C02 can be used in rats and mice as an initial anaesthetic in order to facilitate handling and reduce stress before injections or other kinds of treatment of short duration. Interference with physiological conditions like the energy status of the tissue may reduce its suitability. The present study was undertaken in order to elucidate the effect of C02 treatment of rats on energy charge values in their livers.Rats were anaesthetized with either Briethal (methohexital) alone or C02 + Briethal for 4-5 minutes or C02 + Mebumal (pentobarbital) for 15 minutes and energy charge values were determined in samples from whole liver and in liver celle nuclei, isolated by a non-aqueous method. C02 caused a significant decrease in energy charge which was detected after the short narcosis of 4—5 minutes. After 15 minutes the values had recovered. Energy charge was similar in whole tissue and cell nuclei

Andersson, M

Lewan, L

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Scand. J. Lab Animt Sci, 14.5rgt nr. 4 1987Effects of CO2 pre-treatment at narcosis on energy charge levels incell nuclei and whole tissue of rat liverby M. Andersson and L. Lewan,Department of Zoophysiology, University of Lund, Helgonavagen 3 B, 2233 62 Sweden.INTRODUCTIONCO2 can be used in rats and mice as an initialanaesthetic in order to facilitate handling andreduce stress before injections or other kinds oftreatment of short duration. However, its sui-tability may be reduced by interference withphysiological conditions like the energy statusof the tissue. It is well known from studies con—cerning concentrations of nucleotides and rela-ted compounds in rat liver that several factorsduring tissue sampling are of great importancefor the results. For example, the importance ofrapid freezing of the specimens in order toavoid hypoxia and enzymatic degradation ofthe molecules has been emphasized (Faupel etal. 1972, Eriksson 1980). The effects of stress(Ove et al. 1967, Faupel et al. 1972) and of dif-ferent anaesthetics on nucleotides and othercompounds (Faupel et al. 1972, Eriksson &Strdht 1981, Christensson & Eriksson 1985) ha-ve also been reported.The aim of the present study was to elucidatethe effect of C02 treatment of rats on energycharge levels in their livers. Short term effectsas well as a subsequent recovery during prolon-ged narcosis were investigated. As differentATP pools in nuclei and cytosol have been re-ported (Rapaport 1980) the possible role ofsuch a compartmentalization was taken intoaccount by separate analyses of cell nuclei iso-lated by a non-aqueous technique.MATERIALS AND METHODSChemicalsNucleotides for calibration were purchasedfrom Sigma Chemical Co, USA. Briethal (me-thohexital) was from Lilly, Switzerland, andMebumal (pentobarbital) from ACO, Sweden.Animals and narcosisTwelve male rats, Sprague Dawley (Anticimex,Sweden), weighing about 300 g and given foodand water ad libitum, were used. Three diffe-rent groups of four rats were studied. The firstgroup was anaesthetized with Briethal, 8mg/100 g BW intraperitoneally. With thisshort acting barbiturate the rats were complete-ly anaesthetized after 4-5 minutes, at which ti—me the livers were excised. Each rat 0f the se-cond group was initially anaesthetized in anair-tight cage into which CO2 was introduceduntil the animal was unconscious (about 20-35seconds). The rats were then removed from thecage and given 5 mg/ 100 g BW of Briethal i.p.The livers were excised 4-5 minutes after thestart of the C02 treatment. In the third groupthe animals were given CO2 as described, but inorder to obtain a prolonged narcosis, the sub—sequent anaesthetic was Mebumal (a longer ac-ting barbiturate) given i.p. 5 mg/100 g BW.The livers were excised 15 minutes after thestart of the C02 treatment. In a previous study(not published) we found no differences in thenucleotide profile between rats given Briethaland Mebumal narcosis respectively. Thus nogroup of rats receiving Mebumal only wasused.Tissue sampling.The livers were rapidly freeze-clamped with apair of tongs, precooled in liquid N2. The fro—zen livers were broken in liquid N2, lyophilizedin a vacuum below 10*1 atm. at —50° C for 48hours and stored at —80° C until further use.Isolation of nuclei.Isolation of nuclei was performed in anhy—drous glycerol according to Kirsch et al. (1970)with some modifications (Andersson et al.1987). The purity of the nuclear preparationswas judged from the RNA/DNA ratios (Sie-bert 1967 and 1968, Andersson et al. 1987) andby light microscopy.I57Chemical analyses.Extraction of the acid soluble fraction in coldperchloric acid and the subsequent neutraliza-tion with KOH was performed according toEriksson (1980). For the extraction from wholetissue, the frozen material was ground in amortar and homogenized in cold perchloricacid in an ice bath for about 1 minute using aPotter Elvehjem homogenizer. Extractionfrom nuclear fractions required no such homo-genization. The total extraction time was 20minutes for both kinds of preparations. RNAwas determined according to Munro and Fleck(1966) and DNA according to Scott et al.