Occurrence of Na(+)-Ca2+ exchange in the ciliate Euplotes crassus and its role in Ca2+ homeostasis.

Ciliates possess diverse Ca2+ homeostasis systems, but little is known about the occurrence of a Na(+)-Ca2+ exchanger. We studied Na(+)-Ca2+ exchange in the ciliate Euplotes crassus by digital imaging. Cells were loaded with fura-2/AM or SBF1/AM for fluorescence measurements of cytosolic Ca2+ and Na+ respectively. Ouabain pre-treatment and Na+o substitution in fura-2/AM-loaded cells elicited a bepridil-sensitive [Ca2+]i rise followed by partial recovery, indicating the occurrence of Na(+)-Ca2+ exchanger working in reverse mode. In experiments on prolonged effects, ouabain, Na+o substitution, and bepridil all caused Ca2+o-dependent [Ca2+]i increase, showing a role for Na(+)-Ca2+ exchange in Ca2+ homeostasis. In addition, by comparing the effect of orthovanadate (affecting not only Ca2+ ATPase, but also Na(+)-K+ ATPase and, hence, Na(+)-Ca2+ exchange) to that of bepridil on [Ca2+]i, it was shown that Na(+)-Ca2+ exchange contributes to Ca2+ homeostasis. In electrophysiological experiments, no membrane potential variation was observed after bepridil treatment suggesting compensatory mechanisms for ion effects on cell membrane voltage, which also agrees with membrane potential stability after ouabain treatment. In conclusion, data indicate the presence of a Na(+)-Ca2+ exchanger in the plasma membrane of E. crassus, which is essential for Ca2+ homeostasis, but could also promote Ca2+ entry under specific conditions.

Effects of epidermal growth factor on isolated digestive gland cells from mussels.

Effects of epidermal growth factor (EGF) and possible mechanisms of EGF-mediated signal transduction were investigated in isolated cells of the digestive gland of the mussel (Mytilus galloprovincialis Lam. ). EGF induced a cytosolic Ca2+ transient and subsequently stimulated DNA synthesis; both effects were dose-dependent in the nanomolar range and inhibited by pretreatment with an inhibitor of tyrosine kinase activity, suggesting specific EGFR-like receptors. The EGF-induced cytosolic Ca2+ transient was mainly due to a Ca2+ influx through the plasma membrane, possibly involving voltage-insensitive Ca2+ channels. Such a Ca2+ response was abolished by pretreatment with indomethacin and NDGA, inhibitors of arachidonic acid metabolism; similarly, the EGF-stimulated increase in DNA synthesis was significantly reduced. Indomethacin, a cyclooxygenase inhibitor, had the greatest effect on both EGF-induced responses. Results suggest the presence of EGF-responsive cells in the mussel digestive gland. A possible role for arachidonic acid and its metabolites in mediating the effects of EGF is also indicated.

Effects of free oxygen radicals on Ca2+ release mechanisms in the sarcoplasmic reticulum of scallop (Pecten jacobaeus) adductor muscle.

In vitro oxyradical effects on SR Ca2+ regulation were studied by using a SR-containing cell-free preparation from scallop (Pecten jacobaeus) adductor muscle. Ca2+ variations were fluorimetrically detected after incubation with Fluo-3 in the presence of ATP. Exposure to Fe3+/ascorbate produced dose-dependent Ca2+ release from SR vesicles, eventually leading to massive Ca2+ loss. Exposure to hypoxanthine/xanthine oxidase also caused Ca2+ release but at a much slower rate. Pre-incubations with catalase or with the hydroxyl radical scavenger KMBA led to a significant decrease in the Fe3+/ascorbate-induced Ca2+ release rate and to a delay of massive Ca2+ loss. Pre-incubations with GSH or DTT strongly reduced the Ca2+ release caused by Fe3+/ascorbate and, moreover, they prevented massive Ca2+ loss from SR vesicles. Addition of GSH or DTT after Fe3+/ascorbate promptly reduced the Ca2+ release rate and delayed massive Ca2+ release. Pre-incubation with the SR Ca2+ channel blocker ruthenium red strongly reduced the Ca2+ release caused by Fe3+/ascorbate, and also prevented massive Ca2+ loss. In the presence of ruthenium red, Fe3+/ascorbate treatments followed by Ca2+ addition revealed that Ca2+ uptake inhibition was slower than Ca2+ release. Taken together, data showed that free radicals and, in particular, hydroxyl radicals, affected the scallop SR Ca2+ regulation. This mainly occurred through Ca2+ channel opening, most likely triggered by sulfhydryl oxidation, which eventually led to massive Ca2+ release from SR vesicles. The demonstration of a specific effect of oxyradicals on SR Ca2+ channels is in line with their possible involvement in cell signaling.

