In vitro photochemical and phototoxicological characterization of major constituents in St. John's Wort (Hypericum perforatum) extracts

Extracts from St. John's Wort (SJW: Hypericum perforatum) have been used for the treatment of mild-to-moderate depression. In spite of the high therapeutic potential, orally administered SJW sometimes causes phototoxic skin responses. As such, the present study aimed to clarify the phototoxic mechanisms and to identify the major phototoxins of SJW extract. Photobiochemical properties of SJW extract and 19 known constituents were characterized with focus on generation of reactive oxygen species (ROS), lipid peroxidation, and DNA photocleavage, which are indicative of photosensitive, photoirritant, and photogenotoxic potentials, respectively. ROS assay revealed the photoreactivity of SJW extract and some SJW ingredients as evidenced by type I and/or II photochemical reactions under light exposure. Not all the ROS-generating constituents caused photosensitized peroxidation of linoleic acid and photodynamic cleavage of plasmid DNA, and only hypericin, pseudohypericin, and hyperforin exhibited in vitro photoirritant potential. Concomitant UV exposure of quercitrin, an SJW component with potent UV/Vis absorption, with hyperforin resulted in significant attenuation of photodynamic generation of singlet oxygen from hyperforin, but not with hypericin. In conclusion, our results suggested that hypericin, pseudohypericin, and hyperforin might be responsible for the in vitro phototoxic effects of SJW extract.     

Lipofuscin-mediated photic stress inhibits phagocytic activity of ARPE-19 cells; effect of donors’ age and antioxidants

The risk of chronic oxidative stress in the retinal pigment epithelium (RPE) increases with age due to accumulation of the photoreactive age pigment lipofuscin (LFG). Here, we asked whether sublethal and weakly lethal photic stress, induced by irradiation of ARPE-19 cells containing phagocytised LFG, affected the cell specific phagocytic activity, which is critically important for proper functioning and survival of the retina, and if natural antioxidants could modify the observed outcomes. ARPE-19 cells preloaded with LFG isolated from human donors of different age or containing LFG enriched with zeaxanthin and α-tocopherol (LFG-A), were irradiated with blue light. Phagocytosis of fluorescein-5-isothiocyanate (FITC)-labelled photoreceptor outer segments was determined by flow cytometry. Photoreactivity of LFG and LFG-A was analysed by measuring photoconsumption of oxygen and photogeneration of singlet oxygen mediated by the granules. LFG-mediated photic stress in ARPE-19 cells induced significant inhibition of their specific phagocytosis. The inhibitory effect increased with age of LFG donors and was reduced by enrichment of the granules with antioxidants. Oxygen consumption and generation of singlet oxygen induced by the photoexcited LFG increased with donor’s age and was partially quenched by antioxidants. Although the phototoxic potential of lipofuscin increased with age, natural antioxidants reduced photoreactivity of LFG and their efficiency to induce oxidative stress. This study has demonstrated, for the first time, that mild oxidative stress, mediated by the age pigment lipofuscin, impairs specific phagocytic activity of RPE, and that natural antioxidants can protect this important cellular function by reducing lipofuscin photoreactivity.     

Reliable Screening of Dye Phototoxicity by Using a Caenorhabditis elegans Fast Bioassay

Phototoxicity consists in the capability of certain innocuous molecules to become toxic when subjected to suitable illumination. In order to discover new photoactive drugs or characterize phototoxic pollutants, it would be advantageous to use simple biological tests of phototoxicy. In this work, we present a pilot screening of 37 dyes to test for phototoxic effects in the roundworm Caenorhabditis elegans. Populations of this nematode were treated with different dyes, and subsequently exposed to 30 min of white light. Behavioral outcomes were quantified by recording the global motility using an infrared tracking device (WMicrotracker). Of the tested compounds, 17 dyes were classified as photoactive, being phloxine B, primuline, eosin Y, acridine orange and rose Bengal the most phototoxic. To assess photoactivity after uptake, compounds were retested after washing them out of the medium before light irradiation. Dye uptake into the worms was also analyzed by staining or fluorescence. All the positive drugs were incorporated by animals and produced phototoxic effects after washing. We also tested the stress response being triggered by the treatments through reporter strains. Endoplasmic reticulum stress response (hsp-4::GFP strain) was activated by 22% of phototoxic dyes, and mitochondrial stress response (hsp-6::GFP strain) was induced by 16% of phototoxic dyes. These results point to a phototoxic perturbation of the protein functionality and an oxidative stress similar to that reported in cell cultures. Our work shows for the first time the feasibility of C. elegans for running phototoxic screenings and underscores its application on photoactive drugs and environmental pollutants assessment.     

