Opposite transitions of chick brain catalytically active cytosolic creatine kinase isoenzymes during development

Int. J. Devl Neuroscience 18 (2000) 815 – 823 www.elsevier.com/locate/ijdevneu

Opposite transitions of chick brain catalytically active cytosolic creatine kinase isoenzymes during development Oscar Ramı´rez *, Esperanza Jime´nez Departamento de Bioquı´mica, Centro de In6estigacio´n y de Estudios A6anzados del Instituto Polite´cnico Nacional, A6. Instituto Polite´cnico Nacional, 2508, Mexico, D.F. 07340, Mexico Received 9 August 1999; received in revised form 12 June 2000; accepted 13 June 2000

Abstract Postnatally the rat brain synthesizes catalytic forms of muscle type (MM) and heart type (MB) creatine kinase (CK), besides the supposedly sole type vertebrate brain-specific (BB) CK. We intended to demonstrate that in Rhode Island chicken brain, cytosolic (c) CK isoenzymatic transitions, (for example BB – CK is followed by the appearance of MB – CK and MM – CK during muscle differentiation), can also occur during development and aging. Cytosolic post 125 000 ×g, mitochondrial CK-free, brain samples were obtained for zone electrophoresis separation and identification of catalytically active cCK isoforms. BB –CK was never found during chicken brain ontogeny. Against the accepted view, an opposite isoenzyme transition pattern from MM through BB–CK was found in the chicken embryonic brain from the very early stages of development up to day 2 post-hatching. At very early stages of chicken brain ontogeny constitutive MM – and MB – CK isoenzymes were present before the advent of creatine. It seems to be that typical and atypical brain MM – and MB – CK could be working as ATPases in the absence of creatine before embryonic stage 28 (day 5.5) and/or such CK isoforms may begin to form part of the slow component b in developing early neurons and later in the nuclei of glial cells to be used by the CK/phosphocreatine (PC) system as the neural tissues mature. The post-hatching transition pattern showed simultaneous expression of more than one CK isoenzyme within the same neural sample as in post-natal rat brain, presumably due to regional differential transphosphorylation requirements. Strain-dependent enzymatic specific activities have been reported in several species. Since equivalent values of brain CK specific activity were obtained previously from the embryonic plateau phase of CK activity during White Leghorn development, and those from Rhode Island brain neurons cultured 11 days, we compared if, in vivo, a similar brain CK specific activity pattern was physiologically equivalent during Rhode Island and White Leghorn chicken ontogeny. We found quantitatively different strain-specific CK specific activity patterns during this period. Rhode Island brain CK activity values were approximately 4.5-fold those of White Leghorn ones. This indicates that production of energy from anaerobic metabolism and transphosphorylation by the CK/PC system to synthesize ATP more efficiently is strain-specific. In Rhode Islands, there was an age-dependent increase of CK specific activity, mostly in older animals (440% above the value found during the embryonic plateau), when the Krebs cycle and glycolysis lose capacity. During adult life and aging, under physiological conditions, the three CK isoenzymes may participate in diverse functions of the different cell compartments of brain glia and neurons with regard to their high and fluctuating energy demands that are not completely covered by anaerobic and aerobic glycolisis. © 2000 ISDN. Published by Elsevier Science Ltd. All rights reserved. Keywords: CK specific activity; CK constitutivity; Neural development; Neural aging; Chicken embryo brain; CK isoenzyme opposite transitions

1. Introduction Abbre6iations: AMP, adenosine 5%-monophosphate; BB–CK, brain type CK; C, creatine; CK, creatine kinase; MB–CK, heart type CK; MM–CK, skeletal muscle type CK; PC, phosphocreatine; NADH, nicotinamide adenine dinucleotide, reduced form; NADP+, nicotinamide adenine dinucleotide phosphate, oxidized form. * Corresponding author. Tel.: +52-5-7473800, ext. 5222; fax: + 52-5-7477083. E-mail address: [email protected] (O. Ramı´rez).

