Effect of human interferons on cAMP phosphodiesterase activity in a culture of human ovarian carcinoma cells (CaOv)

Crude and purified human interferons of alpha type exerted 2 step inhibition of cAMP phosphodiesterase activity in CaOv cells: in 4 and 24 hours after cells treatment with interferon. The maximal inhibition was obtained in response to interferon doses 1200-2000 IU/ml. In contrast to natural interferons the human alpha 2 recombinant interferon (20-25000 IU/ml) did not inhibit the cAMP phosphodiesterase activity in CaOv cells.     

Effect of interferon on the cAMP system in cells

The increase in cAMP concentration in CaOv cells affected by alpha-interferon has been found to have a two wave character with the maximums at 4 and 24 h after the effect. The waves are due to the increase in adenylate cyclase activity and to the decrease in the activity of cAMP phosphodiesterase. The described changes were characteristic of the native and partially purified interferon and depended on the concentration of interferon used (optimal effect at 1200 IU/ml-1). It suffices to notice that the described effects were more largely expressed when the preparations of the native alpha-interferon were used. The correlation was noticed between the increase in adenylate cyclase activity, the decrease in cAMP phosphodiesterase and the concentration of the cyclic nucleotide as well as the expression of antiproliferative effect. The correlation was less significant for antiviral effect.     

Effect of modulation of the production of interferon by L-929 cells treated with theophylline

The specific inhibitor of cAMP phosphodiesterase theophylline has been shown to evoke in L929 cells 2.3-fold induction of 2-5A-synthetase activity and 3.5-fold superinduction of the same enzyme activity while acting in combination with actinomycin D. It has been shown also that temporal coincidence of 2-5A-synthetase induction with the active period of interferon production resulted in 8-16 times decrease in the level of interferon production. The result was supported by the experiments of superinduced cells (containing the high stable level of 2-5A-synthetase) fusion with monolayer of poly(I).poly(C)-induced L929 cells (taken at the start of interferon production). In this case the production of interferon was dramatically decreased in comparison with the control. Possible role of 2-5A-synthetase in regulation of interferon production is discussed.     

Cyclic AMP-mediated control of oocyte maturation in the catfish, Clarias batrachus (Bloch): effects of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one and phosphodiesterase inhibitors

An increase in the percentage of germinal vesicle breakdown (GVBD) with a corresponding decrease in cAMP was found in the oocytes which were incubated for 36 hr with different concentrations of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-DP). At its highest concentration (1 microgram/ml), 17 alpha,20 beta-DP induced 91.9 +/- 2.3% GVBD and decreased cAMP level to 0.8 +/- 0.1 pmol/oocyte from 2.9 +/- 0.2 pmol/oocyte (control). The two different known inhibitors of phosphodiesterase viz. 3-isobutyl-1-methyl-xanthine (IBMX) and theophylline inhibited GVBD in vitro and promoted the accumulation of cAMP in a dose-dependent manner irrespective of whether the oocytes were treated for a short duration (2 hr) or for a long duration (36 hr). Evaluation of time course response to 1 mM IBMX or 1 mM theophylline revealed that cAMP levels increased at all the time points when compared with their respective controls and blocked maturation. In contrast, 1 microgram/ml 17 alpha,20 beta-DP not only induced oocyte maturation but also caused an immediate decrease in cAMP within the first 2 hr (from 3.2 +/- 1.3 to 1.3 +/- 0.1 pmol/oocyte) of incubation which was maintained till the end of experiment (36 hr). Likewise, a significant inhibition of GVBD and accumulation of cAMP was recorded even in oocytes pre-stimulated with 1 microgram/ml 17 alpha,20 beta-DP for 6 hr and then treated with different concentrations of IBMX or theophylline. Taken together, these data strongly suggest that in C. batrachus a decrease of oocyte cAMP concentration is a prerequisite for the induction of oocyte maturation, and its increase is associated with the maintenance of meiotic arrest.     