Protection of neuronal cells against reactive oxygen species by carnosine and related compounds

Carnosine and related compounds were compared in terms of their abilities to decrease the levels of reactive oxygen species (ROS) in suspensions of isolated neurons activated by N-methyl-D-aspartic acid (NMDA) using both stationary fluorescence measurements and flow cytometry. Carnosine was found to suppress the fluorescent signal induced by ROS production and decreased the proportion of highly fluorescent neurons, while histidine showed opposite effects. N-Acetylated derivatives of both carnosine and histidine demonstrated weak (statistically indistinguishable) suppressive effects on the ROS signal. N-Methylated derivatives of carnosine suppressed intracellular ROS generation to the same extent as carnosine. This rank of effectiveness is distinct from that previously obtained for the anti-radical ability of CRCs (anserine>carnosine>ophidine). These differences suggest that the similar ability of carnosine and its N-methylated derivatives to protect neuronal cells against the excitotoxic effect of NMDA is not solely related to the antioxidant properties of these compounds.     

Macrophage-colony-stimulating factor-induced activation of extracellular-regulated kinase involves phosphatidylinositol 3-kinase and reactive oxygen species in human monocytes

We previously reported that activation of the phosphatidylinositol (PI) 3-kinase pathway was important in M-CSF-induced monocyte survival. Because M-CSF also induces activation of the mitogen-activated protein (MAP) kinase extracellular-regulated kinase (Erk), we focused on dissecting the mechanism used by M-CSF to induce Erk activation in human monocytes. We found that, in addition to the MAP/Erk kinase inhibitor PD098059, the PI 3-kinase inhibitors LY294002 and wortmannin both suppressed Erk activation in M-CSF-treated monocytes, suggesting that 3-phosphorylated products of PI 3-kinase played a role in Erk activation. Investigating the biochemical pathways regulated by PI 3-kinase to activate Erk, we found that, in response to M-CSF, normal human monocytes induced reactive oxygen species (ROS), which were suppressed by the PI 3-kinase inhibitor wortmannin but not by the solvent control DMSO or the MAP/Erk kinase inhibitor PD098059. We next found that, in the absence of M-CSF, ROS could induce Erk activation in human monocytes. Exogenous H(2)O(2) induced Erk activation in human monocytes, which was suppressed by exogenous catalase. To determine whether ROS induced by M-CSF played a role in Erk activation, we found that N-acetylcysteine and diphenyleneiodonium both suppressed Erk activation in M-CSF-treated monocytes. Erk activation by M-CSF also seemed to play a role in cellular survival in monocytes. These data suggest that, in M-CSF-stimulated human monocytes, PI 3-kinase products and ROS production play a role in Erk activation and monocyte survival.     

Activation of PPARδ counteracts angiotensin II-induced ROS generation by inhibiting rac1 translocation in vascular smooth muscle cells

Angiotensin II (Ang II)-mediated modification of the redox milieu of vascular smooth muscle cells (VSMCs) has been implicated in several pathophysiological processes, including cell proliferation, migration and differentiation. In this study, we demonstrate that the peroxisome proliferator-activated receptor (PPAR) δ counteracts Ang II-induced production of reactive oxygen species (ROS) in VSMCs. Activation of PPARδ by GW501516, a specific ligand for PPARδ, significantly reduced Ang II-induced ROS generation in VSMCs. This effect was, however, reversed in the presence of small interfering (si)RNA against PPARδ. The marked increase in ROS levels induced by Ang II was also eliminated by the inhibition of phosphatidylinositol 3-kinase (PI3K) but not of protein kinase C, suggesting the involvement of the PI3K/Akt signalling pathway in this process. Accordingly, ablation of Akt with siRNA further enhanced the inhibitory effects of GW501516 in Ang II-induced superoxide production. Ligand-activated PPARδ also blocked Ang II-induced translocation of Rac1 to the cell membrane, inhibiting the activation of NADPH oxidases and consequently ROS generation. These results indicate that ligand-activated PPARδ plays an important role in the cellular response to oxidative stress by decreasing ROS generated by Ang II in vascular cells.     