Only in neuromuscular tissues is creatine kinase, [adenosine 5%-triphosphate (ATP)-creatine N-phosphotransferase, EC 2.7.3.2, commonly abbreviated CK], found in high concentrations [46]. Several isoforms of the enzyme have been described in the different cell compartments of tissues with high and fluctuating en-

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ergy demands [14,51]. Experimental data suggest that CK is located near the sites where force generation by motor proteins, ion transport across membranes by ion pumps and other ATP-dependent processes take place. The CK/phosphocreatine (PC) system [2] seems to play a complex multi-faceted role in neuromuscular energy homeostasis. Even though the cellular and skeletal muscle syncytial pools of ATP are rather small (2–5 mM) [19], no significant change in overall ATP levels can be detected during activation of excitable tissues because ATP is efficiently replenished from the large pool(s) of PC through the reaction catalyzed by CK: MgADP− + PC2 − + H+ ? MgATP2 − +creatine (C) In vertebrate tissues, four to five subunit isoforms of CK have been found, an octamere/dimer ratio of mitochondrial CK (Mi – CK) influenced by substrates, pH, ionic strength, and protein content [20,51], and three cytosolic CK (cCK) isoforms [5]. Owing to its dimeric structure, the cCK exists as three main isoenzymes, skeletal muscle (MM), heart (MB), and brain (BB) [7,12]. From ontogeny of heart and skeletal muscles, it is believed that cCK isoenzyme transition is similar among vertebrates. As in skeletal muscle cultures, during differentiation, the appearance of BB – CK is followed by the MB – CK isoenzyme and concludes with the adult MM – CK isoenzyme [11,49]. It is currently accepted that BB – CK is virtually the only isoform present in brain, (tissue-specificity), during the entire life of vertebrates, although a faint trace of MM–CK may appear in older rats [11,34], and in adult human brain [23]. Recently this was confirmed in human brain by means of the polymerase chain reaction [15]. On the other hand, the ontogeny of brain CK activity has been studied in White Leghorn chicks during embryonic development, where the specific activity increased 170% in 10 h from the 2nd to the 3rd day of incubation. This was followed by a plateau phase throughout development and at the end of incubation there was another increase of cytosolic and mitochondrial CK activities. Separation of cCK isoforms was not done since it was assumed that in chicken brain BB – CK remained the sole isoenzyme type [39]. However, evidence was presented by Ramı´rez and Licea [40] showing that, as in muscle tissues, post-natal rat brain synthesizes catalytic forms of MM–CK and MB–CK in addition to the BB – CK brain type in quantities that permit the isoenzymes to be visualized by the nicotinamide adenine dinucleotide phosphate (NADP+) coupled reactions technique. Therefore, as a working hypothesis, it is possible that cCK isoenzymatic transitions may also occur during chicken brain ontogeny and after hatching. In this case, from an enzyme – substrate relationship, it

would be important to correlate the appearance of the different catalytical brain CK isoforms with that of creatine during physiological chicken development. There is accumulated evidence of strain-specificity of enzyme activity in some avian (CK in turkeys) [18], and mammalian tissues, in vivo [8], and in vitro [17]. In pharmacogenetics of centrally acting drugs, there is a large body of evidence regarding the differential sensitivity of different species or strains of animals to drugs such as d-amphetamine, iproniazid, chlorpromazine and nicotine [3]. However, contrary to findings of strain-dependency of different enzymatic specific activities, we have previously found equivalent values of brain CK specific activity from the embryonic plateau phase of CK activity during ontogeny of White Leghorn chicks [39], and Rhode Island chick brain neurons in culture [41]. Therefore, we decided to determine whether or not brain CK specific activity patterns were similar during ontogeny of Rhode Island and White Leghorn chicks in ovo. In the present study it is shown that before hatching quantitatively different strain-specific brain CK specific activity patterns occur. There is also an agedependent increase in CK specific activity during aging. There appear to be ontogenetic transitions of chicken brain cCK isoenzymes which follow a transition pattern in the absence and presence of creatine contrary to what has been previously reported.

2. Experimental procedures

2.1. Incubation conditions Varieties of White Leghorn and Rhode Island chicken (Gallus domesticus) eggs (Armour Hatchery de Me´xico, S.A.) were stored at 18°C after they were laid. They were used within 10 days. Incubation was carried out at 37.5°C in a Jamesway machine with controlled humidity.