(1956) as modified by Hinrichs et a1. (1964).The separation of nucleotides was done byHPLC as described previously (Andersson eta1. 1987). The separation was carried out in aPolyanion S I HR 5/5 column (Pharmacia FineChemicals, Sweden) with a 10-1000 mMNH4H2PO4/(NH4) 2HP04 buffer at pH 6,5using a linear gradient during 15 minutes andan isocratic flow during 2 minutes with the highconcentration (Sjoberg & Kanje 1987).Scand. J. Lab. Anim. Sci. 14. arg. nr. 4 1987Calculations.The adenylate energy charge (ABC) was calcu-lated using adenosine phosphate values fromeach separation directly in the following for-mula:1ABC = ATP + /2ADP(ATP + ADP + AMP)The total amounts of adenosine phosphateswere determined in nmoles/mg DNA.RESULTS AND DISCUSSIONThe total amounts of adenosine phosphates ofwhole tissue were similar in the three groups,about 2300 nmoles/mg DNA. In the nuclei, thevalues decreased in an unlinear fashion in rela-tion to decreasing RNA/DNA ratios as descri-bed by Andersson et al. (1987). Energy chargevalues of cell nuclei and whole tissue are shownin Table 1. The energy charge in the first group(Briethal) was in agreement with previous fin-dings for barbiturates (Christensson & Eriks-son 1985, Andersson et al. 1987). After intro-ductory C02 treatment followed by shortTable l. Adenylate energy charge values of rat liver whole tissue and cell nucleiAnimal treatment Adenylate energy charge Group Narcosis n Whole tissue Cell nucleiNoBriethal1 4-5 minutes 4 O.782t0.041a 0.7651LO.057bC02 + Briethal2 4-5 minutes 4 0667:0020“ 0.67lir0.034-bC0; + Mebumal3 15 minutes 4 0.81lir0.015 0.804i'0.008a: p 5 0.0005b: p< 0.01Table values are mean values + S. D. in each group and are based onvalues of double samples from each rat.Statistics: Student’s t-test.159Scand. J. Lab. Anim, Sci. 14.5rg. nr. 4 1987anaesthesia (4—5 minutes) before tissue sam-pling, a significant decrease in energy chargewas found. When the CO2 treatment was follo-wed by a longer narcosis, however, the energystatus returned to normal before excision ofthe livers. Consequently C02, used as an initialanaesthetic, produces only short-lived effectson the energy status of the liver. All nuclearpreparations of the three different groups hadenergy charge values equal to their correspon-ding whole tissue preparations, although theRNA/DNA ratios were between 0.22 and 1.50,indicating nuclear fractions of various purity(Siebert 1967 and 1968, Andersson et al. 1987)Thus no compartmentalization between nucleiand cytoplasm was found regarding energycharge, which is in agreement with findings byOve et a1. (1967). However, the effects of a de—creased energy status in the cell may be more orless pronounced in different compartments dueto localization of various adenosine phosphatedependent functions (Jones 1984, Aw & Jones1982 and 1985). Furthermore, different adeno—sine phosphate compartments which may beshown by experiments using radioactive pre—cursors (Rapaport 1980), may not be revealedby a pure quantification. Nevertheless, our re-sults indicate that the use of C02 as an initialanaesthetic in order to facilitate the handlingof laboratory animals might be convenient inliver sampling except in cases where short termeffects must be avoided.Summary.C02 can be used in rats and mice as an initial anae-sthetic in order to facilitate handling and reducestress before injections or other kinds of treatment ofshort duration. Interference with physiological con-ditions like the energy status of the tissue may reduceits suitability. The present study was undertaken inorder to elucidate the effect of C02 treatment of ratson energy charge values in their livers.Rats were anaesthetized with either Briethal (metho-hexital) alone or C02 + Briethal for 4-5 minutes orC0; + Mebumal (pentobarbital) for 15 minutes andenergy charge values were determined in samplesfrom whole liver and in liver celle nuclei, isolated bya non—aqueous method. C02 caused a significantdecrease in energy charge which was detected afterthe short narcosis of 4—5 minutes. After 15 minutesthe values had recovered. Energy charge was similarin whole tissue and cell nuclei.160Sammtmdrag.C02 kan anvandas till rattor och mess som en inle-dande narkos fér att underlatta hantering och min—ska stress fore injektioner