Oct 28, 2017 - with ovine p-lipotropin (1520%) on a molar basis. The minimum amount of ovine p-EP detectable was 3 pg/tube at 1:28,000 antiserum dilution.
Vol. 269, No. 43, Issue of October 28, pp. 26697-26705, 1994 Printed in U.S.A.
J o u r u r ~OF~ BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistryand Molecular Biology, Inc. THE
Cyclic AMP and Ethanol Interact to Control Apoptosis and Differentiation in Hypothalamic j3-Endorphin Neurons* (Received forpublication, July 6 , 1994, and in revised form, August 5 , 1994)
Alok De, Nadka Iv. BoyadjievaS, MartinePastorcic, Bhaskar V. Reddyg, and Dipak K. Sarkad From the Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, Washington 99164-6520
In this study we have determined the role of cyclic have been proposed t o function as neurotransmittersor neuroAMP on the function and differentiation of p-endorphin modulators regulating a variety of brain functions, including (P-EP) neurons in rat fetal hypothalamic cell cultures. psychomotor stimulation, positive reinforcement,adaptive procAddition of Bt,cA” or the CAMPelevating agent,for- esses, drinking, eating, sexual behaviors, pituitary function, skolin, in cultures, dose and time dependently increased thermoregulation, nociception, and mood(4-7). Opioid pepP-endorphin secretion. The increased p-EP secretion af- tides, particularlyp-EP, have beenimplicated in prenatal ethater Bt,cAMP or forskolin treatment was associated with nol-associated undernutrition, hypoxia, and acidosis (8, 91, proopiomelanocortingeneexpression,enhancedneugrowthretardation, behavioral abnormalities,andcentral rite growth, and increased neuronal viability. Determi- nervous system damage (lo), defects in learning and memory nation of internucleosomal cleavageof DNA by agarose (8), neuroendocrine abnormalities (10-121, and increased ingel electrophoresis revealed that apoptosis occurred in to be involved in fant mortality(9). Opioid peptides also appear hypothalamic neurons during the first 6-8 days in culsome of the effects of ethanol on dependence and intoxication ture. Addition of Bt,cAMP during this developmental period inhibited DNA degradation in hypothalamic neu- (13).The self-administrationof ethanol in non-human primates of and animals isreduced following treatment with opioid receprons.Furthermore,incubationwithvariousdoses ethanol, whichis known to reduce intracellular levels of torantagonists naloxone or naltrexone (14-16). Naltrexone also has been shown to reduce craving in alcoholic human paBt,cAMP, increased DNA degradation in thesecells. 18). Ethanol and endogenous opioid peptide interactients (17, Ethanol-inducedDNA degradation was blocked by concomitant incubation with Bt,cAMP. Histochemical iden- tion is further evidentby the observations that ethanol treatments acutely increase and chronically decrease p-EPsecretion tification of apoptoticcellsfollowingethanoland from hypothalamic neurons (19). Bt,cAMP treatments further revealed that apoptosis occurred in /3-EP neurons during the developmental peHormone and neurotransmitter control of p-EP secretion riod, and that ethanol increased and Bt,cAMP reduced from the hypothalamus is not well understood. However, studapoptotic B-EP cell numbers. These results suggest thaties on the POMC system of the pituitary revealed that the ethanolneurotoxicityonp-EPneuronsduringearly control of POMC-derived peptide secretion is multifactorial and neuronal differentiation involves an apoptotic process involves variousstimulatoryandinhibitory neuroendocrine and that theCAMPsignaling system plays an importanthormones (20). Also, studies of transduction pathways identirole in controlling apoptosis and differentiation of the fied the adenylylcyclase-CAMPsystem as an importantsecond p-EP neuronal system. messenger system in the regulationof hormone secretion and POMC gene regulation (20, 21). There is evidence that the adenylyl cyclase-CAMP system is involved in regulation of hyThe opioid peptide P-endorphin (p-EP)’ is derived from the pothalamic p-EPsecretion (22,231.Cyclic AMP has beenshown P-EPladrenocorticotropin hormone precursor proopiomelano- to play an important role in neuronal differentiation of various cortin (POMC) (1).The p-EP, producing perikarya are located cell types during development (24, 25) that often involves conregion that trolling programmed