Homocysteine altered ROS generation and NO accumulation in endothelial cells

Mild hyperhomocysteinemia (HHcy) is a risk factor for vascular disease and is closely associated with endothelial dysfunction. Oxidative stress and decreased nitric oxide (NO) bioavailability were reported in HHcy-induced vascular injury; however, the exact relationship is not understood. We thus directly determine the production of reactive oxygen species (ROS) and NO in cultured endothelial cells (HUVECs) to demonstrate the correlated variation between ROS and NO induced by Hcy (homocysteine), Cys (cysteine), another thiol compound, and Met (methionine), precursor of HHcy in animal study. HUVECs were treated with Hcy, Cys, or Met for 0.5 or 22-24 h; ROS generation was detected by DCF fluorescence with flow cytometry and NO by chemiluminescence. In non-cytotoxic (<1.0 mM) concentration ranges, Met exerted no effects on either ROS production or NO concentration, Cys decreased ROS production and increased NO in both short-term (0.5 h) and long-term (22-24 h) treatments; Hcy, however, induced a biphasic effect on ROS production, i.e., inhibitory at 0.5 h but stimulatory at 24 h. The maximal stimulation by Hcy (0.25 mM) was significantly reduced by co-incubation (12 h) with estrogen (1 microM). Hcy caused an early (0.5 h) increase of medium NO which was absent in long-term Hcy treatment. The oxidative stress caused by long-term Hcy incubation could be ameliorated by estrogen, consistent with earlier in vivo observations that estrogen prevents HHcy-induced injury.     

Role of reactive oxygen species and NADPH-oxidase in the development of rat cerebellum

Experimental evidence suggests that reactive oxygen species (ROS) could participate in the regulation of some physiological conditions. In the nervous system, ROS have been suggested to act as signaling molecules involved in several developmental processes including cell differentiation, proliferation and programmed of cell death. Although ROS can be generated by several sources, it has been suggested that NADPH oxidase (NOX) could be critical in the production of ROS acting as a signal in some of these events. It has been reported that ROS production by NOX enzymes participate in neuronal maturation and differentiation during brain development. In the present study, we found that during rat cerebellar development there was a differential ROS generation at different ages and areas of the cerebellum. We also found a differential expression of NOX homologues during rat cerebellar development. When we treated developing rats with an antioxidant or with apocynin, an inhibitor of NOX, we found a marked decrease of the ROS levels in all the cerebellar layers at all the ages tested. Both treatments also induced a significant change in the cerebellar foliation as well as an alteration in motor behavior. These results suggest that both ROS and NOX have a critical role during cerebellar development.     

Synthetic indole Mannich bases: Their ability to modulate in vitro cellular immunity

The synthetic indole Mannich bases 1–13 have been investigated for their ability to modulate immune responses measured in vitro. These activities were based on monitoring their affects on T-lymphocyte proliferation, reactive oxygen species (ROS), IL (interleukin)-2, IL-4, and nitric oxide production. Compound 5 was found to be the most potent immunomodulator in this context. Four of the synthesized compounds, 5, 11, 12, and 13, have significant potent inhibitory effects on T-cell proliferation, IL-4, and nitric oxide production. However, none of the thirteen indole compounds exerted any activity against ROS production.     

H2O2致WB-F344细胞内活性氧的产生及机理

以双氢罗丹明123（DHR123）作为荧光探针,采用激光共聚焦扫描显微镜研究小剂量（800nmol/L)H2O2诱导大鼠肝卵细胞株WB－F344细胞内活性氧产生的动态变化过程及其机理。结果发现：（1）小剂量H2O2的一次作用可以引起胞内活性氧的产生;（2）胞内活性氧清除剂N－乙酰－L－半胱氨酸（NAC）处理2h时后,再加入小剂量H2O2,发现胞内活性氧的产生明显减少;（3）用广谱的蛋白激酶抑制剂2－氨基嘌呤（2－AP）、Ca^2 依赖性蛋白激酶（PKC）抑制剂Bisindolylmaleimide Ⅰ、酷氨酸蛋白激酶（TPK）抑制剂Tyrphostin25分别预处理15min后,H2O2诱导的胞内活性氧的产生现象均消失;（4）细胞在无外钙环境下,小剂量H2O2诱导的胞内活性氧的产生明显减少;（5）细胞在无外钙环境下用NAC预处理后,H2O2诱导的胞内活性氧的产生现象消失。结果表明,H2O2可以通过胞内信号转导系统诱使WB细胞胞内活性氧产生,这可能与小剂量H2O2调控细胞生物学功能（如增殖、转化）相关。     