Reduction of adenine nucleotide content of clone 4 MDCK cells: effects on multiplication, protein synthesis, and morphology

The antitumor agent hadacidin (N-formyl-hydroxyamino-acetic acid), at 4 mM, inhibited the multiplication of clone 4 Madin Darby canine kidney (MDCK) cells within 24 hr. Growth resumed rapidly upon replacement of hadacidin with aspartate, an observation consistent with the drug's action as a competitive inhibitor of adenylosuccinate synthetase, an enzyme in adenine nucleotide biosynthesis. Data indicate that the drug-treated cells were arrested in S phase of the cell cycle. Accompanying inhibition of multiplication was a 16-fold increase in the area occupied by the cells and a refractoriness to release by treatment with trypsin. None of these changes occurred when 0.5 mM adenosine was included in the incubation mixture containing the inhibitor. Hadacidin decreased the adenosine triphosphate (ATP) and cyclic adenosine monophosphate (cAMP) content of the cells as well as the rate at which 3H-leucine was incorporated into protein. In the presence of 1 mM dibutyryl cAMP and theophylline, the drug had no effect on cell division and protein synthesis. The data suggest that, in clone 4 MDCK cells, the effects of hadacidin are mediated by diminishing the level of cAMP.     

Interactions of formylmethionyl-leucyl-phenylalanine, adenosine, and phosphodiesterase inhibitors in human monocytes. Effects on superoxide release, inositol phosphates and cAMP

Cessation of the fMLF-induced burst of human monocyte superoxide release was associated with a rise in cAMP. This was not due to inhibition of phosphodiesterase (PDE), the major form of which was the PDE IV isozyme. The action of burst inhibitors did not correlate with cAMP levels: Rolipram, a PDe IV inhibitor, increased cAMP 6-fold, with minimal effects on the burst; whereas theophylline increased cAMP less than 2-fold but decreased the burst to less than half. Although theophylline and the adenylate cyclase activator, adenosine, inhibited fMLF-induced superoxide release, they did not inhibit production of inositol phosphates. Thus, these studies on inhibition of superoxide release implicated neither cAMP nor inositol phosphates.     

Differences and similarities between guanosine 3',5'-cyclic monophosphate phosphodiesterase and adenosine 3',5'-cyclic monophosphate phosphodiesterase activities in neuroblastoma cells in culture.

There are phosphodiesterase activities in both particulate and supernatant fractions which hydrolyze guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) with an apparent Km of 2-8 muM and with an apparent Km of 44-222 muM. 4-(3-Butoxy-4-methoxybenzyl-2-imidazolidinone (RO20-1724) did not inhibit cGMP phosphodiesterase activity in homogenates of mouse neuroblastoma cells, but markedly inhibited cAMP phosphodiesterase activity. Papaverine and theophylline inhibited both cGMP and cAMP phosphodiesterase activities to about the same extent. The former was more potent than the latter. The specific activity of cGMP phosphodiesterase as a function of protein concentrations first increased and then decreased. The specific activity of cAMP phosphodiesterase decreased under a similar experimental condition.     

Cyclic AMP and theophylline enhance DNA synthesis in L cells stimulated with anti-actin IgG and [(IgG)2protein A]2 complex by recruiting cells into S-phase

Surface binding of anti-actin IgG alone or in a M_r = 716 000 [(IgG)₂Protein A]₂ complex results in a stimulation of DNA synthesis and cell growth in L cells. Cyclic-AMP (0.01–1.0 mM) added to such cell cultures augmented DNA synthesis as measured by incorporation of [³H]thymidine into DNA. Theophylline (0.1–1.0 mM), a phosphodiesterase inhibitor which prevents enzymatic breakdown of cAMP, had similar effects, but cGMP (0.01–1.0 μM) reversed the effects of cAMP and theophylline upon DNA synthesis. Analysis of the cell cycle by flow cytometry revealed that antibody produced a shift (7%) of cells from the G₁-phase to the S-phase (DNA-synthetic) of the cell cycle at 72 hr of incubation. Addition of cAMP (0.5 mM) to cell cultures, however, produced significant shifts of antibody stimulated cells from G₁-phase to S-phase at all time points measured, i.e., 24 (12%),48 (22%),72 hr (23%). Thus, antibody recruited cells into S-phase of the cell cycle and cAMP greatly augmented the effect. These observations suggest that the mechanism of activation of L cell growth by antibody to surface antigens involves a recruitment of cells into the DNA-synthetic phase and that the effect may be mediated by cAMP.     