cell death (PCD; Ref. 26). However, the mainly in theventromedialarcuatenucleus projects to widespread brain structures, including many areas effect of this intracellular transducer in neuronaldifferentiaof the hypothalamus and limbic system (2). In addition to its tion in hypothalamic p-EP neurons is not apparent. Also, it is presence in the central nervous system, large amountsof the not known whether chronic ethanol administration,which has POMC peptides have been detected in both the intermediate been shown to decrease CAMP accumulation in various cells and anterior lobe of the pituitary gland (3). POMC peptides and brain tissues (27,28), alters p-EP neuronal differentiation during the developmental period. Hence, in this study we de* This work was supported by National Institutes of Health Grant AA08757. The costs of publication of this article were defrayed in part termined the effects of CAMPon the function and differentiaby the payment of page charges. Thisarticle must therefore be hereby tion of hypothalamic p-EP neurons using rat fetal hypothamodel marked “advertisement” in accordance with 18 U.S.C.Section 1734 lamic neuronprimarycultureasanexperimental solely to indicate this fact. system. We tested whether thereis any PCD during p-EP neu$ Supported by NINDS International Neurological Science Fellowronal differentiation in culture, and whether ethanol CAMP and ship NS05130. 5 Present address: Dept.of Animal Sciences,School of Life Sciences, alter this cell death and affect morphological and functional differentiation of p-EP neurons. University of Hyderabad, Hyderabad, India. 11 To whom correspondence should be addressed. Tel.: 509-335-2303; Fax: 509-335-4650. EXPERIMENTAL. PROCEDURES The abbreviations used are: Bt,cAMP, dibutyryl cyclic adenosine Cell Culture and Cell Viability-Monolayer fetal hypothalamic cell monophosphate;IR-P-EP, immunoreactive-p-endorphin; POMC, proopiomelanocortin;PCD, programmed cell death; PBS, phosphate-buff- cultures were prepared usingthe procedure described by us previously ered saline; TUNEL, terminal deoxynucleotidyl transferase-mediated (29). In brief, pregnant rats (Sprague-Dawleyfemale rats, 19-21 days ddUTP-biotin labeling;bp, base pair(s). gestation) were sacrificed by decapitation, and the fetuses were re-
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CAMPInhibits and Ethanol Stimulates Apoptosis
moved immediately by aseptic surgical procedure. The mediobasal hypothalami were removed from the brains of these fetuses and kept in ice-cold Hank's balanced salt solution (Sigma)containing 1%Fungi-bact (Irvine Scientific, CA), 0.1% bovineserum albumin (Sigma),and 200 p~ ascorbic acid (Sigma). The hypothalamic cells were washed and then incubated at 37 "C for 10-15 min using the same medium. After incubation, the tissues were resuspended in 25 mM Hepes-buffered DulbecCO'S modified Eagle's medium(4.5 gAiter glucose;Sigma) containing 1% Fungi-bact (Irvine Scientific, CA), 0.1% bovineserum albumin, 200 p~ ascorbic acid (Sigma), and 10% heat inactivated fetal calf serum (HyClone Laboratories, Logan, UT). Tissues were gently dissociated mechanically by passing 7-8 times through an 18-gauge needle fixed toa 20-ml syringe. Following dispersion, the cell viability was determined by trypan blue exclusion: 80-90% cells were viablein all experiments. The cells were then plated at a density of 3.0 x lo6 cells/25-cm2flasks which were previously coated with polyornithine at a concentration of 100 pg/mland incubated overnight at 37 "C. The cells weremaintained in Dulbecco'smodified Eagle's medium at 37"C and 7.5%CO, in a humidified water-jacketed incubator for 2 days. After2 days of plating, the medium was changed at 1-or 2-day intervals with the serum-free, chemically defined medium (consisting of 30 MI selenium, 20 n~ progesterone, 1p~ iron-free human transferrin, 100 p~ putrescine, and 5 pg/ml insulin (Sigma)). Radioimmunoassay-Immunoreactive p-EP was measured by radioimmunoassay as described by us previously (30). This radioimmunoassay system consisted of anti-human p-EP antiserum (YlO), ovinep-EP standard (0-0-EP;this has the same amino acid sequence as rat p-EP) and '251-o-p-EP.The antiserum Y10 cross-reacted with p-EP (100%)and with ovinep-lipotropin (1520%) on a molar basis. The minimum amount of ovine p-EP detectable was 3 pg/tube at 1:28,000 