Induction of murine erythroleukemia cell differentiation by proteinases is inhibited by aromatic poly-amidines

The effects of the tetra benzamidine serine-proteinase inhibitor 1,3-di-(p-amidinophenoxy) -2,2- bis- (p-amidinophenoxymethyl)propane (TAPP-H) and related compounds, including halo-derivatives, were determined on the erythroid differentiation of murine erythroleukemic cells induced by trypsin and kallikrein. These aromatic poly-amidines and their halo derivatives were found to be strong inhibitors of both trypsin and kallikrein mediated induction of commitment of MEL cells to erythroid differentiation, hemoglobin synthesis and accumulation, globin mRNA production. No inhibitory effects were detected by treating proteinase-induced MEL cells with benzamidine. Only slight inhibitory activity was found after treatment of trypsin-induced MEL cells with other antiproteinase compounds widely used in the control of proteinase-dependent functions, including leupeptin, antipain and Bowman-Birk proteinase inhibitor. MEL cells induced to erythroid differentiation by proteinases could be proposed as an experimental system to test the biological activity of proteinase inhibitors.     

Protein Phosphatases but not Reactive Oxygen Species Play Functional Role in Acute Amphetamine-Mediated Dopamine Release

Drug abuse-induced neurodegeneration can be triggered by elevated production of reactive oxygen species (ROS). Involvement of oxidative stress in acute amphetamine (AMPH)-mediated dopamine (DA) release, however, has not been completely understood yet. In order to elucidate the dopaminergic response of PC12 cells to a single dose of 10 μM AMPH, ROS production was measured as related to the extracellular DA level. Due to the spontaneous oxidation of peroxide-sensitive fluorophore 2′,7′-dichlorofluorescin diacetate (DCFH-DA) to 2′,7′-dichlorofluorescein (DCF), the increase in fluorescence could not be unambiguously attributed to AMPH-triggered ROS production. Based on Amplex Red fluorescence, no ROS production was detected after acute AMPH application. Our data strongly suggest that ROS development was not the main triggering factor for immediate DA release after acute AMPH treatment. On the other hand, AMPH-induced elevation of DA levels in rat brain striatal slices was quenched by the water soluble antioxidant, N-acetylcysteine (NAC) at 10 mM. In this study, we also investigated the contribution of protein phosphatases to the AMPH-induced rat brain striatal dopaminergic response. The experimental protocol, double AMPH challenge was applied for screening the effect of NAC and cantharidin on AMPH-mediated DA release. Here we show that AMPH-mediated DA release increased nearly twofold in striatal rat brain slices pretreated for 30 min with 1000 μM cantharidin, a selective PP1 and PP2A inhibitor. These findings prove the lack of ROS inhibitory action on protein phosphatase activity in acute AMPH-mediated DA efflux.     

Activity of grape extracts from Greek varieties of Vitis vinifera against mutagenicity induced by bleomycin and hydrogen peroxide in Salmonella typhimurium strain TA102

Several in vivo and in vitro studies have shown that grape extracts could prevent certain steps in carcinogenesis and a few mechanisms have been proposed for this activity. In this study, the potential antimutagenic activity of methanolic and aqueous extracts from two Greek grape varieties of Vitis vinifera against DNA damage induced by reactive oxygen species (ROS) was assessed as a potential novel chemopreventive mechanism, using Salmonella typhimurium strain TA102. The two grape varieties were Assyrtiko (white grapes) and Mandilaria (red grapes), while the oxidant mutagens used were bleomycin (BLM) and hydrogen peroxide (H(2)O(2)). Since it has been considered that polyphenols present in grapes are their most potent biologically active compounds, we also tested the effects of polyphenol-rich fractions as well as some of the more common grape polyphenols on the activity of the two test mutagens. These polyphenols were quercetin, (+)-catechin, (-)-epicatechin, trans-resveratrol, gallic acid and protocatechuic acid. Almost all extracts showed inhibitory activity against both mutagens. On the other hand, polyphenol-rich fractions as well as individual polyphenols at concentrations found in the extracts either did not diminish or did enhance the activity of the mutagens. These results suggest that the protection of DNA from mutations induced by ROS may be one of the mechanisms accounting for the chemopreventive activity of grape extracts. However, it seems that this protective activity may not be attributed to polyphenols but rather to a synergism of many compounds in the grapes.     

