Quantitative estimation of mercury intake by toxicokinetic modelling based on total mercury levels in humans.

Mercury is a toxic metal that can be disseminated into the environment from both natural and anthropogenic sources. Human exposure to the metal stems mainly from food, and more particularly from the consumption of fish and other seafoods. Examining dietary exposure and measuring mercury levels in body tissues are two ways of estimating exposure to mercury. In this study, we utilized a modelling system consisting of three linear toxicokinetic models for describing the fate of methyl mercury, inorganic mercury, and metallic mercury in the body, in order to estimate daily intake of mercury as measured through total mercury concentrations in the blood. We then compared the results stemming from our modelling system to those of the detailed semi-quantitative food frequency questionnaire (FFQ) of the Norwegian Fish and Game (NFG) Study, a project that focused on dietary mercury exposure. The results indicate that toxicokinetic modelling based on blood levels gave higher daily intake values of mercury compared to those of the FFQ. Furthermore, the former had a wider range of estimates than the latter. The properties of the toxicokinetic model or limitations in the dietary exposure assessment could be posited as reasons for the differences between the respective methods. Moreover, the results may have been influenced by sources of mercury exposure that cannot be described as dietary, such as amalgam fillings.

Maternal dietary exposure to dioxins and polychlorinated biphenyls (PCBs) is associated with language delay in 3year old Norwegian children.

BACKGROUND: Prenatal exposure to dioxins and PCBs is potentially harmful to the developing fetus and may increase the risk of delayed or impaired neurodevelopment. Several studies have reported negative associations between prenatal exposure to these compounds and aspects of cognition related to language in early childhood. OBJECTIVES: The aim was to examine the association between maternal low level dietary exposure to dioxins and PCB during pregnancy and language development in 3year old children in a large group of mother-child pairs participating in the Norwegian Mother and Child Cohort Study (MoBa). METHODS: This study includes 44,092 children of women who were recruited to the Norwegian Mother and Child Cohort Study (MoBa) during the years 2002-2009. Maternal dietary exposure to dioxins and PCBs was estimated based on a validated food frequency questionnaire (FFQ) answered mid-pregnancy and a database of dioxin and PCB concentrations in Norwegian foods. Exposure to dioxins and dioxin-like PCBs (dl-compounds) was expressed in total toxic equivalents (TEQ), and PCB-153 was used as marker for non-dioxin-like PCBs (ndlPCBs). Children's language skills at age 3 were assessed by parental report including a Dale and Bishop grammar rating and questions about communication skills from the Ages and Stages Questionnaire (ASQ). Logistic regression models adjusted for confounders were used to examine the association between maternal dietary exposure to dl-compounds or PCB-153 and language development in children. RESULTS: The maternal dietary exposure to dl-compounds and PCB-153 was generally low, and 98% of women had intakes of dl-compounds ≤14pg TEQ/kg bw/week, which is the tolerable weekly intake set by EU's Scientific Committee for Food (SCF). High maternal exposure (>14pg TEQ/kg bw/week of dl-compounds (median 2.6pg/kg bw/day, range 2-16) or >97.5-percentile intake of PCB-153 (median 11ng/kg bw/day, range 5-28) was associated with higher odds of incomplete grammar (in boys and girls, adjusted ORs 1.1 to 1.3) and severe language delay in girls, adjusted ORs 2.8 [95% CI 1.1, 7.1] for PCB-153 and 2.9 [95% CI 1.4, 5.9] for dl-compounds. Furthermore, high exposure to dl-compounds was associated with moderate language delay 1.4 [95% CI 1.0, 2.0] and lower communication score (ASQ), adjusted OR 1.4 [95% CI 1.1, 1.9] in girls. CONCLUSIONS: The main findings of this study were: 1) Girls born to mothers who exceeded the tolerable weekly intake for dl-compounds or had a PCB-153 intake above the 97.5 percentile in early pregnancy may have increased risk of language delay at age 3years. 2) Negative associations with maternal exposure to dl-compounds or PCB-153 were observed for both boys and girls having incomplete grammar, which is a subtle reduction in language skills. This interesting finding should not be considered as deviant at this age.