In vitro effect of 3,5,3'-triiodothyronine on poly(ADP-ribosyl)ation of DNA topoisomerase I.

DNA topoisomerase I activity (topo I) is known to be inhibited by poly(ADP-ribosyl)ation. Both poly(ADP-ribose)polymerase (pADPRP) and DNA topoisomerase I participate to major biological events, such as DNA transcription, repair and synthesis. It has been shown that thyroid hormones, such as 3,5,3'-triiodothyronine (T3), stimulate DNA transcription and down-regulate pADPRP activity. Using an in vitro model, we have studied the poly(ADP-ribosyl)ation of topo I, in vitro, in the presence of T3. T3 treatment of pADPRP inhibits the enzyme up to 75-80% of control activity. DNA topoisomerase I relaxing activity was determined on supercoiled plasmid DNA, and topoisomers were separated by agarose gel electrophoresis. Poly(ADP-ribosyl)ation completely inhibits the relaxing activity of topo I, with respect to non-ribosylated controls, but the activity remains unaffected when pADPRP is inactivated by heat or treated with specific inhibitors, such as 3-aminobenzamide (3ABA). In this study we show that treatment of pADPRP with T3 reduces the inhibition on topo I. In this system 10(-8) M T3 was effective in maintaining almost all topo I activity, even though modifications in processivity and distributivity of the reaction were noted. These data support a close relationship between pADPRP and topo I in hormone-stimulated DNA transcription.

Hepatic poly(ADP-ribose) polymerase activity in rat is controlled by thyroid hormones.

The in vivo effect of the thyroidal state on poly(ADP-ribose) polymerase activity was investigated in eu- and hypothyroid rats after treatment with L-triiodothyronine. Untreated hypothyroids showed an increased basal rate of the enzyme. The treatment of both eu- and hypothyroid rats with L-triiodothyronine induced a prompt drop of the endogenous activity not due to a reduction of the catalytic protein. This decrease well evident 1 h after treatment was transient, returning to controls values within 8 h. In isolated liver nuclei from euthyroids the in vitro exposure to increasing L-triiodothyronine concentrations from 10(-18) to 10(-6) M resulted in a progressive inhibition of the enzyme. This loss in activity was not derived from a reduction of the total level of the catalytic protein. The pretreatment with the antagonist amiodarone suppressed the hormone effect, suggesting that nuclear receptors could mediate poly(ADP-ribose) polymerase activity.

Effects of heavy metals on the Ca(2+)-ATPase activity present in gill cell plasma-membrane of mussels (Mytilus galloprovincialis Lam.).

1. Heavy metals (Hg2+, Cu2+, Cd2+, Zn2+, Pb2+) at micromolar concentrations strongly inhibit the Ca(2+)-ATPase activity present in the plasma-membrane obtained from the gill cells of Mytilus galloprovincialis Lam. Heavy metals act through inhibition of the formation of the phosphorylated intermediate. 2. All the heavy metals tested inhibit the Ca(2+)-ATPase activity, the effect following the order: Hg2+ > Pb2+ > Cu2+ > Cd2+ > Zn2+; the simultaneous addition of different heavy metals causes a summatory inhibition of the enzyme activity; addition to the reaction mixture of GSH at a final concentration of 0.5 mM, reverses inhibitory effects of heavy metals. 3. The inhibitory effects of Cu2+ on Ca(2+)-ATPase are highly enhanced by addition of ascorbate to the reaction mixture. In the presence of ascorbate (100 microM), copper strongly stimulates the lipid peroxidation damage of the gill plasma-membranes, a result that may explain the high copper cytotoxicity.