2.2. Animals and experimental conditions Living embryos were obtained to be compared with the standard chicken embryonic plates from Hamburger and Hamilton [16]. Every embryo was classified at early stages under the stereomicroscope, considering the main specific features appearing in each reference plate at the following stages beginning at stage 9 (St 9), before liver appearance where creatine biosynthesis occurs, White Leghorn at St 9, n= 4; St 11.5, n =5; St 12, n= 4; St 12.5, n= 4; St 14.5, n=5; St 20, n= 4; St 23.5, n = 4; St 31, n=4; St 36, n= 5; St 43, n= 7; St 46, n=7. Rhode Island embryos were obtained at St 12,

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n= 5; St 14, n=6; St 20, n =6; St 23.5, n = 6; St 30.5, n =9; St 40, n=10; St 46, n =6; at P2 (post-hatching day 2), n = 6; P7, n = 4; P120, n = 4; P240, n = 6, and P547, n = 5. The number of brains utilized in every CK determination appears in parentheses in Section 3.

2.3. Tissue dissection Systematically, glass needles were used to remove the beating embryo heart to avoid myocardial CK contamination before neural structures were dissected. Developing brain was taken by transection above the auditory pit (or vesicle) during the very early embryonic stages and above the first cervical ganglion thereafter. As much as possible, the eye structure or cranial nerves were not included. The brain was carefully transferred through a series of Ringer and/or 145 mM NaCl solutions at 0–4°C, until blood or extraneous loose tissue had disappeared.

2.4. Biochemical procedures 2.4.1. CK assay and protein determination Homogenization of brain tissue was carried out in cold 100 mM KCl using a ground glass type Potter– Elvejhem homogenizer immersed in an ice box. At least two homogenizations of 30-s duration were performed at approximately 1000 mv/min − 1 with an electrically driven Teflon pestle. A sample of the homogenate was routinely observed under the microscope to assure that all cells were disrupted. The suspension was then centrifuged for 30 min at 27 000 ×g, in a Sorvall DC-2B machine at 4°C. The pellet containing heavy and light mitochondria was discarded and the supernatant was used for the CK assay [39]. The decrement of NADH absorbance at 340-nm was registered in a PMQ II Zeiss spectrophotometer. Protein was determined by a conventional assay [24]. The supernatants were further centrifuged 60 min at 125 000× g at 4°C in an OTD65B Sorvall ultracentrifuge, the microsomal pellet discarded, and the cytosol was used immediately for the cCK isoenzymatic separation and identification [40], because preparations cold stored for more than 24-h can produce spurious CK dimeric hybrids. The presence of contaminating Mi – CK in post-116 000× g supernatants has been excluded as determined by protein immunoblot analysis [26]. 2.4.2. Separation and identification of CK isoenzymes Zone electrophoresis on cellulose polyacetate strips, (Sepraphore III from Gelman Sciences Inc. Ann Arbor, MI, USA), was carried out at 250 V using 60 mM sodium barbital buffer, pH 8.6, containing 10 mM 2-mercaptoethanol [33,49]. Samples of the post125 000×g supernatant containing 1 – 5× 10 − 3 U of CK (1 mmol NADH oxidized per min) in 1 – 5 ml were

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applied to the strips on the marked origin. After electrophoresis for 120 min, the strips were stained for CK using overlay reaction gels containing hexokinase and glucose-6-phosphate dehydrogenase as the coupling enzymes in the forward reaction for CK at pH 7.2 [40,49]. To exclude interference by co-migration with other enzymes or nonspecific staining, AMP was used to inhibit adenylate kinase activity [47], and appropriate blanks were prepared from each sample. Treatment of these controls was identical to the experimental samples, except that PC was omitted from the incubation mixture. The chemical reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

3. Results

3.1. Brain CK specific acti6ity Previously, it has been shown that the earliest chicken CK activity is present in the embryonic heart from 27 h of incubation or about St 8.5 [10]. Also, it has been demonstrated that embryonic heart cannot synthesize creatine [36], and the egg contains neither creatine nor the machinery for its biosynthesis [37]. In fowl, the whole creatine pathway is found in liver [50]. The hepatic primordium appears in the chick embryo at the end of the 2nd day of incubation (about St 12) [43], and for creatine biosynthesis, liver glycine amidinotransferase (EC 2.1.4.1) activity is reliably assayed only after day 4 of incubation (about St 23) [38]. PC has not been detected in whole chick embryos before day 6 [39]. Brain CK activity in White Leghorn chick embryos was first found from St 11.5 of Hamburger and Hamilton [16] or after about 42-h incubation. That was the earliest stage at which brain CK activity could be detected based upon the first reliable significant absorbance values in the spectrophotometric recordings, since they were not present at St 9 in spite of the large number (45) of embryonic brains analyzed per experiment. In Fig. 1, brain CK specific activity curves from Rhode Island and White Leghorn strains are compared. At St 14.5, CK specific activity of both strains clearly reached a plateau phase, which persisted until St 40. From this stage onward in Rhode Island chick embryos, and at St 43 in White Leghorn embryos, an increase in brain CK specific activity occurred lasting until hatching at day 21 (St 46). It is worth mentioning that brain CK activity values in Rhode Island chick embryos were approximately 4.5-fold those of White Leghorns although the same CK assay was employed in both strains. Rhode Island brain CK specific activity value at P240 (post-hatching day 240 or month 8), was similar to the values found at P2, P7 and P120 ($D350 nmol PC per min/mg prot.), whereas four-fold more brain CK specific activity was