Consumption of lead-shot cervid meat and blood lead concentrations in a group of adult Norwegians.

Several recent investigations have reported high concentrations of lead in samples of minced cervid meat. This paper describes findings from a Norwegian study performed in 2012 among 147 adults with a wide range of cervid game consumption. The main aim was to assess whether high consumption of lead-shot cervid meat is associated with increased concentration of lead in blood. A second aim was to investigate to what extent factors apart from game consumption explain observed variability in blood lead levels. Median (5 and 95 percentile) blood concentration of lead was 16.6 µg/L (7.5 and 39 µg/L). An optimal multivariate linear regression model for log-transformed blood lead indicated that cervid game meat consumption once a month or more was associated with approximately 31% increase in blood lead concentrations. The increase seemed to be mostly associated with consumption of minced cervid meat, particularly purchased minced meat. However, many participants with high and long-lasting game meat intake had low blood lead concentrations. Cervid meat together with number of bullet shots per year, years with game consumption, self-assembly of bullets, wine consumption and smoking jointly accounted for approximately 25% of the variation in blood lead concentrations, while age and sex accounted for 27% of the variance. Blood lead concentrations increased approximately 18% per decade of age, and men had on average 30% higher blood lead concentrations than women. Hunters who assembled their own ammunition had 52% higher blood lead concentrations than persons not making ammunition. In conjunction with minced cervid meat, wine intake was significantly associated with increased blood lead. Our results indicate that hunting practices such as use of lead-based ammunition, self-assembling of lead containing bullets and inclusion of lead-contaminated meat for mincing to a large extent determine the exposure to lead from cervid game consumption.

Essential and toxic element concentrations in blood and urine and their associations with diet: results from a Norwegian population study including high-consumers of seafood and game.

The first aim of the study was to evaluate calculated dietary intake and concentrations measured in blood or urine of essential and toxic elements in relation to nutritional and toxicological reference values. The second aim was to identify patterns of the element concentrations in blood and urine and to identify possible dietary determinants of the concentrations of these elements. Adults with a known high consumption of environmental contaminants (n=111), and a random sample of controls (n=76) answered a validated food frequency questionnaire (FFQ). Complete data on biological measures were available for 179 individuals. Blood and urine samples were analyzed for selenium, iodine, arsenic, mercury, cadmium and lead. Principal component analysis was used to identify underlying patterns of correlated blood and urine concentrations. The calculated intakes of selenium, iodine, inorganic arsenic and mercury were within guideline levels. For cadmium 24% of the high consumer group and 8% of the control group had intakes above the tolerable weekly intake. Concentrations of lead in blood exceeded the bench-mark dose lower confidence limits for some participants. However, overall, the examined exposures did not give rise to nutritional or toxicological concerns. Game consumption was associated with lead in blood (B(ln) 0.021; 95%CI:0.010, 0.031) and wine consumption. Seafood consumption was associated with urinary cadmium in non-smokers (B(ln) 0.009; 95%CI:0.003, 0.015). A novel finding was a distinct pattern of positively associated biological markers, comprising iodine, selenium, arsenic and mercury (eigenvalue 3.8), reflecting seafood intake (B 0.007; 95%CI:0.004, 0.010). The study clearly demonstrates the significance of seafood as a source of both essential nutrients and toxic elements simultaneously and shows that exposure to various essential and toxic elements can be intertwined.

Determinants of plasma concentrations of perfluoroalkyl substances in pregnant Norwegian women.