Regulation of abalone sperm cyclic AMP concentrations and the acrosome reaction by calcium and methylxanthines

When Ca2+ is added to abalone sperm (Haliotis rufescens) in Ca2+-free artificial seawater (CaFASW) to a final concentration of 9.6 mM a 4-fold elevation in sperm cAMP occurs within 15-30 sec. The methylxanthines, theophylline and 1-methyl-3-isobutylxanthine (MIX), also elevate sperm cAMP concentrations. In CaFASW, either compound causes up to a 3-fold increase in cAMP within 15-30 sec. MIX (150 microM), added to sperm in the presence of 9.6 mM Ca2+, elevates sperm cAMP 100-fold within 15-30 sec and also triggers the acrosome reaction (AR) in the same period. Under identical conditions theophylline (1.67 mM) is much less potent at elevating cAMP and inducing the AR. The effects of methylxanthines on cAMP of sperm incubated in the presence of Ca2+ appear to represent a potentiation by these compounds of the action of Ca2+. Neither compound induces the AR in the absence of Ca2+. All of the observed effects on sperm cAMP and the AR are dependent on Ca2+ and methylxanthine concentrations. Added cyclic nucleotides or their derivatives do not induce the AR in either the absence or presence of Ca2+. Experiments with isolated sperm heads and flagella indicate that the dramatic stimulatory response of sperm cAMP to Ca2+ plus MIX is present in the head region (acrosome, nucleus, midpiece) of the cell. The data suggest that the dramatic elevation of cAMP by MIX in the presence of Ca2+ may occur directly by an inhibition of phosphodiesterase activity and indirectly by an increase in cellular Ca2+. A strong temporal correlation between the cAMP elevation and the abalone AR exists, although cAMP elevation by itself does not act as the primary mediator of this exocytotic event.     

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.     

Antiviral and antiproliferative activity of human interferons and their induction of 2',5'-oligoadenylate synthetase

Native preparations of alpha, beta and gamma-interferons as well as recombinant beta-interferon and purified leukocyte alpha-interferon and purified leukocyte alpha-interferon exert antiviral and antiproliferative activity in CaOv cells. Native interferon preparations were shown to be more antiproliferative than purified interferons per unit of antiviral activity (with EMC as well as with less susceptible VSV used as test viruses). It was shown that level of 2'5' oligoadenylatesynthetase activity induction in general correlates with antiproliferative and pronounced antiviral activity of interferons, besides that, the earlier (by 11 hours) induction of the enzyme activity by beta-interferon correlates with more rapid expression of antiproliferative effects by this interferon in comparison with that of alpha-interferon, the latter inducing the peak of enzyme activity by 24 hours.     

Cyclic AMP and the vascular action of parathyroid hormone

The involvement of tissue cAMP in the vasodilating action of parathyroid hormone (PTH) was investigated. The bovine active fragment bPTH-(1-34) was used in all studies. In anesthetized dogs, theophylline, a phosphodiesterase inhibitor, potentiated the hypotensive action of bPTH-(1-34) at the dose of 1 microgram/kg. The potentiation was related to the dose of theophylline infused. In an in vitro rat tail artery helical strip assay, dibutyryl cAMP produced dose-related relaxation in arginine vasopressin (AVP) constricted blood vessels. bPTH-(1-34) also produced dose-related relaxation in the tail artery constricted by AVP. In the presence of isobutylmethylxanthine, another phosphodiesterase inhibitor, the bPTH-(1-34) dose--response curve was shifted to the left, indicating potentiation. Imidazole, which has phosphodiesterase stimulating activity, significantly decreased the in vitro vasorelaxing effect of bPTH-(1-34). In addition, bPTH-(1-34) increased significantly the rat tail artery cAMP content. b-PTH-(1-34) oxidized with hydrogen peroxide lost its vasorelaxing activity and was also ineffective in increasing the tail artery cAMP content. All these data strongly suggest that cAMP may be involved in eliciting the vasorelaxing action of bPTH-(1-34).     

Chemotactic peptide induces cAMP elevation in human neutrophils by amplification of the adenylate cyclase response to endogenously produced adenosine

The transient increase in human neutrophil cAMP levels induced by the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) is shown to be caused by amplification of adenylate cyclase response to endogenously produced adenosine. The FMLP-stimulated increase in neutrophil cAMP was potentiated markedly by a nonmethylxanthine cAMP phosphodiesterase inhibitor (Ro 20-1724). By inhibiting the degradation of newly formed cAMP, Ro 20-1724 rendered the FMLP-induced cAMP elevation persistent rather than transient. The role of endogenously produced adenosine in this phenomenon is demonstrated by the ability of either adenosine deaminase or theophylline, an adenosine receptor antagonist, to prevent FMLP-stimulated cAMP elevation. The general nature of the FMLP-potentiated cAMP response is indicated by the finding that FMLP-treated neutrophils, in the presence of exogenously supplied adenosine deaminase, exhibited augmented cAMP generation in response to three different types of receptor agonists: 2-chloroadenosine, prostaglandin E1, and L-isoproterenol. Moreover, like the neutrophil cAMP increase caused by FMLP alone, the ability of FMLP to augment cAMP response to 2-chloroadenosine in adenosine deaminase-treated cells was short-lived and declined after 1.0 min of exposure to FMLP. Preincubation of neutrophil suspensions with the adenylate cyclase inhibitor SQ 22,536 completely prevented FMLP-induced cAMP generation. Furthermore, when neutrophil suspensions were preincubated with concentrations of Ro 20-1724, which apparently maximally inhibit cAMP phosphodiesterase, a 30-s incubation with FMLP still resulted in substantially elevated cAMP levels. It therefore appears that FMLP raises cAMP by activating adenylate cyclase rather than inhibiting cAMP phosphodiesterase.     

