Carotenoids in oxidative metabolism of molluscoid neurons

Experimental

CAROTENOIDS

Cell Research 64 (1971) 301-306

IN OXIDATIVE MOLLUSCOID

METABOLISM

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NEURONS

V. N. KARNAUKHOV Laboratory

of Biophysics

of Living Structure, Institute of Biophysics, USSR, Puschino, Moscow Region, USSR

Academy

of Sciences

SUMMARY 1. The absorption and fluorescent spectra of different parts of living mollusc Lymnaea stagnalis giant neurons have been investigated. The existence of a mitochondrial cytochrome c/cytochromoxidase system was proved. 2. The changes of absorption spectra under the action of respiratory inhibitors (amytal, KCN) suggest that the absorption bands at I = 465 and 495 nm are due to carotenoids which participate in oxidative metabolism. 3. The extraction and chemical analysis of the “yellow pigment”, with absorption bands at I = 465 and 495 nm in the neurons, confirmed the carotenoid nature of the pigment. 4. Comparative microspectrophotometrical and electronmicroscopical investigations of the cells showed that the carotenoids, in common with myoglobin, are localized in specific cytoplasmic granules - cytosomes. 5. It is suggested that the carotenoids, which possess many conjugated unsaturated double bonds, in common with myoglobin, provide the intracellular stock of oxygen.

The interrelation between exergonic meta- siological role of the carotenoids in animal bolic oxidation-reduction reactions and en- cells [ 121. In this paper some spectrophotometrical dergonic functional mechanismsof the single evidence of the participation of carotenoids living cell is one of the most real and difficult problems. Close correlation between the in the oxidative metabolism of the mollusc oxidized/reduced ratio of mitochondrial re- Lymnaea stagnalis neurons are presented. spiratory enzymes and their functional states These carotenoids, in common with myo[l], as well as the existence of characteristic globin and unsaturated fatty acids, are loabsorption and fluorescent spectra of the calized in specific cytoplasmic granules. enzymes, allow the use of spectrophotometric techniques to solve the problem [2-71. MATERIAL AND METHODS It is most difficult, in this study, to distinguish flavoproteins from carotenoids [8, Giant neurons of the gastropod mollusc Lymnaea 91 because of the similarity of their absorp- stagnalis were investigated. The size of the cells ran up to 200 or 300 pm in diameter. tion spectra in the visible region. Moreover, The massive stock of glycogen allows the neurons ganglia to retain their electrical acflavoprotein fluorescent spectra [lo] are intivitytheforisolated several days [13]. The absence of haemosimilar to the fluorescent spectra of some globin which interferes with cytochrome spectra is a carotenoids [l 11.These factors have hindered, considerable advantage of Lymnaea stagnalis cells used for spectrophotometric investigation of respirso far, a complete understanding of the phy- atory chains [7]. Exptl

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A modified commercial doublebeam differential microspectrophotometer MY@-5 was used to measure the absorption and scattering spectra of different areas of the cell [4, 5, 13, 141. The areas were 4 ,um in diameter. The construction of the perfusion chamber was adapted to investigations of the action of oxidation inhibitors and substrates on absorption spectra of the cells. A highly sensitive microspectrofluorimeter for measurements in the visible regions (500-700 nm) [15] was used for investigations of self-fluorescence of the living neurons [6]. The dimensions of measured parts of the cell were 3 x 10 ,um. The perfusion chamber was also used. The excitation of cell fluorescence was induced by a mercury arc lamp flf’fff-250 (250 watts). The emission of the lamp at wavelengths of 365 and 405 nm was separated by interference filters. Since the fluorescence level was found to be dependent upon the length of exposure of the preparation to light excitation for protracted measurements, the period of each exposure was confined to 5-10 sec/lO min. Comparative electronmicroscopical observations were also made.