BACKGROUND: Perfluoroalkyl substances (PFASs) are widespread pollutants that have been associated with adverse health effects although not on a consistent basis. Diet has been considered the main source of exposure. The aim of the present study was to identify determinants of four plasma PFASs in pregnant Norwegian women. METHODS: This study is based in the Norwegian Mother and Child Cohort Study (MoBa) conducted by the Norwegian Institute of Public Health. Our sample included 487 women who enrolled in MoBa from 2003 to 2004. A questionnaire regarding sociodemographic, medical, and reproductive history was completed at 17 weeks of gestation and a dietary questionnaire was completed at 22 weeks of gestation. Maternal plasma samples were obtained around 17 weeks of gestation. Plasma concentrations of four PFASs (perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), perfluorohexane sulfonate (PFHxS), and perfluorononanoate (PFNA)) were examined in relation to demographic, lifestyle, dietary, and pregnancy-related covariates. Predictors were identified by optimizing multiple linear regression models using Akaike's information criterion (AIC). RESULTS: Parity was the determinant with the largest influence on plasma PFAS concentrations, with r(2) between 0.09 and 0.32 in simple regression models. In optimal multivariate models, when compared to nulliparous women, parous women had 46%, 70%, 19%, and 62% lower concentrations of PFOS, PFOA, PFHxS, and PFNA respectively (p<0.001 except for PFHxS, p<0.01). In all these models, duration of breastfeeding was associated with reduced PFAS levels. PFOA showed the largest reduction from breastfeeding, with a 2-3% reduction per month of breastfeeding in typical cases. Levels of PFOS, PFOA, and PFNA increased with time since most recent pregnancy. While pregnancy-related factors were the most important predictors, diet was a significant factor explaining up to 4% of the variance. One quartile increase in estimated dietary PFAS intake was associated with plasma PFOS, PFOA, PFHxS, and PFNA concentration increases of 7.2%, 3.3%, 5.8% and 9.8%, respectively, resulting in small, although non-trivial absolute changes in PFAS concentrations. CONCLUSION: Previous pregnancies and breastfeeding duration were the most important determinants of PFASs in this sample of pregnant women.

Dietary mercury exposure in a population with a wide range of fish consumption--self-capture of fish and regional differences are important determinants of mercury in blood.

Human, low level, chronic exposure to mercury (Hg) from fish is of concern because of potential neurodevelopmental and cardiovascular toxicity. The purpose of the study was to 1) measure total mercury (THg) in blood and estimate dietary exposure in a population group with a wide range of seafood consumption, 2) assess the intake and blood concentration in relation to tolerable intake values, 3) characterise dietary sources, and 4) to investigate the relationship between dietary THg with THg in blood (BTHg), including factors that can explain the variance in BTHg concentrations. The participants (n=184) filled in an extensive food frequency questionnaire which was combined with a database on THg concentrations in Norwegian food, and donated blood and urine. Median consumption of seafood was 65 g/day (range 4 to 341 g/day). The calculated mean dietary THg exposure was 0.35 (median 0.30) µg/kg body weight/week. Seafood contributed on average 95% to the exposure. The JECFA Provisional Tolerable Weekly Intake (PTWI) of 1.6 µg MeHg/kg bw/week was not exceeded by any of the participants. BTHg ranged from 0.6 to 30 µg/L, with a mean of 5.3 (median 4.0 µg/L). There was a strong relationship between total seafood consumption and BTHg concentrations (r=0.58 95%CI: 0.48, 0.67) and between estimated THg dietary exposure and BTHg (r=0.46 95%CI: 0.35, 0.57). Fish consumption, sex, catching >50% of their seafood themselves, and living in coastal municipalities were significant factors in linear regression models with lnBTHg. Including urinary Hg in the regression model increased the explained variance from 54% to 65%. In a toxicokinetic model, the calculated dietary intake appeared to moderately underestimate the measured BTHg among the participants with the highest BTHg. Only two of the participants had BTHg slightly above a value equivalent to the JECFA PTWI, but none of them were women in fertile age.

Cyclic AMP regulates expression of the RI alpha subunit of cAMP-dependent protein kinase through an alternatively spliced 5' UTR.

