output and oxidation of endogenous 14C-labelled substrates. 7. These data suggest that stimulation of glycolysis from endogenous stores of glycogen is sufficient to provoke insulin release even in glucose-deprived islets, as if the binding of extracellular glucose to hypothetical plasma-membrane glucoreceptors is not an essential feature of the stimulus-secretion coupling process.     

"Superinduction" of tyrosine aminotransferase by actinomycin D: a reevaluation.

Reexamination of the effects of actinomycin D (AMD) on the intracellular level and rate of synthesis of tyrosine aminotransferase (TAT) in hepatoma tissue culture (HTC) cells reveals that much apparent controversy can be resolved with acknowledgment of the multi-faceted nature of this inhibitor's action. AMD can slow overall protein synthesis and inhibit the degradation of both TAT and its mRNA as well as block the synthesis of RNA. The extent of these secondary actions of the inhibitor depend somewhat upon the growth condition of the cells. The effects of cordycepin (3'-deoxyadenosine) on the metabolism of TAT and its mRNA are also complex, but differ in several respects from those of AMD.     

Effect of theophylline on ion transport in the lizard colon

1. The effect of theophylline on ion transport was examined using an in vitro short-circuited preparation of lizard colon. 2. Theophylline increased short circuit current (Isc) and transmural potential difference (PD). This increase caused by theophylline was accompanied by a small increase in transmural conductance (Gt). 3. Theophylline did not inhibit the absorption of Na+ but reversed Cl- absorption to secretion. This latter effect was due to an increase of the serosal-to-mucosal flux of Cl-. 4. Ion substitution experiments revealed that the effect of theophylline was Na+- and HCO3(-)-dependent and that these ions were required in the bathing solution. 5. These results with lizard colon are compared with those reported for mammalian colon and the mechanism of theophylline-induced Cl- secretion in these epithelia is discussed.     

The effect of heat-stable Escherichia coli enterotoxin, theophylline and forskolin on cyclic nucleotide levels and mucosal surface (acid microclimate) pH in rat proximal jejunum in vivo

The normally acidic mucosal surface pH of 6.24 +/- 0.02(30) in rat proximal jejunum in vivo is effectively neutralised by 30 min exposure to heat-stable Escherichia coli (STa) enterotoxin (14 micrograms/ml) to 6.80 +/- 0.07 (n = 5) or to a forskolin/theophylline combination (1 mM:20 mM) to 7.10 +/- 0.07(7) while perfusion with Krebs-phosphate buffer alone without glucose left the mucosal surface pH unchanged at a pH of 6.21 +/- 0.02(9). Forskolin alone had no effect, and 20 mM theophylline moderately elevated the surface pH to 6.52 +/- 0.03(5). Theophylline, forskolin and their combination all elevated cAMP levels per mg tissue DNA above control values while STa enterotoxin was without effect. In contrast, all agents elevated cGMP levels per mg tissue DNA above control levels. These findings indicate that surface pH is only moderately affected by changes in cAMP levels and is affected to a much greater extent by altered cGMP levels.     

Inhibition by theophylline of phagocytosis in Acanthamoeba castellanii.

Theophylline inhibited the phagocytosis of latex beads by Acanthamoeba castellanii (Neff). The cells recovered the ability to engulf beads after 1-2 hr of exposure to theophylline. Cells which have been exposed to 25 mM theophylline for a period of inhibition and recovery were not inhibited further by incubation with a fresh medium containing the same concentration of theophylline. However, the medium in which the cells recovered was as effective as a fresh medium in inhibiting phagocytosis in a fresh batch of cells, suggesting that the development of insensitivity to theophylline inhibition resides with the cells themselves. Dibutyryl cyclic AMP also inhibited bead uptake.     

Uptake of cyclic guanosine-3',5'-monophosphate and its metabolism in 3T6 cells

Uptake of 3H-cGMP by cultured murine 3T6 cells was studied. The cells were shown to contain radioactivity 1 minute after its addition, with the level of radioactivity increasing during a 3 hour incubation period. By this time, the intracellular non-metabolized cGMP corresponded to 1-5% of the whole intracellular radioactivity. In the presence of theophylline the uptake of 3H-cGMP by cells was seen decreasing, however, the portion of non-metabolized cGMP reached 45-50% of the whole intracellular radioactivity. Thus, the presence of theophylline made it possible to maintain the high level of intracellular cGMP. It is concluded that the incubation of cell cultures in the medium with cGMP may be useful for achieving an elevating intracellular cGMP concentration and for studying the biological effect of cyclic nucleotide.     

Cellular cyclic AMP in dispersed mucosal cells from guinea pig stomach.

In dispersed mucosal cells from guinea pig stomach cyclic AMP was increased 4-fold by theophylline, 5-fold by prostaglandin E2, and 10- to 15-fold by histamine. Theophylline augmented the increase in cellular cyclic AMP caused by histamine or prostaglandin E1 and the actions of histamine and prostaglandin E1 were additive. Cellular cyclic AMP was not altered by carbachol, gastrin, secretin, vasoactive intestinal peptide, glucagon, insulin or the octapeptide of cholecystokinin. Metiamide or diphenhydramine but not atropine inhibited the increase in cellular cyclic AMP caused by histamine, but did not alter the concentration of cyclic AMP in control cells or in cells incubated with theophylline or prostaglandin E1.     

Tyrosine aminotransferase sensitivity to bromodeoxyuridine during restricted intervals of S phase in hepatoma cells

Synchronized hepatoma tissue culture (HTC) cells, accumulated at the G1/S boundary with aminopterin, were released into S phase with either thymidine or 5-bromodeoxyuridine (BUdR). Tyrosine aminotransferase (TAT) activity was found to be unaffected by BUdR over the initial 3 h of S phase, but then to rapidly decline to a new basal level of 40% of control by 9 h. There was no corresponding response in the activities of alcohol dehydrogenase, malate dehydrogenase, acid phosphatase, and alkaline phosphatase, or in the rate of protein and RNA synthesis. If BUdR incorporation was restricted to limited periods of S phase, TAT was found to be maximally suppressed by incorporation into the initial 40% of the DNA. Incorporation of the analogue into the latter 60% of DNA synthesized during S phase had no effect on TAT. This is the first report that the effect of BUdR on TAT in HTC cells is associated with incorporation of the analog into DNA synthesized during a specific interval of S phase.