antiserum dilution. Immunoreactive p-EP characterization by the gel chromatography from hypothalamic cell and medium extracts showed a major p-EP component and a small 0-lipoprotein component (29), suggesting that the values obtained by this radioimmunoassay represented mostly p-EP peptide. The cellular content of DNA was measured according to the method of Labarca and Paigen (31). RNA Isolation and Quantification of POMC Danscripts by Ribonuclease Protection Assay-Nuclear and cytoplasmic RNA were prepared as described previously(32,33) with the following modifications. At the end of treatment, the culture medium was removed and stored for IR-p-EP radioimmunoassay. Cells were washed 3 times with PBS and lysed with 1ml ofbufferA(l0 m~ Tris, pH 8.0,3 mM CaCl,, 2 mM MgCl,, 0.5 mM dithiothreitol, 0.15% Triton X-100) containing 0.3 M sucrose/25cm2 flask. Cells were collected by scrapping and the suspension was pushed 3 times through a 22-gauge syringe needle to complete the lysis. The lysate was loaded onto a 300-pl cushion of lysis buffer containing 0.4 M sucrose and centrifuged at 3000 x g for 10 min. Eight hundred microliters of the supernatant which contains the cytoplasmic fraction was transferred to a new tube. Ninety microliters of 10 x SET buffer (10% SDS, 50 m~ EDTA, 100 m~ Tris, pH 8.0) were added with 10 pl of 10 mg/ml proteinase K, and samples were incubated for 1h at 45 "C. After phenol extraction RNA was precipitated with 2-propanol. The nuclear pellet was washed with 1 ml of 0.4 M sucrose buffer and resuspended into 200 p1of guanidinium isothiocyanate-phenol solution t o extract the RNA (34). All steps were carried out at 4 "C. Nuclear and cytoplasmic RNA were analyzed by RNase protection assay as describedpreviously (35, 36). Both the antisense-labeled POMC probe and the (+)-RNAstandard were synthesized from a 470-bp fragment of the POMC gene subclonedinto PBS(+)(37). The fragment include 20 bp ofintron A, the entire180-bp exon 2, and 270 bp of intron B. The cyclophilin probewas synthesized using prpXCYC/BS (37). After synthesis, the probes were purified on a 5% acrylamide, 7 M urea gel. Specific activities were lo9 c p d p g for POMCprobe and 2-5 x 10' c p d p g for the cyclophilin probe. Total RNA was added to 30-pl hybridization mixtures containing 60% formamide, 10 m~ Hepes, pH 7.5,600 mM NaC1,2 mM EDTA, together with 0.5 ng of antisense POMC RNA probe (5 x lo5 cpm), cyclophilin probe (2 x lo5cpm), and tRNAin order to keep the totalamount of RNA to 20 pg in each sample. Hybridizations were carried out at 56 "C for 16-20 h. Reactions weretreated with 2.5 pg/ml RNaseA and processed as described previously(36). RNA samples were analyzed on 5% acrylamide, 7 M urea gels. Autoradiography was performed a t -80 "C with two intensifying screens. RNA was quantitated by counting the countdmin in each gel band crushed into scintillation fluid. We calculated the ratio of POMC mRNA level in treated cells overPOMC mRNA in control cellsin each experiment. The numbers were normalized using cyclophilin as an internal control. The picogram amounts of POMC transcripts were estimated by
in p-EP Neurons
comparing counts/min to a standard curve generated by linear regression of counts/min from hybridization products versus picograms of (+)-sense RNA standard. Amounts of hybridization products were converted into amounts of full-length cellular RNA by multiplying by the ratio between the full-length species and the size of the protected fragment. We used 6 kilobases for the full-length primary transcript, 4.2 kilobases for the processing intermediate, and 1.2 kilobases for the mRNA. The numbers in thetext indicate the means f S.E. of the ratios calculated in four to six independent experiments. Analysis ofDNA Fragmentation on Agarose Gels-DNA was prepared from treated and untreated cultures according to the method described previously(38). The cells were lysedin 100 pl of lysis buffer (100 mM Tris, pH 8.0, 20 mM EDTA, 0.5% SDS). Following treatment with 50 pg/ml DNase-free RNase at 37"C for 30 min and 100 pg/ml proteinase K for 2 h at 50 "C, extraction with phenolkhloroform and chloroform and precipitation in 3 volumes of ethanol, the DNA (1pg/ lane) was electrophoresed on 1%agarose gels. Molecular weightstandards were run concurrently. The gels were stained with ethidium bromide and