The present study examines novel mechanisms that regulate levels of the RI alpha subunit of cAMP-dependent protein kinase. We found that RI alpha protein is induced threefold by 8-(4-chlorophenyl)thio-cAMP in hormone responsive rat Sertoli cells, while total RI alpha mRNA is not correspondingly induced. Two RI alpha mRNA isoforms with different 5' untranslated sequences (RI alpha 1a and RI alpha 1b) are produced from the RI alpha gene in Sertoli cells. Deletion/mutation analysis of the cAMP-response-element-containing promoter upstream of the RI alpha exon 1b revealed that while mutation of the cAMP response element had no effects on cAMP-mediated induction, a 73-bp region of the RI alpha exon 1b itself conferred a fivefold to eightfold induction of reporter activity to homologous and heterologous promoters. The responsiveness of this region was dependent on a sense orientation downstream of the promoter start sites and had no effect on reporter mRNA, indicating that the cAMP-mediated induction occurs at the post-transcriptional level. Modeling of the RI alpha 1b 5' UTR secondary structure revealed a 5' CAP-proximal, strong stem-loop presenting an element similar to multiple start-site element downstream-1 (GCTCGG) in the loop region. RNA-EMSAs performed with the labeled RI alpha 1b 5' UTR showed stabilization of a protein/RNA complex in extracts from 8-(4-chlorophenyl)thio-cAMP stimulated Sertoli cells. This complex was abolished by mutation of the multiple start-site element downstream-1-like element. Our findings indicate that there is a cAMP-mediated induction of RI alpha expression at the post-transcriptional level, dependent on the 5' UTR of RI alpha 1b mRNA.

Method of transfection affects the cAMP-mediated induction of the RIIbeta subunit of protein kinase A in Sertoli cells: inhibition of response by increase in intracellullar calcium.

mRNA for the regulatory subunit RIIbeta of cAMP-dependent protein kinase is stimulated more than 50-fold by cAMP in primary cultures of rat Sertoli cells. We have previously shown that this induction involves regulation of transcriptional activation as well as mRNA stabilization. The rat RIIbeta gene contains no cAMP response element (CRE), and the induction of RIIbeta mRNA is slow and requires on-going protein synthesis. When a construct containing the 5'-flanking region of the RIIbeta gene upstream of a CAT reporter was transfected into Sertoli cells by the calcium phosphate method, low and variable responses to cAMP (three- to fivefold) were observed, whereas a 15- to 20-fold increase in reporter activity by cAMP was observed after lipofectamine transfection. Interestingly, when a vector containing CRE elements upstream of a reporter gene was transfected into Sertoli cells, the responses to cAMP were similar regardless of the transfection method used. We have also demonstrated that increased intracellular levels of calcium by A23187 and thapsigargin dramatically inhibit cAMP-mediated induction of RIIbeta mRNA, but not the mRNA for the CRE-containing RIalpha gene. Furthermore, decreased cAMP responsiveness of endogenous RIIbetamRNA (but not RIalpha) was also observed in calcium phosphate-transfected Sertoli cells but not in lipofectamine-transfected cells. Thus, calcium-mediated reduction in cAMP response appears to be a gene-specific phenomenon.

Isoform-specific regulation of the CCAAT/enhancer-binding protein family of transcription factors by 3',5'-cyclic adenosine monophosphate in Sertoli cells.