which have characteristic oxidized myoglobin. The maxima of the spectra (fig. 1c) occur at 418 nm (y-band), 580 and 540 nm (x- and p-bands, respectively). If the cells were kept in a hermetic chamber for long (30 min), the y-band shifted at 436 nm, and a wide band at 560 nm replaced the c(- and ,!Ibands (fig. 1~2). Such behaviour of the absorption spectra under anaerobic conditions is peculiar to myoglobin [18]. In the apical parts of the neurons, large numbers of cytoplasmic granules (running up to 2-3 pm in diameter) were observed, which were of yellow and yellow-orange colour. The absorption spectra (fig. 1b) of this part contained bands of myoglobin (,I =418, 540, 580 nm) and “yellow pigment” (A=465 and 495 nm). After 10 min of KCN (10 mM) RESULTS action the spontaneous electrical activity of the neurons disappeared completely and it The comparative microspectrophotometrical and electronmicroscopical investigations [ 171 was accompanied by an increase in the reshowed that areas of the giant nerve cells duced form of haemoproteins. At the same which have different morphology and func- time, the increase of intensity of “yellow tions also have different absorption spectra. pigment” absorption bands was observed The ultrastructure of giant neurons and ab- within the range of 4655495nm [7] (fig. 1b2). Such behavior of these bands suggests sorption spectra of different areas of the cell that they are due to carotenoids, but not to are presented in fig. 1. The spectra of the proximal part of the flavoproteins, the absorption bands of which cell, especially rich in mitochondria, are in the same range must decrease under the typical of the mitochondria respiratory system conditions [9]. Extraction and chemical analysis of the cytochrome c/cytochromoxidase (fig. 1d). Their maxima lie in wavelengths I =415, 445, “yellow pigment” of mollusc neurons were 455 nm (y-bands of the reduced cytochromes carried out [l l] and confirmed the suggestion c and a +as) and near to 550 and 520 nm about the carotenoid nature of the pigment. (a and p-bands of cytochromes, respectively) The carotenoids were extracted from nerve Fig. 1 d(2) also shows the effect of amytal tissue and tissue of salivary gland, the cells (5 mM) on the absorption spectra of the of which are characterized by high concentraproximal part of the cell. There is a shift of tions of “yellow pigment”, and by low conthe y-band at 407 nm and an increase of the centrations of myoglobin (fig. 2a). oxidation/reduction ratio of cytochromes, as The absorption spectra unsaponifiable shown by the calculated differential spectrum fractions of acetone extracts [19] of these (fig. 1d3). tissues in light petroleum are presented in Absorption spectra were observed in the fig. 2b. There are two groups of absorption neuron cytoplasm, especially in axon hilok, bands in the visible and ultraviolet regions Exptl

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Fig. 1. (a) Scheme of ultrastructural organisation of mollusc Lymnaea stagnalis neuron. Ordinate: (b-d) O.D. (6) Absorption spectra of neuron apical part. I, before KCN action; 2, after 10 min KCN (10 nM) action; 3, calculated differential spectrum. (c) Absorption spectra of neuron akson hilok. 1, aerobiosis; 2, anaerobiosis (30 min). (d) Absorption spectra of neuron proximal part. I, before amytal action; 2, after amytal(5 nM) action; 3, calculated differential spectrum (2-l). (e) Spectrum of neuron apical part self-fluorescence (intensity in arbitrary units).

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Fig. 2. Ordinate: O.D. (a) Absorption spectra of the mollusc Lymnaea stagnalis living cells. I, neuron apical part; 24, salivary gland cells. (b) Absorption spectra of acetone extracts (in light petroleum). I, nervous tissue; 2, salivary gland tissue.

of the spectrum. The variability of their relative intensity in the spectra shows that they are due to two different compounds. The position of the long-wave absorption bands (usually three) of the carotenoids is a function of the number of conjugated double bonds in the molecule. If the central absorption band of carotenoids with 9-10 conjugated double bonds lies in the region 440-450 nm, the same band of the carotenoids with 3-4 conjugated double bonds lies in the region 270-280 nm [20]. Fig. 2b shows that the concentration of carotenoids with low (3-4) and high (9-10) numbers of conjugated double bonds differs in cells with differing metabolic activity. It was shown that selffluorescence of the cells with a maximum at 560-550 nm (fig. le) was also due to the carotenoids. The fluorescence level of living neurons was found to be dependent upon the oxygen concentration in the media. The level changes under the action of inhibitors and substrates of oxidative metabolism in nerve cells [6]. The intensity of the carotenoid absorption bands in the spectra of living cells was found to be dependent upon the physiological state of the mollusc. It increases reversibly during Exptl