photographed. Degradation ofDNA was quantitated by scanning the photographs using a laser fast-scan image analyzer (Image Quant Software Version3.22,MolecularDynamics, Sunnyvale, CA), and integrating the area under the curve (66). The range of molecular weights used during gel quantitation were 9400 to 90 bp. The relative DNA degradation value was calculated by substracting the integrated area for the degraded DNA of control sample from the integrated area of treated sample. AHpaII digest of KS(+) (Stratagene, CA) used as molecular weight standards. Immunocytochemistry-On the day of the study, culture media were removed and the cells were washedwith cold PBS, pH 7.2, followed by Bouin's fixation for 4 h at room temperature. The cellswere then washed in PBS and treatedwith 1:lOO dilution of normal goat serum for 30 min at room temperature. The staining of IR-p-EPwas carried out by incubating the cultured cells sequentially, first with a rabbit anti p-EP antiserum a t 1:lOOO dilution in PBS containing 0.1% Triton X-100 at 4 "C overnight.After washing with PBS, the cells wereincubated for 20 min at room temperature with biotinylated anti-rabbit immunoglobulins. The cells werethen washed with PBS and incubated with alkaline phosphatase-conjugated streptavidin for 20 min at room temperature. After washing, color was developed using Naphtol phosphate as a substrate. The number of positively stained cells forIR-p-EP was counted. The cells were counterstained with Nissl stain to identify fragmented nuclei in IR-p-EP neuron. Terminal Deoxynucleotidyl Dansferase-mediated ddUTP-biotin Labeling (TUNEL)-On the day of the study, culture media were removed. The cells were fixedin 4% neutral buffered formalin for 30 minat room temperature, post-fixed in ethanol/acetic acid (2:l) for 5 min at -20 "C, washed in PBS, pH 7.2. The endogenous peroxidase activity was prevented by treating the cells with 3% H,O, in PBS for 5 min at room temperature. The cells were then incubated in reaction buffer (25 mM Tris, pH 7.6,200 mM potassium cacodylate, 5 mM CoCI,, and 0.25 mg/ml bovine serum albumin) for 5-10 min and thenincubated with terminal transferase (1 uniff5 pl), digoxigenin-dUTP (10.0 nM), and ddATP (45 p ~ at) 37 "C for 1 h. The ddNTPs wereused to prevent "tailing" and to allow the incorporation of one nucleotide per free DNA 3'-end. After washing in Tris buffer the cells were incubated with anti-digoxigenin antibody conjugated to peroxidase (1:lOOO)overnight at 4 "C. The cells were then washed in PBS and the color was developed using diaminobenzidine as chromogen and H,O, as substrate. The cells were then stained using IR-p-EP primary antibody as described earlier to identify the apoptotic cells in p-EP neurons. Statistics-The data shown in the figures and text are mean * S.E. Data were analyzed using one-way or two-wayANOVA with repeated measures on time, as appropriate. Post-hoc test following ANOVA involved Student-Newmann-Keulstest. Avalue ofp < 0.05 was considered significant. RESULTS
Effect of CAMP on IR-p-EPRelease--In order to test whether the hypothalamic cells in primary cultures are responsive to agents elevating the intracellular levels of CAMP, the effects of various doses of Bt,cAMP and adenylyl cyclase stimulator forskolin were tested. As shown in Fig. 1, treatment with 1, 10, and 100 PM Bt,cAMP (A) or 1, 10, and 100 PM forskolin ( B )for 4 days in cultures produced dose- and time-dependent effects on IR-p-EP release from hypothalamic neurons in primarycultures. Thedoses of Bt,cAMP and forskolin effective in inducing
CAMP Inhibits and Ethanol Stimulates Apoptosis in p-EP Neurons
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FIG.1. Time- and dose-dependent effects of dibutyryl C A M P (dbcAMP) or forskolin ( F S K ) on IR-P-EP secretion from fetal hypothalamic neurons in primary cultures. Hypothalamic cells were dissociated and culturedfor 2 days in the presenceof 10% serum as described under “Experimental Procedures.” After this period, the cultures were fed with medium containing serum supplement anddifferent doses of dbcAh4P ( A ) and forskolin ( B )at 1-day intervals for 4 days. Media from the cultures were obtained daily and measured for IR-p-EP. Data are the mean * S.E. from eight independent observations. * , p < 0.05, significantly differentfrom the vehicle-treated control group.