Effects of okadaic acid on insulin-sensitive cAMP phosphodiesterase in rat adipocytes. Evidence that insulin may stimulate the enzyme by phosphorylation.

Okadaic acid, a potent inhibitor of Type 1 and Type 2A protein phosphatases, was used to investigate the mechanism of insulin action on membrane-bound low Km cAMP phosphodiesterase in rat adipocytes. Upon incubation of cells with 1 microM okadaic acid for 20 min, phosphodiesterase was stimulated 3.7- to 3.9-fold. This stimulation was larger than that elicited by insulin (2.5- to 3.0-fold). Although okadaic acid enhanced the effect of insulin, the maximum effects of the two agents were not additive. When cells were pretreated with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), the level of phosphodiesterase stimulation by okadaic acid was rendered smaller, similar to that attained by insulin. In cells that had been treated with 2 mM KCN, okadaic acid (like insulin) failed to stimulate phosphodiesterase, suggesting that ATP was essential. Also, as reported previously, the effect of insulin on phosphodiesterase was reversed upon exposure of hormone-treated cells to KCN. This deactivation of previously-stimulated phosphodiesterase was blocked by okadaic acid, but not by insulin. The above KCN experiments were carried out with cells in which A-kinase activity was minimized by pretreatment with H-7. Okadaic acid mildly stimulated basal glucose transport and, at the same time, strongly inhibited the action of insulin thereon. It is suggested that insulin may stimulate phosphodiesterase by promoting its phosphorylation and that the hormonal effect may be reversed by a protein phosphatase which is sensitive to okadaic acid. The hypothetical protein kinase thought to be involved in the insulin-dependent stimulation of phosphodiesterase appears to be more H-7-resistant than A-kinase.     

Regulation of prostaglandin E2 binding to a murine macrophage cell line, P388D1, by insulin.

Preincubation of murine macrophage-like P388D1 cells with physiological amounts of insulin resulted in an increase in prostaglandin E2 binding to these cells, by approximately 2-fold, when compared to untreated cells. Scatchard analysis of the binding of PGE2 to insulin-treated cells indicated that the enhanced binding was due to an increase in receptor number (from 0.30 +/- 0.02 to 0.63 +/- 0.03 fmol/10(6) cells for the high affinity receptor binding sites, and from 2.4 +/- 0.31 to 5.0 +/- 0.41 fmol/10(6) cells for the low affinity receptor binding sites) rather than to an increase in the affinity of the binding sites. The insulin-stimulation of PGE2 binding appeared to be associated with a lowering of the cAMP level in these cells; treatment of cells with insulin lowered the cAMP level by increasing the cAMP phosphodiesterase activity of both the membrane and cytosolic fractions. However, enhanced PGE2 binding to the cells resulted in an increase in cAMP level in the cells. This increase in cAMP level may help to enhance the immunosuppressive action of this prostanoid, as PGE2 is known to suppress many steps in the immune response, including interleukin-1 expression, by raising cAMP levels via activation of receptor-linked adenylate cyclase. Our data suggest that insulin at physiological concentrations may enhance the immunosuppressive action of PGE2.     

A similar pool of cyclic AMP phosphodiesterase in Xenopus oocytes is stimulated by insulin, insulin-like growth factor 1, and [Val12,Thr59]Ha-ras protein