Activation of PPARδ counteracts angiotensin II-induced ROS generation by inhibiting rac1 translocation in vascular smooth muscle cells

Abstract

Angiotensin II (Ang II)-mediated modification of the redox milieu of vascular smooth muscle cells (VSMCs) has been implicated in several pathophysiological processes, including cell proliferation, migration and differentiation. In this study, we demonstrate that the peroxisome proliferator-activated receptor (PPAR) δ counteracts Ang II-induced production of reactive oxygen species (ROS) in VSMCs. Activation of PPARδ by GW501516, a specific ligand for PPARδ, significantly reduced Ang II-induced ROS generation in VSMCs. This effect was, however, reversed in the presence of small interfering (si)RNA against PPARδ. The marked increase in ROS levels induced by Ang II was also eliminated by the inhibition of phosphatidylinositol 3-kinase (PI3K) but not of protein kinase C, suggesting the involvement of the PI3K/Akt signalling pathway in this process. Accordingly, ablation of Akt with siRNA further enhanced the inhibitory effects of GW501516 in Ang II-induced superoxide production. Ligand-activated PPARδ also blocked Ang II-induced translocation of Rac1 to the cell membrane, inhibiting the activation of NADPH oxidases and consequently ROS generation. These results indicate that ligand-activated PPARδ plays an important role in the cellular response to oxidative stress by decreasing ROS generated by Ang II in vascular cells.     

Induction of oxidative stress by humic acid through increasing intracellular iron: a possible mechanism leading to atherothrombotic vascular disorder in blackfoot disease

Humic acid (HA), a potential toxin that has penetrated the drinking well water of blackfoot disease-endemic areas in Taiwan, has been implicated as an etiological factor of this disease. In this study, we investigated the effects of HA on the generation of reactive oxygen species (ROS) in cultured human umbilical vein endothelial cells (HUVECs). The generation of ROS was monitored by flow cytometry. Pretreatment of HUVECs with HA induced reactive oxygen species in a dose- and time-dependent manner. Xanthine oxidase inhibitor (Allopurinol), NADPH oxidase inhibitor (diphenylene iodomium) and calcium chelator (BAPTA) could not reduce the generation of ROS. Protein kinase C inhibitor (H7) could reduce the generation of ROS slightly, but the intracellular antioxidant glutathione monoethyl ester and the iron chelator desferrioxamine (DFO) could inhibit the generation of ROS completely. HA also enhanced the expression of ferritin and induced intracellular chelatable iron; however, HA reduced the expression of transferrin receptor. Pretreatment with DFO inhibited HA-mediated increases of ferritin synthesis and intracellular chelatable iron, but caused recovery of the inhibitory effect on transferrin receptor. Cotreatment with iron and HA induced more ROS and intracellular chelatable iron than iron or HA treatment alone. Furthermore, HA enhanced the accumulation of iron in endothelial cells. These data demonstrate that HA can increase the generation of ROS through enhancing the accumulation of intracellular iron. Taken together, our findings suggest that iron mediates HA-associated oxidative stress in endothelial cells, which may be a possible mechanism leading to atherothrombotic vascular injury observed for patients with blackfoot disease.     

PI3Kgamma controls oxidative bursts in neutrophils via interactions with PKCalpha and p47phox

Neutrophils release reactive oxygen species (ROS) as part of the innate inflammatory immune response. Phosphoinositide 3-kinase gamma (PI3Kgamma), which is induced by the bacterial peptide N-formylmethionyl-leucyl-phenylalanine (fMLP), has been identified as an essential intracellular mediator of ROS production. However, the complex signalling reactions that link PI3Kgamma with ROS synthesis by NADPH oxidase have not yet been described in detail. We found that activation of neutrophils by fMLP triggers the association of PI3Kgamma with protein kinase Calpha (PKCalpha). Specific inhibition of PI3Kgamma suppresses fMLP-mediated activation of PKCalpha activity and ROS production, suggesting that the protein kinase activity of PI3Kgamma is involved. Our data suggest that the direct interaction of PI3Kgamma with PKCalpha forms a discrete regulatory module of fMLP-dependent ROS production in neutrophils.     