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found in hens at 1.5-year-old (P547), the highest for the Rhode Island strain.

3.2. Separation and identification of brain CK isoenzymes In order to classify chick brain catalytical cCK isoforms by zone electrophoresis, as was originally done [11,49], it was necessary to screen a large number of cytosolic post-125 000 × g samples from chick brain, heart, and skeletal muscle. According to the migration distance, in cm from the origin, the distribution pattern of chicken catalytical cCK isoforms is shown in Fig. 2. Besides, the typical migrating chicken cCK isoenzymes found in brain, heart and skeletal muscle, several atypical ones were also found. They were tentatively named with superscripts after the original dimer. The closest to the origin and most catodic of all cytosolic isoforms (non Mi–CK) was considered to be as the MM¦ –CK isoenzyme. The ones appearing between the typical adult MM– and MB – CK isoenzymes were classified as MM% –CK. In a similar manner, the CK isoforms appearing between the typical adult MB – and BB–CK isoenzymes were classified as MB% – CK. Their corresponding range of migration is indicated in Fig. 3.

Contrary to the current view that brain tissue does not show isoenzymatic transitions from BB–CK during the development of the Rhode Island chick it was found that transitions of brain cCK isoenzymes occur during pre- and post-hatching neural differentiation (Figs. 3 and 4). Remarkably, the pre-hatching pattern was practically opposite to the universally accepted one for rat and chicken heart and skeletal muscles (BB–CK isoenzyme followed by MB–CK isoform to conclude with MM–CK ‘mature’ isoenzyme). In Figs. 3 and 4, it is shown that typical BB–CK was never present during chicken brain ontogeny. It is also shown that in the absence of creatine at St 12 and 14 (from about 46 h of incubation), developing chicken brain initiated with typical MM– and MB–CK as well as with atypical MB% –CK isoforms (more anodic than the standard MB–CK isoenzyme). With creatine in brain, the atypical MB% –CK type was presented from the St 30–40. Immediately after hatching CK isoforms migrating within the range of MB% and BB were expressed. Hens at about P240 (month 8) and P547 (1.5 years of post-hatching) did show the presence of several typical and atypical variants of the cCK isoforms within the same brain tissue sample. With about 40% more brain CK specific activity, as compared with the

Fig. 1. Comparative development of brain cCK activity from Rhode Island and White Leghorn chicken strains. Specific activity is the ordinate. Upper abscissa indicates embryonic stages according to Hamburger and Hamilton [15], whereas lower abscissa indicates pre- and post-hatching days of development and aging. Arrows along the lower abscissa indicate three important embryonic events, (a) the first detection of heart CK activity; (b) the appearance of the hepatic primordium; and (c) the first determination of creatine in the embryo. () Earliest embryonic heart CK activity value [9]. CK specific activity data shown are the mean 9 S.D. of the experiments performed in White Leghorn (open circles); St 9, n = 4; St 11.5, n = 5; St 12, n =4; St 12.5, n= 4; St 14.5, n = 5; St 20, n =4; St 23.5, n =4; St 31, n =4; St 36, n = 5; St 43, n = 7; St 46, n = 7; and Rhode Island (filled circles), St 12, n= 5; St 14, n= 6; St 20, n= 6; St 23.5, n = 6; St 30.5, n = 9; St 40, n = 10; St 46, n = 6; P2, n =6; P7, n = 4; P120, n =4; P240, n =6; and P547, n= 5. The number of brains utilized in every CK determination appears in parentheses.

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atypical muscular MM¦ –CK isoforms were found at about P547, coinciding with the highest brain CK specific activity ever measured in Rhode Island chicks.

