[International Review of Cytology] Volume 49 || Cytosomes (Yellow Pigment Granules) of Molluscs as...

Cytosomes (Yellow Pigment Granules) of Molluscs as Cell Organelles of

Anoxic Energy Production

I. 11.

111. IV.

V.

VI.

VII.

VIII. IX. X.

IMUIRE ZS.-NAGY'

Center for Cytology, I.N.R.C.A., Ancona, ltaly

Introduction. . . . . . . . . Histology of Cytosomes . . . . . . Ultrastructure ofCytosomes . . . . . Respiratory Enzyme Activity in Cytosomes . . A. Light Microscope Histochemistry of Cytochrome

Oxidase and Succinic Dehydrogenase . . . B. Electron Microscope Histochemistry of

Succinic Dehydrogenase . . . . . The Effect of Anoxia on Cytosomes . . . . A. Experimental Anoxia . . . . . . B. Anoxic Tolerance and the Pigmentation of

the Ganglia . . . . . . . . C. Structural Changes in Cytosomes during

AnoxiaandReoxygenation . . . . . D. Histochemical Changes in Cytosomal Lipids

during Anoxia. . . . . . . . E. Changes in Acid Phosphatase Activity in

Cytosomes during Anoxia . . . . . F. Electron Microscope Visualization of Energy

Production in Cytosomes . . . . . Electron Acceptor Properties of the Cytosomal Lipochrome Pigment . . . . . . . Adenosine Phosphate Concentrations of Tissues during Anoxia . . . . . . . . Carbohydrate Consumption of Tissues during Anoxia Fatty Acid Composition of Total Lipids during Anoxia General Discussion . . . . . . . A. Cytosomes as Cell Organelles . . . . B. Oxygen Storage as a Basis for Anoxic Energy Production C. Anaerobic Glyco- or Clycogenolysis in Anoxia . . D. A Possible Mechanism of Cytosomal Energy Production

XI. Summary and Conclusions . . . . . . . References . . . . . . . . . . Note Added in Proof . . . . . . . .

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' Permanent address: Biological Institute, Medical University, H-4012 Debrecen, Hungary.

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I. Introduction Numerous bivalves and snails have many tissues that are remark-

ably pigmented. Their nervous systems are especially rich in yellow or reddish-yellow pigments, as noted earlier by nineteenth-century workers (Schultze, 1879; Rawitz, 1887; Bochenek, 1905). Different carotenoids have been found in the pigments of marine species (Goodwin, 1952), while in the freshwater clam Anodonta cygnea the main colored component is p-carotene (Libos et al., 1966). The pigment is localized in special granules recognizable in many cases even with light microscopy, and always occurs intracellularly.

Several hypotheses had been presented regarding the function of these pigment granules. One can find reports describing them (1) as aging pigment, that is, lipofuscin (Nagy, 1962, 1965), (2) as neurosecretory material, because they are paraldehyde-fuchsin-positive (Gabe, 1955; Krause, 1960; Fahrmann, 1961; Jungsstand, 1962; Anth- eunisse, 1963; Baranyi and Salinki, 1963; Kuhlmann, 1963; Baranyi, 1964; Zs.-Nagy, 1964, 1965), and (3) as lysosomes (Meek and Lane, 1964; Baranyi, 1966; Lane, 1966). However, the first hypothesis has proved to be completely erroneous (Ebos et al., 1966), and the second one could not be confirmed by later investigations (see Section 11). The lysosome theory seems to be correct, by definition, since some of the pigment granules contain acid phosphatase activity. How- ever, numerous other characteristics differ from those of common lysosomes. This problem is discussed in Section X,A.

In 1965 two groups of investigators (Nolte et al., 1965; Sakharov et al., 1965) independently expressed the opinion that the pigment and neurosecretory granules are not identical. This has also been accepted by Gabe (1966), however, the pigment granules were sporadically interpreted as neurosecretion even thereafter (Baranyi and Salinki, 1967; Benjamin and Peat, 1968). Nolte and her associates (1965) also changed the terminology and called the pigment granules cytosomes, the term used by Lindner (1957). This term is also used in this article, in spite of its rather confusing character, since it has been used for designation of “almost any cytoplasmic structure lined by a single unit membrane and of dubious identity” (De Duve and Wattiaux, 1966). It should be emphasized that in this report the term cytosome refers to the yellow pigment granules of molluscs, and that this article is in- tended to summarize the results indicating that cytosomes perform a much more complex function than lysosomes. Nolte et al. (1965) attributed a certain “metabolic depot” function to cytosomes, however, this function was not defined more exactly.