Inhibition of the respiratory burst by resveratrol in human monocytes: correlation with inhibition of PI3K signaling

trans-Resveratrol (t-RVT) has been shown to have a wide range of anti-inflammatory properties, some of which have been suggested to contribute to the molecular explanation of the French Paradox, a possible reason for the low incidence of heart disease in France. The ability of t-RVT to inhibit the production of reactive oxygen species (ROS) from monocytes (differentiated U937) was investigated using isoluminol, luminol, lucigenin, and 2',7'-dichlorofluorescein (DCF). t-RVT (0.1-50 microM) was found to significantly inhibit cellular ROS production stimulated by f-Met-Leu-Phe (fMLP), 12-phorbol 13-myristate, and arachidonic acid after a 1-h preincubation. The efficacy of t-RVT could be increased if it was added directly into the assay. NADPH-dependent superoxide production was measured in cell homogenates and t-RVT (10-50 microM) was found to have no effect on this activity. The majority of these redox probes require a peroxidase to be oxidized; therefore, the inhibitory effect of t-RVT on ROS measured by these probes is complicated by its ability to be oxidized by peroxidase enzymes and thus compete with the probe. t-RVT, known to be oxidized by the horseradish peroxidase (HRP)/H(2)O(2) system, was found to inhibit the HRP-dependent oxidation of the fluorescent probe DCF and the chemiluminescent probe isoluminol. However, using a redox probe that did not require oxidation by a peroxidase (lucigenin), significant inhibition was still observed. Moreover, the inhibitory effects of t-RVT on fMLP-induced ROS production correlated with significant inhibitory effects on fMLP-induced phosphatidylinositol 3-kinase (PI3K) activity at 50 microM and Akt phosphorylation (10-50 microM). Other known inhibitors of both PI3K and Akt were also found to inhibit this response. Therefore, inhibition of signaling through the PI3K to NADPH oxidase by t-RVT might represent an important anti-inflammatory mechanism.     

Effect of curcumin-nanoemulsion associated with photodynamic therapy in breast adenocarcinoma cell line

Curcumin, a natural compound has several antineoplastic activities and is a promising natural photosensitizer used in photodynamic therapy. However, its low solubility in physiological medium limit the clinical use of curcumin. This study aimed to analyze the action of curcumin-nanoemulsion, a new and well-designed Drug Delivery System (DDS+) molecule, used as a photosensitizing agent in photodynamic therapy in an in vitro breast cancer model, MCF-7 cells. The empty nanoemulsion fulfils all necessary requirements to be an excellent DDS. Furthermore, the use of curcumin-nanoemulsion in photodynamic therapy resulted in a high phototoxic effect after activation at 440?nm, decreasing to <10% viable tumor cells after two irradiations and increasing the reactive oxygen species (ROS) production. The use of curcumin-nanoemulsion associated with photodynamic therapy resulted in an increase in the levels of caspase 3/7 activity for the studied MCF-7 cell model, indicating that this therapy triggers a cascade of events that lead to cell death, such as cellular apoptosis. In conclusion, curcumin-nanoemulsion proved to be efficient as a photosensitizing agent, had phototoxic effects, significantly decreased the proliferation of MCF-7 cells and stimulating the ROS production in combination with photodynamic therapy, so, this formulation has a great potential for use in treatment of breast cancer.     

The inhibitory effect of cyclic AMP on phosphatidylinositol kinase is not mediated by the cAMP dependent protein kinase

Addition of cAMP to cells has been shown to inhibit phosphatidylinositol (PI) metabolism. cAMP has been reported to inhibit an enzyme in this pathway, PI kinase and it has been suggested that this inhibition is due to phosphorylation of PI kinase by the cAMP dependent protein kinase (PKA). In the present study we directly investigated if the inhibitory effect of cAMP was mediated by PKA. In membranes derived from murine hepatocytes we found that cAMP inhibited PI kinase but other adenine derivatives were more potent inhibitors. Moreover, it was found that the effects of the derivatives were unlikely to be due secondarily to the production of cAMP via their interaction with adenosine receptors. Through studies employing an inhibitor of PKA, mutant cells lacking PKA, and addition of purified catalytic subunit of PKA, we found that the inhibitory effect of cAMP was not mediated by PKA. In addition, the inhibitory effect of cAMP and adenosine was retained upon partial purification of PI kinase. Pulse chase experiments affirmed that the inhibitory effect was not due to breakdown of PI but rather to inhibition of its synthesis. We conclude that the inhibitory effect of cAMP and related compounds on PI kinase is not mediated by PKA dependent phosphorylation but rather appears to be a direct effect of these agents.