IR-P-EP release ranged between 10 and 100 p~ and 1and 100 p ~ respectively. , Treatment with a 100 VM dose ofBt,cAMP significantly raised ( p < 0.001) media levels of P-EP by day 1, and the levels of IR-P-EP in media reached a maximum between days 2 and 4.Treatments with 10 PM doses of Bt,cAMP caused a significant ( p < 0.05) increase of IR-P-EP release only on day 4, as compared t o the control. As shown in Fig. 1B, cultures treated with 100 PM forskolin showed a significant ( p < 0.01) increase of IR-p-EP secretion after 1 day and reached
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FIG.2. Effect of Bt,cA” or forskolin on POMC gene expression. The autoradiograms showing the standard curve and the protected hybridscytoplasmic (A) and nuclear fraction ( B )of RNAsamples. Hypothalamic cells were maintained in cultures in the presence of 10% serum for 2 days, and then in the presenceof serum supplement for 6 days. The cells were then treated withvehicle (C), 10 p~ forskolin ( F ) , or 10 and 100 p~ BfcAMP (CAMP)for 24 h. RNA was isolated and 5 pg of cytoplasmic RNA or 2.5 pg nuclear RNA were analyzed by RNase protection assay. The top and bottom arrows in B denote the primary transcript and the nuclear processing intermediate, respectively. The mature transcripts are indicated by “POMC.”The 111-nucleotide long cyclophilin fragment (C) was used to normalize the level of mRNA. POMC standards show amounts of probe protected by a seriesof known quantities of (+)-RNA control. The size in nucleotides of molecular weight markers ( M )is indicated alongside the gel. Autoradiograms were exposed for 6 h for A and 48 h for B.
plateau after 2 days of treatment. Cultures treated with 10 PM forskolin showed gradual increase of IR-p-EP release over time for a period of 4 days, whereas cultures treated with 1J ~ M forskolin showed a significant increase of IR-p-EP release only on days 3 and 4. Effect of CAMP on POMC Gene Panscription-As p-EP is derived from the precursor POMC (11, we determined how much of the observed effects of forskolin and Bt,cAMP on P-EP secretion reflect the changesof the expression of POMC mRNA. We have examined the effects of Bt,cAMP (10 and 100 mM) and forskolin (10 PM) on the transcription of the POMC gene. We have used an RNase protection assay to monitor the level of POMC mRNAin cytoplasmic RNA. The level of POMC primary transcripts in nuclearRNA was also examined, since changes
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CAMPInhibits and Ethanol Stimulates Apoptosis
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FIG. 4. Agarose gel electrophoresisshowing theeffects of ethano1 and Bt,cAMP on DNA fragmentation in hypothalamic neurons in cultures.Hypothalamic cells were dissociated and cultured in the presence of 10% serum as described under “Experimental Procedures.”After 2 days the medium was replaced with serum supplement and treated with 100 p~ Bt,cAMP (a),100 PM Bt+AMPand 200 mM ethanol ( b ) , 200 mM ethanol ( c f , or vehicle ( d ) for 6 days. DNA was isolated and electrophoresed on 1% agarose gel. The size in base pairs of the molecular weight markers (lane 5 )is indicated alongside the gel. The number on the right side indicates the length in nucleotide for several of the bands.
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