4. Discussion

4.1. Brain CK strain-specificity

Fig. 2. Representative samples of all typical and atypical cCK isoenzymes that can be presented in Rhode Island chicken neuromuscular tissues. The atypical catalytically active cCK isoforms were tentatively named with superscripts after the original dimer. O, origin; MD, migration distance from O in cm.

embryonic plateau, BB – CK was present at P2, whereas typical and atypical (more anodic) MB – CK isoforms were found at P7 and P120. Typical BB – and MB–CK isoenzymes, as well as atypical MB%-, MM%-, and the most catodic of all isoforms, MM¦ – CK were present at about P240. Finally, typical BB – CK isoenzyme and the

In order to analyze animal findings properly, the difference in chicken strains must be stressed. The data shown in Fig. 1 demonstrate that regarding brain CK specific activity, Rhode Island chicks were more than four-fold better endowed than their White Leghorn counterparts throughout the embryonic period to play the anaerobic and complex role in the neuromuscular energy homeostasis by means of the CK/PC system. Our working hypothesis, namely that the expected strain-dependent differences in brain CK specific activities is supported by these data. This is consistent with recent findings regarding strain-dependendent specificity of enzyme activity among species [8,17,18]. From an enzyme–substrate relationship, it is evident in both strains of chicks, that brain catalytical CK was synthesized as a constitutive enzyme during critical early periods of neuroblast multiplication (St 11.5–12.5) and initiation of glial proliferation [9], since creatine is not present in the whole embryo until about stage 23 ( $4 day of incubation) [38], and the chick embryo does not receive creatine or PC from the egg, i.e. the yolk sac or albumen [37]. Reported values of brain PC during chicken embryo development [44], plotted semi logarithmically, do show that extrapolation of the first PC slope to the earliest stage of development supports the idea that brain PC begins to be detected shortly after the 6th day of incubation [39]. This finding fits well with the fact that no PC was present in whole embryo before St 28–29 (day 6 of incubation) [37]. So, any type of catalytical CK present in developing chick tissues before St 28–29, must be considered as non-functional in spite of the availability of creatine since St 23 (day 4 of incubation), in the whole embryo . In the absence of a rapid increase in brain mass before hatching [39], increases in brain PC as well as brain Mi–CK and CK specific activities are likely due to an increased energy demand associated with neuronal and glial maturation, and increased neuromuscular activity of the precocious chick. Equivalent findings on CK and adenylate kinase increased activities associated with neuronal maturation in altricial mammals such as the mouse, occur during the 1st month after birth [21]. Manos et al. have suggested a role for CK in myelinogenesis from the studies in cultured rat oligodendrocytes [26].

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Brain CK specific activity at hatching of Rhode Island chicks was about 44% higher than that found during the embryonic plateau phase. However, it underwent a dramatic increase of about 440% in 1.5-yearold hens. Decreasing blood supply to the brain of senescent birds diminishes oxygen for aerobic metabolism, thus affecting the efficiency of ATP generation. Also, lactate and H+ cannot escape rapidly from the neural tissue into the blood stream for better buffering capacity based on the hydrogen carbonate ion. All of this leads to the production of energy from anaerobic metabolism and transphosphorylation by the CK/ PC system that utilizes H+ to synthesize ATP. It is also known that the Krebs cycle and glycolysis lose capacity with increasing age and with a chronic decrease in physical activity [31]. It has been postulated that the error frequency in protein biosynthesis rises with the age of the animal to the point that the resulting protein molecules may be rendered less active or even inactive. This hypothesis has been confirmed. Although, the total amount of immunologically detectable aldolase remained unchanged in the muscles of aged animals, the isolated aldolase was only one-third as active cata-

lytically as aldolase isolated from the young animals [22]. Previous study has also shown that large quantities of antigenically CK isoforms are catalytically inactive and can be separated from the CK catalytical ones [1]. So, it seems that ATP must be generated vicariously from phosphagen by means of the active CK/PC system in the brain of 1.5-year-old hens.