Biochemical characterization of a phosphatidylinositol 4,5-bisphosphate-specific phospholipase C activity in gills and digestive gland of the marine mussel Mytilus galloprovincialis Lam.

1. Polyphosphoinositide-specific phosphodiesterase (phospholipase C, PLC) activity against phosphatidylinositol 4,5-bisphosphate, present in gill and digestive gland homogenates of mussel (Mytilus galloprovincialis Lam.), has been biochemically characterized. 2. The enzyme was strictly modulated by free calcium ion concentration in both tissues and maximally activated at 10(-5) M Ca2+ (19 +/- 4 and 11 +/- 2 nmol phosphatidylinositol 4,5-bisphosphate hydrolysed/min/mg of protein for gill and digestive gland PLC, respectively, at 19 degrees C). Optimum pH at 10(-5) M Ca2+ was around 7.0 in both cases. The Ca(2+)-stimulated PLC activity showed high specificity for PIP2; the KMa for PIP2 were 150 and 170 microM for the gills and digestive gland, respectively. 3. Good substrate dispersion was obtained in the presence of sodium deoxycholate; the concentration routinely used in the assay (0.08%) produced a 9-fold activation of both gill and digestive gland PLC, consistent with previous reports. 4. The possible biochemical and physiological role of the enzyme in mussel tissues is discussed.

Influence of poly(ADP ribose) polymerase depletion on promotion of liver carcinogenesis.

In previous studies we demonstrated that liver poly(ADP ribose) polymerase (pADPRP) activity was lost in animals exposed to N-2-acetylaminofluorene (2AAF) according to the Teebor and Becker experimental model (Cancer Res 31:1-3, 1971). In addition, we used the resistant hepatocyte model of Solt and Farber (Nature 263:702-703, 1976) to further investigate pADPRP activity during the multistep process of liver carcinogenesis. A marked depletion of the catalytic protein was evidenced after 2AAF exposure, confirming previous results and indicating a specific effect of 2AAF on this nuclear enzyme that controls conformational changes of chromatin and regulates several catalytic activities in the nucleus. The levels of pADPRP mRNA, measured by northern blot analysis using both experimental models, indicate that the enzyme depletion is not due to a loss of transcript. Moreover, these data indicate that pADPRP depletion, caused by 2AAF, was also maintained during liver compensatory growth, which is known to induce a rapid and marked increase in pADPRP activity and protein level. Treatment of 2AAF-exposed animals with N-acetyl-L-cysteine not only efficiently protected against DNA damage, but also prevented a rapid depletion of the catalytic protein. Interestingly, these data indicate that the marked loss of liver pADPRP occurred during the promotion step induced by 2AAF feeding and that this loss was observed using different models for experimental hepatocarcinogenesis. This phenomenon can be ascribed to a highly defective transcript that cannot be correctly translated into the specific protein or to a rapid degradation of the translated protein.

Effect of N-2-acetylaminofluorene on poly(ADP-ribose) polymerase activity and DNA synthesis in cultured rat hepatocytes exposed to epidermal growth factor.

The nuclear enzyme poly(ADP-ribose) polymerase is involved in basic cellular processes such as DNA replication and repair, cell differentiation and transformation, gene expression. We have studied the effect of 2AAF, a genotoxic aromatic amine, on pADPRP activity during DNA synthesis stimulated by EGF, using the cultured rat hepatocytes model. DNA synthesis was measured as [3H]thymidine incorporated/microgram DNA while pADPRP activity was expressed in pmol[32P]NAD incorporated/min/microgram DNA. Our results show that 2AAF treatment of EGF-stimulated rat hepatocytes induces a full block of DNA replication which is preceded and accompanied by a net inhibition of endogenous and total pADPRP activity, respectively. A block in pADPRP activity in normal hepatocytes, exposed to 2AAF in vitro or in vivo, could play a key role in cell transformation. Our data add further information on the possible involvement of this nuclear catalytic activity during DNA replication.

Relationship between DNA topoisomerase I activity and DNA synthesis in cultured hepatocytes: effects of orotic acid.