The C/EBP (CCAAT/enhancer-binding protein) family of transcription factors is important for differentiation, lipid biosynthesis, and metabolism. Here, we demonstrate for the first time the presence of C/EBP alpha, beta, delta, and zeta messenger RNA (mRNA) and protein in Sertoli cell primary cultures. Treatment with FSH or 8-CPTcAMP strongly induced C/EBP beta mRNA above basal levels with rapid and transient kinetics in Sertoli cell primary cultures as well as in whole testes from hypophysectomized rats. Whereas C/EBP beta mRNA was induced approximately 50-fold, C/EBP delta mRNA was induced 5- to 8-fold by cAMP in Sertoli cells. Messenger RNA for C/EBP beta and delta were induced by inhibition of protein synthesis with cycloheximide and cycloheximide acted synergistically with cAMP. Immunoblots with C/EBP antibodies demonstrated a strong induction of C/EBP beta, delta, and zeta by cAMP. Electrophoretic mobility shift analysis of nuclear proteins from cAMP-treated Sertoli cells using a C/EBP consensus oligonucleotide and antibodies revealed specific binding of C/EBP/DNA complexes, the majority of which were supershifted by C/EBP beta antibody. Transfections of Sertoli cells with a C/EBP reporter construct showed approximately 3-fold induction of reporter gene activity by cAMP. In contrast, the reporter gene vector with a mutated form of the C/EBP binding site, was almost unresponsive to cAMP in transfections of Sertoli cells. Furthermore, C/EBP beta expression increased the activities of two promoters known to be cAMP-responsive in Sertoli cells. Thus, the early induction of C/EBP isoforms by cAMP may play a role in FSH-dependent regulation of late response genes in Sertoli cells.

Characterization of the 5'-flanking region of the gene for the cAMP-inducible protein kinase A subunit, RIIbeta, in Sertoli cells.

Activation of cyclic AMP-dependent protein kinases (protein kinase A, PKA) by gonadotropins and cyclic AMP (cAMP) plays an important role in the regulation of testicular functions. A regulatory subunit, RIIbeta, of PKA is transcriptionally induced in rat Sertoli cells in response to treatment with cAMP. The present study addresses regulatory mechanisms leading to increased transcription of the rat RIIbeta gene. We have localized a footprint which overlaps one of the major transcription initiation sites in the basal promoter (-293 to -123). One of the proteins binding this sequence belongs to the NF-1 family of transcription factors. We also observed binding to a basic helix-loop-helix (bHLH) response element. Furthermore, transfection studies of various 5'-deletions of the rat RIIbeta gene in primary cultures of rat Sertoli cells and in peritubular cells revealed the presence of an upstream region (-723 to -395, cAMP-responsive region) inhibiting basal expression from the rat RIIbeta gene only in Sertoli cells. This region was found to enhance cAMP responsiveness in Sertoli cells but not in peritubular cells. Interactions with downstream elements seemed to be important for the function of the cAMP-responsive region. Although some short stretches reveal homology to the cAMP-responsive regions of other slowly cAMP-responding genes, and an AP-1-like element is present, no strong resemblance to any known regulatory element responsive to cAMP is found.

Structure, function, and regulation of human cAMP-dependent protein kinases.

A large number of hormones, neurotransmitters, and other signaling substances that bind to G-protein-coupled cell-surface receptors have their signals converge at one sole second messenger, cAMP. The question of how specificity can be maintained in a signal-transduction system in which many extracellular signals leading to a vast array of intracellular responses are all mediated through one second-messenger system has been the subject of thorough investigation and a great deal of speculation. An increasing number of cAK isozymes, consisting of homo- or heterodimers of R subunits (RIalpha, RIbeta, RIIalpha, RIIbeta) with associated catalytic subunits (C alpha, Cbeta, Cgamma), may, at least in part, explain this specificity. The various cAK isozymes display distinct biochemical properties, and the heterogeneous subunits of cAK reveal cell-specific expression and differential regulation at the level of gene transcription, mRNA stability, and protein stability in response to a wide range of hormones and other signaling substances. The existence of a number of anchoring proteins specific to either RIIalpha or RIIbeta, and which localize cAKII isozymes toward distinct substrates at defined subcellular loci, strongly supports the idea that specific functions can be assigned to the various cAK isozymes. The demonstration that selective activation of cAKI is necessary and sufficient for cAMP-mediated inhibition of T-cell proliferation, and the observation that T-cell activation is associated with redistribution and colocalization of cAKI to the TCR, is also compatible with the notion of isozyme-specific effects.

Regulation of protein kinase A subunits by cyclic adenosine 3',5'-monophosphate in a mouse Sertoli cell line (MSC-1): induction of RII beta messenger ribonucleic acid is independent of continuous protein synthesis.