It is also a well-known fact that certain molluscs are able to tolerate

CYTOSOMES AND ANAEROBIOSIS OF MOLLUSCS 333

long periods of anoxia (Brand, 1946), however, the nature of anoxic energy production is not completely clear. The great disproportion between the amount of carbohydrate consumed and the lactic acid produced, as well as the low level or almost complete absence of the Pasteur effect, seem to exclude the possibility that the only source of energy is anaerobic glycolysis or glycogenolysis (Chapheau, 1932; Brand et al., 1950; Meenakshi, 1956, 1958; Martin, 1961; Goddard and Martin, 1966). Other known anoxic energy-producing mechanisms, such as succinate and alanine production (for references, see Bueding, 1962; Hochachka et al., 1973), may contribute to a certain extent, however, neither of these pathways can explain all the phenomena of anoxic survival of molluscs.

Several experimental results suggested a possible correlation between the presence of cytosomes and the anoxic tolerance. For example, Baranyi and Saltinki (1967), although starting from the neurosecretory theory of cytosomes, showed that a certain depletion of the PAF-positive material can be induced by prolonged anoxia. Further- more, electron microscope analyses of cytosomes carried out during 1967-1968 (Zs.-Nagy, 1969) revealed considerable structural alteration of cytosomes after several days of anoxia. However, some for- merly described hypotheses postulating that the pigment granules may be of mitochondria1 origin (Lacy and Home, 1956; Fahrmann, 1961) have obtained further support from the fact that some respiratory enzyme activity was found in cytosomes (Zs.-Nagy, 1967a). These observations called our attention to the possible role of the pigment granules in anoxic energy-producing processes. This article summarizes the results and gives a general interpretation of anoxic phenomena on this basis.

11. Histology of Cytosomes In the freshwater mussel A. cygnea the ganglia show the highest

level of pigmentation among all the tissues. In untreated cryostat sections one can see numerous yellow pigment granules in the nerve cell soma, which are absent from the neuropile. The pigment content of the granules is soluble in organic solvents (Zs.-Nagy, 1967a). After fixation with different fixatives, the solubility persists to a great extent, although some part of the pigment remains even in paraffin- embedded materials. The reason for conhsion with neurosecretory substances is that the pigment granules show intense staining with PAF. This property is connected with the presence of the pigment which is of lipid character and has nothing to do with true neurosecre-

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tion (Sakharov et al., 1965; L5bos et al., 1966; Zs.-Nagy, 1967a; Zs.- Nagy and Csuks, 1969). In the neurons of Helix pomatia the highest level of pigmentation was found in the axon hillock (Chalazonitis and Arvanitaki, 1956; Chalazonitis and Gola, 1964).

Both histological and chemical analyses showed unanimously that the pigment is a lipochrome (Pearse, 1972), i.e., its colored component is a carotenoid which dissolves in lipids (Goodwin, 1952; Ltibos et al., 1966). The main lipid components are neutral and phospholipids (Section V,D). It is known that the solubility of lipochrome pigments in organic solvents is influenced to a great extent by their binding to proteins (Pearse, 1972). Therefore, the “seasonal changes of neurosecretory material” observed by Antheunisse (1963) and Baranyi (1964) are due most probably to changes in lipochrome-protein interactions. A carotenoprotein has been isolated from the ganglia of Lymnaea stagnalis (Benjamin and Walker, 1972).

Besides carotenoids, the extracted pigment of A. cygnea contains another chromophore (Libos et al., 1966) which may correspond to the heme protein described by Chalazonitis (1961) in an ApZysi~ species and in L. stagnalis (Benjamin and Walker, 1972). The presence of myoglobins in the cytosomes of L. stagnalis has also been observed (Kamaukhov, 1970). The complexity of cytosomes is further shown by a yellow autofluorescence seen with ultraviolet excitation, especially in freeze-dried material (Dahl et al., 1962, 1966; Zs.-Nagy, 1967b), which is also present in the extracted pigment (Ubos et al., 1966) but absent in solutions of authentic P-carotene.