Previous studies have demonstrated that DNA topoisomerase I activity can be closely related to DNA replication and active transcription in different experimental models. This relationship was further investigated by studying the time course of DNA topoisomerase I activity in cultured rat hepatocytes stimulated by epidermal growth factor. This mitogen has been shown to stimulate DNA synthesis in liver cells both in vivo and in vitro. DNA topoisomerase I activity was assayed quantifying ATP-independent relaxation of a negatively supercoiled plasmid, substrate for the enzyme. Incubation of cellular extracts was carried out at 30 degrees C for 15'. Products were electrophoresed and analyzed by densitometry. DNA synthesis was measured as [3H]thymidine incorporation in the hepatocytes, after a 2 hrs pulse labelling. An increase in DNA topoisomerase I activity was observed early after epidermal growth factor addition, before the onset of DNA synthesis. Concomitant administration of orotic acid, which has recently shown mitoinhibitory effect, abolished the EGF-induced activity as well as DNA synthesis. Taken together these data indicate that, in cultured hepatocytes, the induction of DNA synthesis is supported by DNA topoisomerase I with a precise time schedule. Orotic acid administration abolishes this response and causes an overall loss in DNA topoisomerase I activity.

A rapid method for detecting DNA strand breaks in Mytilus galloprovincialis Lam. induced by genotoxic xenobiotic chemicals.

1. In this study, DNA from haemolymph cells of Mytilus galloprovincialis Lam., as well as from L1210 (murine leukemia) mouse cells was investigated utilizing the technique of the alkaline unwinding of the double stranded DNA molecule. 2. The data show that DNA of haemolymph cells from the marine invertebrate has an unwinding time and, therefore, a molecular weight considerably lower than that of DNA of mammalian cells. 3. The exposure of the cells from mussel haemolymph and from mouse L1210 to a genotoxic compound such as dimethylsulfate results in DNA damage and consequently in a reduction of the unwinding time. 4. These results suggest that the fluorimetric DNA unwinding assay can be used in studies concerning the damage of DNA of marine organisms induced by genotoxic compounds or environmental factors.

Differential assay and biological significance of poly(ADP-ribose) polymerase activity in isolated liver nuclei.

Poly(ADP-ribosyl)ation of nuclear proteins is catalyzed by poly(ADP-ribose) polymerase. This enzyme is involved in the regulation of basic cellular functions of DNA metabolism. DNA breaks induced by DNA-damaging agents trigger the activation of poly(ADP-ribose) polymerase increasing its endogenous level. This increase modifies the pattern of poly(ADP-ribosyl)ated chromatin proteins. In this paper we describe a procedure for the isolation of intact nuclei from rat liver to be used for the endogenous activity assay. Artifactual activation of the enzyme was avoided since a very low level of DNA-strand breaks occurs during the isolation of nuclei. We present a series of experiments which prove the ability of this procedure to detect increases in endogenous liver activity without modification of the total level. The application of this technique can be useful for a better understanding of the role of early changes in poly(ADP-ribose) polymerase level in physiological conditions and during exposure to DNA-damaging agents.

Changes in activity and mRNA levels of poly(ADP-ribose) polymerase during rat liver regeneration.

ADP-ribosylation of nuclear proteins, catalysed by the enzyme poly(ADP-ribose) polymerase, is involved in the regulation of different cellular processes of DNA metabolism. To further clarify the role of the enzyme during proliferating activity of mammalian cells, we have studied the control of gene expression in regenerating rat liver. The changes in activity and mRNA levels were analysed during the early and late phases of the compensatory model. When enzyme activity was measured in isolated liver nuclei obtained at different times after hepatectomy, two different phases were observed: an early wave occurring before the onset of DNA synthesis, and a second one, starting several hours after the onset of DNA synthesis and returning to control values at later times. The evaluation of the enzymatic level in nuclear extracts and by activity gel analysis showed a more gradual increase starting 1 day after hepatectomy, in concomitance with the peak of DNA synthesis. By using a specific murine cDNA probe, a significant enhancement of mRNA levels for poly(ADP-ribose) polymerase was observed during liver regeneration, slightly preceding the onset of DNA synthesis. The results obtained show that changes in poly(ADP-ribose) polymerase activity, during liver regeneration, are associated both to early events preceding the increase in DNA synthesis and to later phases of the cell proliferation process.