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the transition of mollusc from winter lethargy to spring activity [21, 221. The samechanges of carotenoid bands at A=465 and 496 nm were observed with temperature changes of media, where molluscs were kept for several days. The dependence of neuron apical part absorption spectra upon changes of the environmental temperature is presented in fig. 3. An increase in temperature leads to a growth of the carotenoid bands (A= 465 and 495 nm) and a reduced form of the myoglobin band (A=436 nm). It is likely that the increase in metabolic activity in poikilothermal animal cells, accompanied by the increase in oxygen consumption [23] is responsible for this reversible growth of carotenoid absorption bands. Comparative microspectrophotometrical and electronmicroscopical investigations of Lymnaea stagnalis neurons showed that carotenoids, in common with myoglobin, are localized in special cytoplasmic granules which are characterized by a complicated ultrastructural organisation [17]. Similar granules in molluscoid neurons have been described under different names: compound granules [24], complicated particles [24], coloured granules [26], “pigment grains” [27, 281 and cytosomes [29, 301.

Carotenoids in oxidative metabolism of neurons

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3. Ordinate: O.D. The dependence of typical absorption spectra of neuron apical parts under the temperature of mollusc environment. 1, t = 4°C; 2, t = 15°C; 3, t = 22°C. Fig.

The existence of carotenoids in the granules was shown by Cain [26] and Arvanitaki & Chalazonitis [27, 281 who demonstrated the presence of haemoproteins in such granules. In addition, Nolte and co-workers [29] and Zs-Nagy [30] reported that the specific mitochondrial respiratory enzymes cytochromeoxidase and succinate-dehydrogenase are also localized in these granules. DISCUSSION The experimental data evidenced that carotenoids take part in oxidative metabolism of molluscoid neurons. Under conditions of oxygen concentration decrease, terminal oxidase inhibition, or cell activity increase, a reversible increase of the carotenoid absorption bands in the visible region of spectrum (465 and 495 nm) was observed. A reversible change in the number of conjugated unsaturated double bonds in carotenoids may produce such a reversible change of absorption bands. Apparently, the role of carotenoids in oxidative metabolism is to provide a large number of unsaturated double bonds as an intracellular oxygen reserve. A scheme for a possible pathway for oxygen transport in the

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molluscoid body, with carotenoid oxygen stock in evidence, is shown in fig. 4. Oxygen is transported by haemocyanin mainly in tissues containing myoglobin (the nervous system, radular and pharyngial muscles), since myoglobin has a greater oxygen affinity than has haemocyanin [32]. Under conditions of low metabolic activity of cells and, respectively, low oxygen consumption by them, a superfluous part of the oxygen is stocked up by the use of carotenoid unsaturated double bonds. The decrease in number of conjugated double bonds leads to a decrease of the absorption intensity at a=465 and 495 nm. Apparently myoglobin may serve as an oxidase of unsaturated double bonds in that process [33]. It is very difficult to state now anything about the type (peroxide, aldehyde or other type) of oxygen bond with the carotenoids. Under conditions when metabolic cell activity increases (electrical stimulation, spring season, or high temperature), when oxygen diffusion is insufficient, withdrawal of oxygen from the stock takes place. This leads to an increase in the number of conjugated double bonds in chromophore groups of carotenoids, and is accompanied by an increase of absorption band intensity at A=465 and 495 nm in living neuron spectra. It is of interest that the inhibition of the respiratory chain terminal oxidase a3 by KCN leads to withdrawal of oxygen from the stock, whereas the amytal block does not produce a similar effect. It is evidenced that the oxygen stock is connected with the respiratory chain in the region flavoprotein +Q-b-+c,-+-c+a. It is possible that unsaturated carotenoids serve as oxygenase [34], forming with oxygen unstable peroxides (or aldehydes). The extra oxygen of these organic peroxides may be used by peroxidase (or by methmyoglobin [35]) for oxidation in the respiratory chain. Exptl