4.2. Brain CK isoenzyme transitions in the chick Regarding the electrophoretic results, the most striking findings shown in Fig. 3 confirm our working hypothesis that more than one catalytical variant of cCK isoforms appears to be present in the chick brain during the ontogenetic period as well as in adulthood and old age (Fig. 4). The current view predicts that BB–CK must be the quantitatively predominant, if not the only, isoform present in brain during the entire life of vertebrates, assuming that the BB–CK isoform is quasi-tissue-specific marker [11,34]. During avian neural ontogeny, it seems that transphosphorylation can cope with the high-energy needs for neuronal requirements, and perhaps for glial

Fig. 3. Transitions of chicken brain cCK isoenzymes during ontogeny, early post-hatching development, adulthood and senescence. Arrows along the lower abscissa indicate three embryonic events, (a) the appearance of the hepatic primordium; (b) the first specific determination of creatine in the embryo; and (c) the first detection of PC in chicken embryonic brain. The right ordinate indicates migration distance and range of typical and atypical cCK isoenzymes. Each experimental point represents the mean 9S.D. of migration distance. The number of pooled (St 12–40) and individual chicken brains that showed a particular cCK isoenzyme appears in parentheses; St 12, n =5; St 14, n = 6; St 30, n =6; St 40, n= 5; St 46, n = 6; P2, n=4; P7, n= 4; P120, n= 4; P240, n= 4, and P547, n =5. Some hens had more than one CK isoenzyme band. Notice that contrarily to the common view that brain tissue does not show cCK isoenzymatic transitions from BB – CK, during the development of Rhode Island chicken, it can be seen that transitions of brain cCK isoenzymes occur during pre- and post-hatching neural differentiation. The pre-hatching pattern was practically opposite to the universally accepted one for rat and chicken heart and skeletal muscles.

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Fig. 4. Chicken brain cCK isoenzymes found along development and at post-hatching ages. Notice that catalytic MM and MB – CK dimers were not faint traces in the chicken neural tissue. More than one CK isoenzyme can be present within the same sample of adult chicken brain. Three different cCK isoenzymes were found in distinct samples of pooled embryonic brains at St 14. O is the origin and MD is migration distance in cm.

proliferation and myelin synthesis during development [26,27]. Since the CK isoenzymatic pattern (Fig. 3) is practically opposite to the one found in heart and skeletal muscles of mammalian and avian origin in vivo and in vitro [11,49], it must be stressed that the earlier presence of brain MM – and MB – CK isoenzymes occurs in the absence of creatine up to St 23 (about day 4 of incubation), as shown in Fig. 3. If functional, these muscle CK isoenzymes would form ATP less actively

since both show larger Kms, for PC and ADP, as compared with BB–CK [30]. Regarding the atypical MB% –CK isoenzyme it seems to be associated with the sudden increase of brain PC content up to St 44 (day 18). The most anodic MB% –CK isoform present at hatching coincided with a large increase in CK specific activity. Until proven otherwise, atypical MB–CK isoenzymes could show smaller Kms, for PC and ADP since they are more anodic than the typical MB–CK

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(Fig. 2). The atypical MB – CK variants observed at stage 14, are perhaps phosphorylated by protein kinase C in the B monomer [6]. The presence of atypical CK isoforms may arise by protein post-translational modification(s) of the enzyme [6,35], as well as by differential splicing of an unique CK gene [52], or by differences in the transcription starting point of the CK promoter [28,29], and from alternative ribosomal initiation [48]. The simultaneous presence of more than one cCK isoenzyme could be associated with increased local energy demands not completely covered by aerobic and anaerobic glycolisis. ATP can be regionally synthesized by the CK/PC system [14,27], at different speeds depending on the Kms, for PC and ADP of the several CK isoenzymes (Fig. 2). The catalytical MM and MB – CK isoforms, physiologically present before St 28 – 29, must be considered as operationally constitutive since they do not participate in PC biosynthesis in any chicken organ. This fact poses a question regarding the catalytical role of CK as a transphosphorylase. Speculatively, two alternatives seem to be possible, (a) typical and atypical brain MM and MB–CK as dual isoforms may be working as ATPases in the absence of creatine before St 28 (day 5.5) [45], and (b) such cCK isoforms being synthesized before the advent of creatine begin to form part of complex structures, such as the slow component b [4,32] in developing early neurons and later in glial cells to be used in the CK/PC system as the neural tissues mature [27]. Lyons et al. [25] have not found MCK mRNAs in chicken embryonic muscle before St 19. In the brain, it is likely that CMD1, the chicken homologue of MyoD1 is expressed at St 13, and myogenin, first detected at St 15 in chicken embryonic muscle, can also be present in the chicken embryonic brain at comparable earlier stages. It has been proposed that the myogenic factors CMD1 and myogenin directly regulate MCK gene expression by binding to its enhancer. However, these factors by themselves seem to be not sufficient to initiate MCK gene expression in muscle at comparable chicken stages. Moreover, it has been suggested that a 4.8-kb MCK genic upstream region is sufficient to modulate MCK mRNA expression in certain neural cells and may be regulated independently from other muscle genes [15]. Estrogen can induce BB – CK synthesis in rat uterus [42,53]. Steroids are synthesized in the chicken embryo not earlier than St 23 – 24 [13]. However, in the absence of steroids at early stage 14 during ontogeny, there is the expression of BCK containing isoforms. Under physiological conditions, the avian neural tissues are capable of expressing simultaneously both types of MCK and BCK polypeptide-containing isoenzymes. The three cCK typical isoenzymes and their