111. Ultrastructure of Cytosomes Cytosomes display a rather variable structure. Their size varies

between 0.3 and 12.0 pm. Their frequency in different tissues is variable. One can find them most frequently in the cytoplasm of the nerve cells, therefore the description given here is of neural cytosomes (Nolte et al., 1965; Sakharov et al., 1965; Chalazonitis et al., 1966; Zs.-Nagy, 1967a, 1968a, 1969; Zs.-Nagy and Borovyagin, 1972).

Cytosomes are composed of three basic structural elements, namely, membranes, a fine granulated matrix, and lipid droplets of various density (Fig. 1).

As regards the membranes, one can distinguish an outer limiting membrane and internal ones. The outer membrane is about 8.0- 8.5 nm thick and displays a regular unit membrane structure. The internal membranes are found within the granulated matrix. They are


localized mostly in the peripheral part of the cytosome. In some places they are confluent with the lipid droplets (Fig. 1). As a rule, in the cytosomes of normal animals more than half the area is occupied by lipid droplets, and the greater part of the rest is filled in with the granular matrix.

By using the method of Napolitano et al. (1967) for normal cytosomes, it has been shown that the outer membrane and also the greater part of the internal ones are of lipoprotein character (Zs.-Nagy and Borovyagin, 1972).

The fine granulated matrix is of medium electron density, con- sisting of granules smaller than ribosomes. This matrix fills in the space between the lipid droplets and the internal membranes. Since this matrix remains almost completely unchanged after the treatment of Napolitano et al. (1967), its lipid component must be insignificant. Most probably it is composed of protein substances.

The occurrence of cytosomes was also studied in numerous non- nervous tissues ofA. cygnea (Zs.-Nagy, 1973a). It has been revealed that cytosomes can generally be found in all basic tissues of this animal. The epithelial tissue is rich in cytosomes in certain areas, for example, in the intestine and kidney as well as in the statocyst, whereas other zones such as the syphon, certain channels of the gonads, the mantle, the pedal glands, and the hepatopancreas contain a lower number of cytosomes.

Among the muscle tissues, the highest frequency of cytosomes was found in the heart muscle, however, the smooth muscle cells of numerous organs also frequently contain cytosomes (Fig. 2). At the same time, the adductor muscles contain only a few cytosomes or none at all. It should be noted that the heart muscle of the snail L. stagnalis, in contrast to that of A. cygnea, contains few cytosomes (Zs.-Nagy and Rbzsa, 1970).

The connective tissue of molluscs is rather deficient in cells; it consists mainly of thin, atypical collagen fibers. Nevertheless, the cells of the connective tissue contained cytosomes in almost all the organs inves tigated (Zs.-Nagy, 1973a).

All the results mentioned above suggest that cytosomes should be considered cell organelles of general occurrence, with few exceptions, in molluscan tissues. The main structural components of cytosomes are identical in all the tissues, only their proportion varies to a certain extent from one organ to another. Therefore, the results obtained in the nervous system may be generalized to the other tissues and, vice versa, the results obtained for the whole body of molluscs may be correlated with the function of cytosomes.

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FIG. 2. Detail of a muscle cell from the intestinal wall of A. cygnea under normal conditions. Double fixation. CY, Cytosome; MY, myofilaments; SR, sacroplasmic reticulum. From Zs.-Nagy (1973a).

IV. Respiratory Enzyme Activity in Cytosomes

A. LIGHT MICROSCOPE HISTOCHEMISTRY OF CYTOCHROME OXIDASE AND

SUCCINIC DEHYDROGENASE

Chalazonitis and Arvanitaki (1956) and Chalazonitis and Gola (1964) first published the surprising observation that in molluscan neurons the highest respiratory enzyme activity was revealed by mi- crospectrophotometry to be in the same areas where the highest pigment concentration was observed (the axon hillock).

FIG. 1. General structure of cytosomes of the cerebral ganglion ofA. cygnea under normal conditions. Double (osmium tetroxide plus glutaraldehyde) fixation. L, Lipid droplets; M, internal membranes; GM, granular matrix. From Zs.-Nagy and Borov- yagin (1972), reproduced in Ttssue 6 Cell, by permission of Longman Group Ltd. London.