Relationship between poly(ADP-ribose) polymerase activity and DNA synthesis in cultured hepatocytes.

Previous studies have demonstrated that an increase in poly(ADP-ribose) polymerase activity could be closely related to DNA replication during liver regeneration and to DNA repair synthesis in different experimental systems. This relationship was further investigated by studying the time course of endogenous and total poly(ADP-ribose) polymerase activity in cultured rat hepatocytes stimulated by epidermal growth factor. This mitogen has been shown to stimulate DNA synthesis in liver cells both in vivo and in vitro. A 6-fold increase in endogenous activity was observed early after epidermal growth factor addition, just before DNA synthesis. A subsequent 4-fold increment in total enzyme activity, concomitant with DNA synthesis, was detected. Orotic acid, which has recently shown mitoinhibitory effect, abolished the epidermal-growth-factor-induced increase in endogenous and total poly(ADP-ribose) polymerase activity, as well as DNA synthesis. On the contrary, 3-aminobenzamide inhibitor of poly(ADP-ribose) polymerase completely suppressed the endogenous activity but only partially modified the increase in total catalytic level and the overall pattern of thymidine incorporation. Taken together, these data indicate that, in cultured hepatocytes, the induction of DNA synthesis is supported by an increased poly(ADP-ribose) polymerase activity.

Effect of triiodo-L-thyronine treatment on Ca2+ sequestration in rat liver microsomes.

T3 administration to rats exerts quite different effects on enzyme activities associated to liver microsomal membranes such as G-6-Pase, Mg ATPase and Ca2(+)-dependent ATPase: in fact G-6-Pase activity is significantly enhanced, Mg ATPase is not affected whereas Ca2(+)-dependent ATPase is drastically inhibited. The T3 induced decrease in Ca2(+)-dependent ATPase activity is associated with a net reduction (to about 50% with respect to controls) of the Ca2+ sequestration in liver microsomal vesicles. The enhanced level of inorganic phosphate in the endoplasmic reticulum due to the stimulation of G-6-Pase activity does not significantly affect the uptake of calcium in microsomal vesicles. The decreased Ca2(+)-dependent ATPase activity is associated to an enhanced level of the enzyme in the phosphorylated form (E-P). This suggests that in liver preparations from T3 treated rats the turnover of ATP and cleavage of E-P is reduced, thus resulting in the accumulation of the phosphorylated intermediate. The accumulation of E-P is in agreement with the inhibition of the calcium sequestration since the active transport of this cation in microsomal membranes requires the hydrolysis of the E-P complex.

Biochemical characterization of the DNA polymerase activity present in isolated nuclei from mussel tissues: a possible system for the evaluation of the rate of DNA repair.

1. The nuclear DNA polymerase activity in mussel digestive glands was characterized regarding Mg++ requirement (2 mM), ATP concentration (4 mM), pH (8.4), and ionic strength (50 mM). 2. Most of the enzymatic activity is aphidicolin insensitive, probably due to DNA polymerase beta-like activity. 3. The exposure of mussels to a genotoxic organic compound such as dimethylsulfate (DMS) resulted in increases of about 250% and 100% in the DNA polymerase activity in nuclei isolated from the gills and digestive glands, respectively. 4. These data indicate that the isolated nuclei from mussel tissues may be utilized for the evaluation of the rate of DNA repair synthesis that reflects the DNA damage induced in vivo by exposure of the animals to genotoxic compounds.

Depletion of adenosine diphosphate-ribosyl transferase activity in rat liver during exposure to N-2-acetylaminofluorene: effect of thiols.