variants may participate in the diverse functions of the different cell compartments of brain glial and neuronal cells, given their high and fluctuating energy demands not completely covered by anaerobic and aerobic glycolysis. Acknowledgements We thank Ana Marı´a Cibria´n and Jorge Martı´nez for excellent technical assistance and Gloria E. Arroyo for typing the manuscript. References [1] Armstrong, J. B., Lowden, J. A. and Sherwin, A. L., Brain isoenzyme of creatine kinase II. Species specificity of enzyme and presence of inactive form in striated muscle of rabbit and man. J. Biol. Chem., 1977, 252, 3112 – 3116. [2] Bessman, S. P. and Carpenter, C. L., The creatine –creatine phosphate energy shuttle. Ann. Re6 Biochem., 1985, 54, 831–862. [3] Bovet, D., Nature, nurture and the psychological approach to learning. In Brain and Human Beha6ior, eds A. G. Karczmar and J. C. Eccles, Springer, Berlin, 1972, pp. 335 – 336. [4] Brady, S. T. and Lasek, R. J., Nerve-specific enolase and creatine phosphokinase in axonal transport: soluble proteins and the axoplasmic matrix. Cell, 1981, 23, 515 – 523. [5] Burger, A., Richterich, P. and Aebi, H., Die Heterogenita¨t der kreatine – kinase. Biochem. Z., 1964, 339, 305 – 314. [6] Chida, K., Tsunenaga, M., Kasahara, K., Kohno, Y. and Kuroki, T., Regulation of creatine phosphokinase B activity by protein kinase C. Biochem. Biophys. Res. Commun., 1990, 173, 346 – 350. [7] Dawson, D. M., Eppenberger, H. M. and Kaplan, N. O., The comparative enzymology of creatine kinase. II physical and chemical properties. J. Biol. Chem., 1967, 242, 210 – 217. [8] Dimai, H. P., Linkhart, T. A., Linkhart, S. G., Donahue, L. R., Beamer, W. G., Rosen, C. J., Farley, J. R. and Baylink, D. J., Alkaline phosphatase levels and osteoprogenitor cell numbers suggest bone formation may contribute to peak bone density differences between two inbred strains of mice. Bone, 1998, 22, 211 – 216. [9] Dobbing, J. and Sands, J., Timing of neuroblast multiplication in developing human brain. Nature, 1970, 226, 639 – 640. [10] Eisenberg, S. and Ramirez, J., The role of phosphotransferases in the respiratory control of the embryonic heart. J. Physiol. London, 1963, 169, 799 – 815. [11] Eppenberger, H. M., Eppenberger, M., Richterich, R. and Aebi, H., The ontogeny of creatine kinase isoenzymes. De6. Biol., 1964, 10, 1 – 16. [12] Eppenberger, H. M., Dawson, D. M. and Kaplan, N. O., The comparative enzymology of creatine kinase. I. Isolation and characterization from chicken and rabbit tissues. J. Biol. Chem., 1967, 242, 204 – 209. [13] Freeman, B. M. and Vince, M. A., De6elopment of the A6ian Embryo, Chapman & Hill, London, 1974, pp. 228 – 230. [14] Friedman, D. L. and Roberts, R., Compartmentation of B-type creatine kinase and ubiquitous mitochondial CK in neurons. Evidence for a creatine phosphate energy shuttle in adult rat brain. J. Comp. Neurol., 1994, 343, 500 – 511. [15] Hamburg, R. J., Friedman, D. L., Olson, E. N., Ma, T. S., Cortez, M. D., Goodman, C., Puleo, P. R. and Perryman, B., Muscle creatine kinase isoenzyme expression in adult human brain. J. Biol. Chem., 1990, 265, 6403 – 6409.

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