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The histochemical method of Burstone (1959) showed in the ganglia of A. cygnea that, apart from the mitochondria, cytochrome oxidase (CyO) activity is also localized in the cytosomes. The inhibitors potassium cyanide and sodium azide, in concentrations of M, completely blocked this reaction, whereas peroxidase activity could be excluded (Zs.-Nagy, 1967a).

Succinic dehydrogenase (SDH) activity was studied using the method of Nachlas et al. (1957) with nitro-BT (Zs.-Nagy, 1967a). The reaction product was also found in the cytosomes. These results agreed with the observations of Chalazonitis and Arvanitaki (1956) and Chalazonitis and Gola (1964) and seemed to support the mitochondrial origin of cytosomes suggested by others (Lacy and Home, 1956; Fahrmann, 1961). However, there were some doubts as regards the cytosomal SDH activity, because of some properties called lipoid substantivity of nitro-BT (Pearse and Hess, 1961).

B. ELECTRON MICROSCOPE HISTOCHEMISTRY OF SUCCINIC DEHYDROGENASE

The method of Kerpel-Fronius and Hajbs (1968) was used with the ganglia of A. cygnea (Zs.-Nagy and Kerpel-Fronius, 1970a). In this reaction the sites of enzyme activity are indicated by a copper ferro- cyanide precipitate of high electron density. This method revealed the following results.

In the mitochondria the internal membranes showed a strong reaction (Fig. 3), whereas the mitochondria1 matrix always remained inactive. Mitochondria with different levels of activity were observed in the same cells as well. The mitochondria of the nerve terminals were as a rule inactive, as in vertebrate nervous tissue (Hajos and Kerpel- Fronius, 1969).

Certain cytosomes also showed rather strong SDH activity. This was localized on the internal cytosomal membranes (Fig. 3). Numer- ous cytosomes, however, proved to be inactive in normal animals.

Control experiments with inhibitors confirmed that the reaction observed in the cytosomes was true SDH activity. Therefore one has to accept that at least several cytosomes display some true respiratory enzyme activity.

FIG. 3. Detail of a nerve cell perikaryon from the cerebral ganglion of A. cygnea under normal conditions. CY, Cytosome exhibiting strong SDH activity; M, Mitochon- drion with strong SDH activity; G, Golgi apparatus. From Zs.-Nagy and Kerpel- Fronius (1970a), reproduced in Acta B i d . Acad. Sci. Hung., by permission of Akadkmiai Kiad6, Budapest.

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V. The Effect of Anoxia on Cytosomes

A. EXPERIMENTAL ANOXIA

Anoxic conditions were created gradually as follows. The animals were placed in an amount of water corresponding to eight times their total body weight (including the shell), and then the water surface was covered by a paraffin oil layer 1-2 cm in thickness. Under such circumstances the oxygen content of the water is consumed by 10-15 hours at room temperature. In the case of A. cygnea the water contained no polarographically measurable oxygen by 17 hours. This animal showed an oxygen consumption during the first 5 hours of 0.020 mg/gm fresh weight per hour, calculated for the total body excluding the shell (Zs.-Nagy, 1974). Eventual oxygen diffusion into the water was excluded by polarographic controls. Water in which the oxygen had been exchanged for nitrogen and then covered with paraffin oil contained no polarographically measurable oxygen even after 48 hours. When not otherwise stated, in this article the term anoxia always refers to gradually created anoxia (Zs.-Nagy, 1971a, 197313; etc.).

OF THE GANGLIA Table I summarizes the anoxic tolerance of the animals studied. At

room temperature the highest anoxic tolerance was shown by A.

B. ANOXIC TOLERANCE AND THE PIGMENTATION

TABLE I PIGMENTATION AND SURVIVAL CMACITY DURING

ANOJKIA OF DIFFERENT MOLLUSC~O

Degree of pigmentation in Survival time Species central nervous system ( hours)b

Pelecypoda Anodonta cygnea Mytilus galloprovincialis Venerupis decussata Tapes decussatus Pecten Jacobaeus

Murex trunculus Aplysia limacina

Octopus vulgaris

Gastropoda

Cephalopoda

Strong Moderate Moderate Weak Unpigmented

Strong Moderate

Unpigmented

150-240 24-110 50-70 40-50

2-3

100-200 10-20

2-3

From Zs.-Nagy (1971a). Survival time refers to the total period after covering the water surface with paraffin

oil (Section V,A).