We report the basal and cAMP-regulated expression of protein kinase A (PKA) subunits in a mouse Sertoli cell line (MSC-1). Of the PKA subunits expressed by these cells (RI alpha, RII alpha, RII beta, C alpha, C beta), only RII beta was regulated by cAMP. An approximately 8-fold induction of RII beta mRNA and a 3-fold induction of RII beta protein was observed during 48 h of cAMP-stimulation. This cAMP-mediated RII beta mRNA induction, reaching maximal levels after approximately 12 h, did not require ongoing protein synthesis. Fairly rapid decay of maximally induced RII beta mRNA was observed after removal of cAMP (t1/2 approximately 5 h). Further, ongoing transcription and translation were necessary for rapid degradation of RII beta mRNA. Thus, the MSC-1 cells expressed all the PKA subunits present in primary cultures of Sertoli cells and responded to cAMP with increased levels of RII beta at both mRNA and protein levels. Although the nature of some of these responses distinguished the observations in MSC-1 cells from previously described responses in primary cultures, these cells may prove to be useful in future studies addressing cAMP-mediated gene regulation.

Serum factors induce messenger ribonucleic acid levels for cellular retinol-binding protein in rat Sertoli cells.

This report shows that serum factors dramatically increase the levels of mRNA for cellular retinol-binding protein (CRBP) in cultured rat Sertoli cells. Incubation of rat Sertoli cells (0-24 h) with 10% fetal calf serum (FCS) was associated with a time-dependent increase in CRBP mRNA levels. A significant increase (6-fold) was observed after 3 h of incubation. Maximal levels (15- to 50-fold) were reached after 9-12 h and were maintained for as long as serum was present. The effect was concentration dependent, with maximal induction at 10% FCS. Removal of FCS resulted in a decline in the CRBP mRNA levels, with a t1/2 of approximately 7 h. The CRBP mRNA stimulating activity (CMSA) was completely removed from FCS by precipitation with 5% trichloroacetic acid, but was only partly (50%) inhibited by heating at 100 C or trypsin treatment. Removal of retinol from FCS by repeated ether extractions did not alter the CMSA of FCS. Both the induction and degradation of CRBP mRNA were inhibited by the protein synthesis inhibitor cycloheximide. A nuclear protein binding to the 5'-flanking region of the CRBP gene was detected in nuclear extracts from untreated Sertoli cells, but not in nuclear extracts from Sertoli cells treated with 10% FCS for 3 h. Thus, serum factors, different from retinoids, dramatically stimulate the levels of CRBP mRNA in rat Sertoli cells. This is associated with the loss of protein binding to the 5'-flanking region of the CRBP gene.

Down-regulation of messenger ribonucleic acid (mRNA) for the estrogen receptor (ER) by phorbol ester requires ongoing RNA synthesis but not protein synthesis. Is hormonal control of ER mRNA degradation mediated by an RNA molecule?

Treatment of MCF-7 cells, a human mammary adenocarcinoma cell line, with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (10-7 M) was associated with a time-dependent reduction in the level of estrogen receptor (ER) mRNA (half-life approximately 3 h). In the presence of the RNA synthesis inhibitor actinomycin D [5.0 micrograms/ml (4.0 microM)], half-life of ER mRNA was much longer (approximately 12 h). Furthermore, the TPA-dependent down-regulation of ER mRNA was abolished by actinomycin D. Similar effects were observed with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (150 microM), an inhibitor of RNA polymerase. Inhibition of protein synthesis by cycloheximide (50 microM) or puromycin [50 micrograms/ml (92 microM)] did not alter the steady state level of ER mRNA during a period of 10-12 h. Furthermore, these inhibitors of protein synthesis did not prevent the down-regulation of ER mRNA in the presence of TPA. Our studies show that degradation of ER mRNA by TPA in MCF-7 cells is dependent on ongoing RNA synthesis but not on protein synthesis. This indicates that an RNA molecule with rapid turnover, which does not require translation, might be involved in the TPA-dependent ER mRNA decay.