Pharmacological analysis of in vivo cAMP phosphodiesterase in Xenopus oocytes using the nonselective enzyme inhibitors 3-isobutyl-1-methylxanthine (IBMX), theophylline, and papaverine, demonstrated inhibition of insulin- and insulin-like growth factor-1-induced maturation at concentrations that were 17-60-fold lower than those required to inhibit progesterone-induced germinal vesicle breakdown. The abilities of the phosphodiesterase inhibitors to block the maturation response showed the same rank order of potencies for each hormone: papaverine greater than IBMX greater than theophylline. Insulin-induced oocyte maturation that was accelerated by 0.01 microM progesterone was also inhibited by low micromolar concentrations of IBMX, demonstrating that the accelerated time course was due to a synergistic potentiation of insulin action by progesterone. Both insulin-induced maturation and insulin-stimulated phosphodiesterase activity displayed similar sensitivities to inhibition by IBMX, suggesting that hormone-stimulated phosphodiesterase activity is required for the peptide hormone action. Furthermore, microinjection of the transforming ras gene product [Val12,Thr59]Ha induced oocyte maturation and stimulated oocyte phosphodiesterase activity by approximately 50%, and both of these actions were inhibited by IBMX. These results suggest that oocyte maturation induced by insulin, insulin-like growth factor 1, and transforming ras protein involves stimulation of a similar phosphodiesterase.     

A study of adenosine 3':5'-cyclic monophosphate, sodium butyrate and cortisol as inducers of HeLa alkaline phosphatase

The role of adenosine 3′:5′-cyclic monophosphate in the cortisol-mediated induction of HeLa 65 alkaline phosphatase was investigated. Although growth of these cells with 0.5–1.0 mmN₆,O²′-dibutyryl adenosine 3′:5′-cyclic monophosphate induces a 5- to 8-fold increase in cellular phosphatase activity after 72 hr, neither cAMP nor theophylline induce at concentrations up to 1 mm. Sodium butyrate induces the enzyme as well as dibutyryl cAMP. Moreover, induction kinetics show sodium butyrate to be a more efficient inducer than dibutyryl cAMP, inducing activity as quickly as cortisol. This suggests that the butyric acid cleaved from dibutyryl cAMP by HeLa cells is the mediator of induction when the cyclic nucleotide derivative is used.     

Characteristics of cyclic AMP enhancement of retinoic acid induction of increased transglutaminase activity in HL60 cells

When the human myeloid leukemia cell line (HL60) is induced to differentiate with retinoic acid (RA), there is a concentration-dependent increase in transglutaminase (TGase) activity which peaks on day 5. While dibutyryl 3',5'-cyclic adenosine monophosphate (db-cAMP) alone produced only a slight increase in TGase activity in HL60 cells, the concomitant addition of db-cAMP (100 microM) with RA (10(-12)-10(-4) M) potentiates RA induction of TGase activity. Maximal increases in TGase activity (2- to 10-fold) were observed with 10(-4)-10(-7) M RA and when db-cAMP was present from 24 to 48 h after the addition of RA. The cyclic nucleotide enhancement was dose-dependent from 10 to 100 microM of cAMP. Less marked increases were observed with 8-bromo-cAMP and with the phosphodiesterase inhibitor theophylline. Although the simultaneous addition of PGE1 or PGE2 (10(-8)-10(-6) M) produced no enhancement of RA-induced TGase activity, adding PGE1 or PGE2 24 or 48 h following RA treatments produced an enhancement of TGase activity. The phosphodiesterase inhibitor potentiated the increases produced by db-cAMP and the prostaglandins. Dibutyryl cAMP enhanced the ability of RA to induce the cells to reduce nitroblue tetrazolium (NBT), a functional measure of differentiation, at lower concentrations of RA and with shorter treatment durations. cAMP potentiates RA-induced TGase activity in HL60 cells and the combination appears to be associated with enhanced RA-induced differentiation.     

Cell-mediated cytotoxicity against virus-infected target cells in humans. II. Interferon induction and activation of natural killer cells.

The mechanisms by which human lymphocytes lyse virus-infected allogeneic fibroblast cultures were analyzed with particular consideration of the role of anti-viral antibodies and interferon. Human cells infected with viruses were able to induce high levels of interferon upon contact with human lymphocytes. Interferon, whether produced by lymphocytes after direct infection with virus or induced upon exposure of lymphocytes to virus-infected fibroblasts, appeared to be responsible for enhancing the cytotoxic efficiency of the natural killer cell against the infected target. Activation of cytotoxic lymphocytes occurred as early as 6 hr after addition of interferon and increased up to 24 hr. Antibody-dependent cell-mediated cytotoxicity (Ab-CMC) could be easily induced by sensitization of infected target cells with antiviral antibodies and could be detected at 4 hr from the beginning of the cytotoxic test, before the effect of interferon on the natural killer cell was evident. However, the antibody-dependent effector cell was inactive after 4 hr of incubation. F(ab')2 fragments of rabbit anti-human IgG completely inhibited Ab-CMC but did not at all affect the spontaneous cytotoxic activity of the effector cells against virus-infected target.