CYTOSOMES AND ANAEROBIOSIS OF MOLLUSCS 34 1

cygnea (7-10 days), whereas the other species showed shorter survival times. It is remarkable that a defined correlation exists between the degree of pigmentation of the ganglia and anoxic tolerance. Species having less intensely pigmented ganglia displayed shorter anoxic survival times, whereas those having an unpigmented central nervous system did not tolerate anoxia at all. Pecten Jacobaeus and Octopus uulgaris died 2-3 hours subsequent to the covering of the water surface with paraffin oil (Zs.-Nagy, 1971a). As regards the anoxic tolerance ofApZysia lirnacina (Table I), it should be noted that survival times of 10-20 hours were obtained only when the water was continuously purified in a closed pumping system containing active charcoal. Without purification the survival times amounted to only 6-7 hours, indicating that the accumulation of certain metabolic products is also of importance in the anoxic tolerance of this species. An- other problem with Aplysia is that it has no shell; a volume of water eight times its body weight offered a relatively lower amount of oxygen at the beginning of the experiment. Therefore the anoxia tolerance of this species is not comparable in every respect to that of the others.

The low anoxic tolerance of P. Jacobaeus was also observed by Salhki (1966), and that ofoctopus is also a generally known phenomenon. The tissues of Cephalopoda contain low amounts of carotenoids or none at all (Fox, 1966); that is, the cytosomal lipochrome pigment is missing from these animals (Young, 1963).

If animals are placed in water initially devoid of oxygen (immediate anoxia), even those of otherwise high anoxic tolerance die quickly (Zs.-Nagy and Ermini, 1972b; Zs.-Nagy, 1974). This fact indicates that the mechanism protecting the animals against anoxia begins to function only if total anoxia occurs gradually. Under the natural conditions of the environment the gradual formation of anoxia is more probable than immediate formation, at least in the case of freshwater species; therefore one can consider the anoxic tolerance of the animals an adaptation to environmental conditions.

C. STRUCTURAL CHANGES IN CYTOSOMES DURING ANOXIA AND FhOXYGENATION

During prolonged anoxia an ever-increasing portion of cytosomes shows considerable structural alteration. On the fifth day of anoxia about half the cytosomes display an altered structure in the ganglia of A. cygnea, namely, the size of the lipid droplets decreases and intense membrane proliferation can be observed (Fig. 4) (Zs.-Nagy and Borov- yagin, 1972). Numerous new membranes are formed virtually at the expense of the lipid droplets. Between the newly formed membranes

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there is no matrix, and the membrane thickness is about 7.0-7.5 nm. This transformation leads to serious decomposition of cytosomes by the seventh or eighth day of anoxia (Zs.-Nagy, 1969) (Figs. 5 and 6). The outer membrane breaks, and the cytosomal contents seem to pour out into the cytoplasm. Although one cannot exclude that these phenomena are partially due to inadequate fixation, i.e., fixation adequate for normal cells but inadequate for cells made fragile by anoxia, the altered morphology reflects changed membrane characteristics in uiuo resulting from anoxia. In another form of cytosomal alteration occurring during anoxia, the outer membrane seems to be intact and the-inside of the cytosomes is filled with numerous “dense-cored” vesicles about 200-500 nm in size. These cytosomes as a rule contain no lipid droplets (Zs.-Nagy and Borovyagin, 1972).

With the method of Napolitano et al . (1967) the newly formed cyto- soma1 membranes proved to be almost exclusively of lipid composition. These results testify that the great majority of cytosomes undergo an irreversible transformation during prolonged anoxia; they are “used up” during anaerobiosis, especially during the terminal phase.