Half-lives of different sized mRNAs for the PKA subunit RI alpha are regulated differently in response to inhibition of transcription and translation.

The RI alpha mRNA level is induced 3-5 times by FSH or cAMP analogs in primary cultures of rat Sertoli cells. In rat tissues, the RI alpha gene gives rise to three different mRNAs of different size: 3.2, 2.9 and 1.7 kb. In the present study we report that the 1.7 kb transcript has a shorter half-life than the two other mRNAs. In cells which had been pre-stimulated with a cAMP analog, inhibition of transcription stabilizes the two larger, but not the smaller sized RI alpha mRNA. However, in contrast, inhibition of protein synthesis stabilizes all the RI alpha mRNAs. Thus, degradation of various mRNAs coding for the same protein reveals different dependencies on transcription and translation.

Inhibitors of RNA and protein synthesis stabilize messenger RNA for the RII beta subunit of protein kinase A in different cellular compartments.

Messenger RNA for RII beta is transiently induced (greater than 50-fold) by cAMP analogs in primary cultures of rat Sertoli cells. The induction is dependent on protein synthesis. We have previously shown that mRNA for RII beta is stabilized by cAMP, as well as inhibitors of transcription and translation. This indicated that rapid degradation of RII beta mRNA involved a protein with a rapid turnover and its corresponding mRNA. The two RNA synthesis inhibitors used in the present study stabilized both nuclear and cytoplasmic RII beta mRNA, whereas inhibition of protein synthesis stabilized RII beta mRNA in the cytoplasm only. These results indicate that only cytoplasmic degradation of RII beta mRNA is dependent on a protein with high turnover. In contrast, nuclear degradation appears to be dependent on an RNA with a short half-life, not involving protein synthesis.

Protein kinase C activation by 12-O-tetradecanoylphorbol 13-acetate modulates messenger ribonucleic acid levels for two of the regulatory subunits of 3',5'-cyclic adenosine monophosphate-dependent protein kinases (RII beta and RI alpha) via multiple and distinct mechanisms.

Messenger RNAs (mRNA) for two of the regulatory subunits of cAMP-dependent protein kinases (PKA), RII beta and RI alpha, are transiently (maximal levels at 6 h) stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) in cultured rat Sertoli cells in a time- and concentration-dependent manner. Whereas TPA (10(-7) M) stimulated RII beta mRNA 11 +/- 2.8 fold (mean +/- SEM), mRNA levels for RI alpha increased only 2.5 +/- 0.6-fold (mean +/- SEM). No effects of TPA on the other subunits of PKA (RII alpha, C alpha) were observed. TPA-dependent accumulation of mRNAs for RII beta and RI alpha was observed to the same extent in nucleus and cytoplasm. We have previously shown that mRNA levels for all the PKA subunits are increased by cAMP, particularly that of RII beta (greater than 50-fold). TPA modulated the stimulatory effects of cAMP on RII beta and RI alpha mRNAs in opposite directions. Whereas treatment with both 8-CPTcAMP and TPA gave an additive effect on RI alpha mRNA, TPA reduced the cAMP-dependent increase in RII beta mRNA. Although the mRNA for RII beta had returned to basal levels after 24 h of incubation with TPA, the presence of TPA still inhibited cAMP-dependent induction of mRNA for RII beta. In contrast, similar TPA treatment did not influence the subsequent cAMP-dependent stimulation of RI alpha mRNA. Preincubation with 8-CPTcAMP did not influence TPA-dependent stimulation of mRNAs for either RII beta or RI alpha. TPA induction of RII beta mRNA was completely blocked by cycloheximide (an inhibitor of protein synthesis), whereas that of RI alpha was not. The inhibitory effect of TPA on cAMP stimulation of RII beta mRNA was independent of ongoing protein synthesis. These results indicate that TPA induction of mRNAs for RI alpha and RII beta involves multiple and distinct mechanisms. The stimulatory effect of TPA on RI alpha mRNA levels and the inhibitory effect of TPA on cAMP-stimulated RII beta mRNA expression are probably mediated through stable factors, whereas proteins with rapid turnover or factors induced by TPA are involved in the stimulatory effect of TPA on RII beta mRNA.