The exposure of rats to a feeding regimen containing N-2-acetylaminofluorene (2AAF) causes an accumulation of lesions on liver DNA and a progressive impairment in DNA repair capacity. We used the in vivo experimental model of Teebor and Becker (Cancer Res., 31:1-3, 1971) with the carcinogen given to rats during four consecutive cycles, each one composed of 3 weeks of treatment and 1 week of recovery. The extent of DNA damage and repair was determined during each cycle by the alkaline elution technique. The results obtained showed that the number of alkalilabile sites in DNA is significantly enhanced after the first cycle and remains increased during following cycles. Since ADP-ribosyl transferase (ADPRT) is known to play a central role in the response to DNA damage, we investigated the effect of 2AAF on this enzyme during the carcinogenic process. The activity and the structure of ADPRT were analyzed using the activity gel and Western blot techniques. The catalytic band with a molecular weight of 116,000, clearly evident in liver extracts of control rats, was no longer detectable after one cycle of exposure to 2AAF returning progressively to an almost normal level within the last two cycles. When the aminothiol N-acetyl-L-cysteine (NAC) was added to the 2AAF diet, the extent of DNA damage was drastically reduced, and DNA repair activity preserved for a longer period. In addition, the loss of ADPRT was not observed after the first cycle, but delayed to the end of the second, indicating that NAC exerts a protective effect on DNA and on ADPRT. Such effect was not evident when NAC was substituted by glutathione. The analysis of liver extracts on Western blot showed that the ADPRT immunoreactive band was almost undetectable after the first cycle suggesting that the loss in enzyme activity could be due to a block in de novo synthesis of the enzyme and not to an inhibition of its activity.

Effects of aminothiols in 2-acetylaminofluorene-treated rats. I. Damage and repair of liver DNA, hyperplastic foci, and Zymbal gland tumors.

One hundred and seventy-nine male Wistar rats were divided into 6 groups and fed with a standard diet supplemented with 0.05% 2-acetylaminofluorene (2AAF) and/or 0.1% glutathione (GSH) or N-acetyl-L-cysteine (NAC). Each treatment cycle lasted for 3 weeks, followed by 1 week of standard meal. After 4 cycles, survival was 100% in the 3 control groups, and 86.0, 100 and 91.7%, in the groups receiving 2AAF, 2AAF plus GSH, and 2AAF plus NAC, respectively. After an additional 4-8 weeks, all the 5 surviving rats fed with 2AAF exhibited deforming ear tumors, which on histological examination were classified as sebaceous squamocellular carcinomas of Zymbal glands. No such tumors were detectable in control groups, nor in the 16 surviving rats fed with 2AAF plus GSH or NAC. In the liver, 2AAF produced significant DNA damage at the 3rd week of each cycle, which was partially repaired during the week of standard meal feeding. Moreover, 2AAF determined the appearance of gamma-glutamyl transpeptidase-positive foci, which tended to increase with time both in number and in size. GSH and NAC exerted similar protective effects on these phenomena, but only at early stages of the experimental model used.

Effects of aminothiols in 2-acetylaminofluorene-treated rats. III. Metabolic activation of aromatic amines.

Six groups of Wistar rats received a standard diet supplemented with 0.05% 2-acetylaminofluorene and/or 0.1% natural (reduced glutathione) or synthetic (N-acetyl-L-cysteine) aminothiols. The discontinuous feeding regimen consisted of 4 cycles, each composed of 3 weeks of treatment followed by withdrawal for 1 week. At the 3rd and 4th week of each cycle, the liver was removed from 4-5 rats within each group, and pools of S-12 fractions were assayed for the ability to activate 2-acetylaminofluorene and other aromatic amines, either structurally related (i.e. 4-acetylaminofluorene and 2-aminofluorene) or unrelated (i.e. 2-naphtylamine and benzidine) to mutagenic metabolites in strain TA98 of S. typhimurium. In untreated rats, there was a consistent and marked trend to an age-dependent loss of metabolic activation of all test compounds during the 16 weeks of the experiment. Feeding of 2-acetylaminofluorene resulted in an evident autoinduction of metabolism which was continuously amplified with time, even during the withdrawal weeks. In the same animals, activation of the other amines was initially inhibited but then progressively shifted to a mild cross-induction which, in the case of the structurally related compounds, became significant at the end of the 4-cycle treatment with 2-acetylaminofluorene. The metabolic effects of the two aminothiols were broadly variable, depending on the thiol, on its co-administration with 2AAF, on the week and cycle of treatment, and on promutagens tested.

A rapid HPLC method for determination of adenylate energy charge.

A simple procedure is described for separation and analysis of adenine nucleotides in tissue extracts, utilizing anion exchange HPLC. Determination of AMP, ADP, and ATP takes 10 min per sample.