och andra slag av kortvari—ga behandlingar. Anvandbarheten kan dock min-skas genom att C02 kan tankas paverka fysiologiskaférhallanden Lex. vavnadens energitillstand. Dennaundersokning har gjorts for att belysa effekten avCOz-behandling av réttor pa energy charge varden ilevern.Réttor sdvdes med antingen enbart Briethal (metho—hexithal) eller C0; + Briethal 14—5 minuter eller C02Mebumal (pentobarbital) i 15 minuter, och energycharge bestamdes i prov frén hel lever och levercell-karnor, som isolerats med vattenfri teknik. C02 fer-orsakade en signifikant sankning av energy charge 4-5 minuter efter behandlingen. Efter 15 minuter hadevardena éterstallts. Vardena var lika i hela celler ochcellkarnor.theenvelo/K. PelkonenHiirille ja rotille voidaan kayttaa esinukutusaine-eksi hiilidioksidia helpottamaan kasittelya ja va-hentamaa6n stressiéi ennen injektioita tai muitalyhytaikaisia kasittelyjéi. Menetelman vaikutuksetfysiologisiin muuttujiin, kuten kudosten energizati-laan, saattavat va'hentaa' sen kayttokelpoisuutta.Tama'n tutkimuksen tarkoitus oli valottaa C02 —vaikutusta rotan maksan energiatilaan.Rottia anestetisoitiin joko Brietalilla (methohexi-tal) yksin tai C02 + Brietalilla tai C02 + Mebu-malilla (pentobarbital) 15 minuutin ajaksi ja ener—giatasot mitattiin koko maksataa ja maksasolujentumista, jotka oli eristetty vedettomalla menetel-malla. C02 aiheutti merkittéivéin energiatason la-skun, joka oli havaittavissa lyhyen 4—5 minuuttiakestava'n narkoosin ja'lkeen. 15 m'inuutin kuluttuaarvot olivat palautuneet. Energiavaraus oli yhden-mukainen koko kudoksessa ja tumissa.ReferencesAndersson, M., P. 1. Christensson, L. Lewan & U.Stenram: Isotachophoretic and HPLC determi—nation of nucleotides in rat liver cell nuclei isola-ted by non-aqueous technique. Int. J. Biochem.1987, 19(7), 641-647.Aw, T. Y. & D. P. Jones: Secondary bioenergetic hy—poxia. Inhibition of sulfation and glucuronida-tion reactions in isolated hepatocytes at low 02concentration. J. Biol. Chem. 1982, 257 (15),8997-9004.Aw, T. Y. & D. P. Jones: ATP concentration gra-dients in cytosol of liver cells during hypoxia.Am. J. Physiol. 1985, 249 (18), C 385-392.Christensson, P. I. & G. Eriksson: Effects of sixanaesthetic agents on UDP—glucuronic acid andother nucleotides in rat liver. Acta Anaesth.Scand. 1985, 29. 629—631.Eriksson, G: A method for measuring nucleotides inthe liver of rat with isotachophoresis. Analyt.Biochem. 1980, 109, 239-249.Eriksson, G. & D. Strdth: Decreased UDP-glucuro—nic acid in rat liver after ether narcosis. An isota—chophoretic study. FEBSLett. 1981, 124, (1), 39-42.Faupel, R. P., H. J. Seitz, W. Tarnovski, V. Thie-mann & C. Weiss: The problem of tissue sam-pling from experimental animals with respect tofreezing technique, anoxia, stress and narcosis.Arch. Biochem. Biophys. 1972, 148, 509-522.Hinrichs, H. R., R. O. Petersen & R. Baserga: Incor-poration of thymidine into DNA of mouse or—gans. Arch. Pathol. 1964, 78, 245-253.Jones, D. P.: Benzylamine metabolism at low 02concentrations. Relative sensitivities of mono—amineoxidase, aldehyde dehydrogenase and hip—purate synthesis to hypoxia. Biochem. Pharma-col. 1984, 33, (3), 413-417.Kirsch, W. M., J. W. Leitner, M. Gainey, D.Schultz, R. Lasher & P. Nakane: Bulk isolationin non—aqueous media of nuclei from lyophilizedcells. Science 1970, 168, 1592-1595.Scand. J. Lab. Anim. Sci. l4.éirg.nr,41987Munro, H. W. & . Fleck: Recent developments in themeasurement of nucleic acids in biological mate-rials. Analyst 1966, 91, 78-88.Ove, T., S. Takai, T. Umeda & I. Lieberman: Ade-nosine triphosphate in liver after partial hepatec-tomy and acute stress. J. Biol. Chem. 1967, 242,No 21, 4963-4971.Rapaport, E.: Compartmentalized ATP pools pro—duced from adenosine are nuclear pools. J. Cell.Physiol. 1980, 105, 267-274.Scott, J. F. A. P. Fraccastoro & E. B. Taft: Studiesin histochemistry 1. Determination of nucleicacids in microgram amounts of tissue. J. Histo-chem. Cytochem. 1956, 4, 1-10.Siebert, 0.: Isolation in non-aqueous media (Beh-rens principle) Meth. Cancer Res. 1967, 3, 287-301.Siebert, G.: Nucleus. In: Comprehensive bioehemi—stry, Ed. by J . Florkin and E. Stoltz, Elsevier.Amsterdam. 1968,23, 1-37.Sjoberg, J. & M. Kanje: Incororation of (32P)phos-phate into nucleotides of the dorsal root ganglia0f regenerating rat sciatic nerve. Brain Res. 1987,415, 270-274.161

Effects of CO2 pre-treatment at narcosis on energy charge levels in cell nuclei and whole tissue of rat liver

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Effects of CO2 pre-treatment at narcosis on energy charge levels in cell nuclei and whole tissue of rat liver

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