Adenosine 3',5'-monophosphate-dependent stabilization of messenger ribonucleic acids (mRNAs) for protein kinase-A (PKA) subunits in rat Sertoli cells: rapid degradation of mRNAs for PKA subunits is dependent on ongoing RNA and protein synthesis.

cAMP treatment of primary cultures of Sertoli cells is associated with a transient stimulatory effect on mRNA levels for various protein kinase-A (PKA) subunits. We have previously shown that the induction of mRNA for regulatory subunit II beta (RII beta) is due at least partly to transcriptional activation. In the present study we investigate possible regulatory effects of (Bu)2cAMP on the degradation of mRNAs for various PKA subunits in rat Sertoli cells. We demonstrate subunit specific differences in the decay of mRNAs for the various PKA subunits. When (Bu)2cAMP was removed from Sertoli cell cultures after 6 h of stimulation, there was a rapid decay of mRNAs for both RII beta and RI alpha (half-lives, approximately 3 h). In contrast, mRNA levels for RII alpha continued to increase. Removal of (Bu)2cAMP after a longer period of treatment revealed a similar decay of mRNAs for all of the PKA subunits, with half-lives of approximately 3 h. Incubation of Sertoli cells for 12 h with (Bu)2cAMP, followed by continued incubation in the absence and presence of (Bu)2cAMP as well as in the presence of actinomycin-D (an inhibitor of RNA synthesis), revealed (Bu)2cAMP mediated stabilization of mRNA for the RII beta subunit. Interestingly, actinomycin-D as such stabilized mRNAs for all PKA subunits. Similar treatment with cycloheximide (an inhibitor of protein synthesis) revealed distinct differences between the RI alpha and C alpha subunits vs. the RII subunits; cycloheximide reduced the decay of both RII beta and RII alpha mRNAs, whereas steady state levels of mRNAs for RI alpha and C alpha actually increased after cycloheximide treatment of previously (Bu)2cAMP-stimulated cultures. Cycloheximide treatment also increased basal levels of mRNAs for RI alpha and C alpha, whereas basal levels of RII beta and RII alpha mRNAs were not influenced. These studies indicate that the degradation of mRNAs for the various PKA subunits is subject to different regulation by (Bu)2cAMP, and that ongoing RNA and protein synthesis is required for rapid degradation of all PKA subunits.

Different mechanisms are involved in cAMP-mediated induction of mRNAs for subunits of cAMP-dependent protein kinases.

The present study addresses possible mechanisms through which cAMP mediates its effects on mRNA levels for the subunits of protein kinase A (PKA) and the cellular protooncogene, c-fos. Messenger RNAs for the PKA subunits (RI alpha, RII alpha, RII beta, and C alpha) were regulated by cAMP with similar kinetics in Sertoli cells. However, effects of cAMP on the PKA mRNAs were slow compared to a well characterized cAMP responsive gene, c-fos. The magnitude of stimulation was dramatically different between the various PKA subunits, in that RII beta mRNA increased more than 50-fold while the mRNAs for the other subunits were induced only two to four times. Separation of nuclear and cytoplasmic RNA demonstrated that mRNAs for PKA subunits were stimulated to the same extent in these two cellular compartments. The more rapid induction of c-fos mRNA by cAMP, compared to the mRNA for RII beta, was also seen at the level of transcription. Maximal transcription rate for c-fos, RI alpha, and C alpha were observed after 30 min, whereas that for RII beta was increasing during the 2-h period examined. Transcriptional activation of the RI alpha gene also appeared faster than that for RII beta. When Sertoli cells were incubated with 8-(4-chlorophenylthio) cAMP and cycloheximide, a potent inhibitor of protein synthesis, we observed a super-induction of the mRNAs for c-fos (10-fold) and RI alpha (2-fold).(ABSTRACT TRUNCATED AT 250 WORDS)