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REFERENCES 1. Chance, B & Williams, G R, Adv enzymol 17 (1956) 65. 2. Thorell, B & Chance, B, Exptl cell res 20 (1960) 43. 3. Terzuolo, C A, Chance, B, Handelman, E, Rossini L & Schmelzer, P, Biochim biophys acta 126 (1966) 361. 4. Karnaukhov, V N, Biophysika 13 (1968) 622. 5. - Neurosci transl 8 (1968-69) 876. 6. Karnaukhov, V N & Melnikova, E V, Biophysika 14 (1969) 280. I. Karnaukhov, V N, Structure and functions of macromolecules and macromolecular systems, p. 200. Moscow (1969). 8. Keilin, D K & Smith, E L, Nature 143 (1949) 333. 9. Chance, B, Flavins and flavoproteins, p. 505. Amsterdam, New York, London (1966). 10. Chance, B & Schoener, B, Flavins and flavoproteins, p. 510. Amsterdam, New York, London ,.^,,\ (IYbb). 11. Karnaukhov, V N, Medvedev, A I, Abdurachmanov, A & Fin, R T, Biophysika of living cell. Puschino (1970). 12. Goodwin, T W, The comparative biochemistry of the carotenoids (1952). 13. Veprintsev, B N & Rosanov, S I, Symposium on neurobiology of invertebrates, p. 413. Hungarian acad sci, Budapest (1967). 14. Karnaukhov, V N, Biophysika of living cell. Puschino (1970). 15. Karnaukhov, V N, Rozonov, S I & Svoren, V A, Biophysika 11 (1966) 1085. 16. Karnaukhov, V N, Kulakov, V I, Melnikova, E V & Jshin, V A, Cytologij 10 (1968) 654. 17. Karnaukhov, V N & Varton, S S, Biophysika of living cell. Puschino (1970). 18. Go& M & Chalazo&tis,’ N, Compt rend sot biol 159 (1965) 1777.

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Fig. 4. Scheme of carotenoid participation in oxidative metabolism of the mollusc Lymnaea stagnalis neurons.

19. Davies, B H, Chemistry and biochemistry of plant pigments (ed T W Goodwin) p. 489. Academic Press, London, New York (1965). 20. Weedon, B C L, Chemistry and biochemistry of plant pigments (ed T W Goodwin) p. 75. Academic Press, London, New York (1965’) 21. Karnaukhov, Melnikova, E V, Svoren, V A & Fin, R T, Biophysika 13 (1968) 477. 22. Karnaukhov, V N, Abstracts of the 5th meeting of FEBS, Praha, p. 242 (1968). 23. von Brandt, T, Nolan, M 0 & Mann, E R, Biol bull 95 (1948) 199. 24. McGee-Russell, B, Quart j microscop sot 105 (1964) 139. 25. Borovjgin, V L & Sakharov, D A, Ultrastructure giant neurons of Tritonia. Nauka, Moscow (1968). 26. Cain, A J, Quart j microscop sot 89 (1950) 421. 27. Arvanitaki, A & Chalazonitis, N, Bull inst ockanograph Monaco 57 (1960) 1164. 28. Chalazonitis, N & Arvanitaki, A, Bull inst odanograph Monaco 61 (1963) 1282. 29. Nolte, A, Breucker, H & Kuhlmann, D, Z Zellforsch 68 (1965) 1. 30. Zs-Nagy, I, Ann biol Tihany 34 (1967) 25. 31. Karnaukhov, V N, Biophysika of living cell. Puschino (1970). 32. Manwell, C, J cell camp physiol 52 (1958) 341. 33. Michlin, D M, Biochemistry of cell respiratory, p. 271. Acad sci of USSR, Moscow (1960). 34. Bach. A & Schodat. R. Ber deut them Ges 35 (192i) 2466. ’ ’ 35. George, P & Irvine, D H, Biochem j 52 (1952) 511. Received March 12, 1970 Revised version received September 24, 1970