The effects of reoxygenation after prolonged anoxia, as well as the regeneration of cytosomes after reserpine treatment, have also been studied (Zs.-Nagy, 196813, 1969). (Reserpine treatment, like anoxia, also induces structural changes in cytosomes. This can be a real anoxic effect, since because of monoamine depletion a respiratory block comes into being; nevertheless, other factors may also play a role, e.g., increased metabolic activity of the nerve cells in order to eliminate the reserpine effect.) In both cases one can observe the accumulation of a granular substance in numerous mitochondria, resembling morphologically the cytosomal matrix. These mitochondria then are enclosed by the membrane profiles of the rough endoplasmic reticulum which later become blurred, and in other forms one can observe a new outer membrane. Parallel with these phenomena, lipid accumulation takes place inside, membrane proliferation can be seen near the limiting membrane, and the structure gradually begins to resemble that of cytosomes (Zs.-Nagy, 1969). Similar “precytosomes” were found

FIG. 4. Detail ofa cytosome from the cerebral ganglion on A. cygnea on the fifth day of anoxia. Note the apparent “continuity” between the lipid droplets (L) and the membranes (M) (some overlap due to section thickness may also be involved), the absence of the granular matrix in the intermembranous space, and the somewhat higher electron density of the limiting membrane (LM) of the cytosome. Double fixation. From Zs.- Nagy and Borovyagin (1972), reproduced in Tissue t Cell by permission of Longman Group Ltd. London.

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FIG. 6. An almost completely destroyed cytosome (CY) from the cerebral ganglion of A. cygnea on the eighth day of anoxia. L, Lipid droplet.

during the embryonal development of ganglion cells in the glochidia of A. cygnea (Zs.-Nagy and LBbos, 1969).

The “transforming” mitochondria described above also can be found in normal animals, however, rather rarely. Their increased frequency observed during reoxygenation after prolonged anoxia indicates that the formation of cytosomes probably takes place when the oxygen supply is sufficient, forming in this way a reserve which will be used up during a subsequent oxygen shortage. The mitochondrial origin of cytosomes could explain the cytosomal localization of mitochondrial enzymes such as CyO and SDH (Section IV). However, since the above-mentioned mitochondrial transformation has many morphological features in common with cellular autophagy (Novikoff, 1961; 1973; De Duve and Wattiaux, 1966), one cannot exclude the

FIG. 5. Detail of a nerve cell perikaryon from the cerebral ganglion ofA. cygnea on the eighth day of anoxia. Note intense membrane proliferation within the cytosomes (CY) and the morphological manifestations of the lytic processes (arrows).

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possibility that it represents only the elimination process of mitochondria damaged by anoxia, and that cytosomes are formed in some other way. It is noted that, although the interpretation differs from ours, morphologically similar cytosome formation was also observed in Aplysia (Arvanitaki and Chalazonitis, 1966) and in Helix (Chala- zonitis and GrassC, 1968).

D. HISTOCHEMICAL CHANGES IN CYTOSOMAL LPIDS DURING ANOXIA

Kreps et al. (1968) showed that the central nervous system of molluscs contains relatively high concentrations of phospholipids. How- ever, the lipid content of cytosomes is not known. In order to obtain some information in this area, the lipid content of cytosomes was analyzed under normal and anoxic conditions in the ganglia ofA. cygnea, using numerous methods of light microscope lipid histochemistry (Zs.-Nagy and Csukas, 1969).

In normal animals cytosomes contain high concentrations of phospholipids and neutral lipids, and during the summer season acidic lipids are also abundant. Acidic lipids are absent during the autumn and winter seasons even from normal cytosomes. Certain cytosomes and the neuropile contain some acetal lipids. Glycolipids and choles- terol were not found with the methods used. Both the neutral and phospholipid contents decrease in the cytosomes after 4-7 days of anoxia. Acidic lipids disappear even during the summer season with the effects of anoxia. Acetal lipids showed no considerable change.

It should be noted that the above-mentioned histochemical observations are based on qualitative methods, i.e., a decrease indicates only that the cytosomes show less intense staining, or that the occurrence of vacuolated cytosomes becomes more frequent, and so on.

E. CHANGES ACID PHOSPHATASE ACTIVITY IN CYTOSOMES DURING ANOXIA

Acid phosphatase activity of cytosomes has been studied by light and electron microscope methods (Meek and Lane, 1964; Lane, 1966; Baranyi, 1966; Zs.-Nagy and Borovyagin, 1972).

In normal animals only some of the cytosomes show acid phosphatase activity. Baranyi (1966) has reported that this enzyme is completely absent from the nerve cells of A. cygnea during the winter season. Our light microscope investigations revealed acid phosphatase activity only in a few cytosomes in normal animals, using the pararosaniline method of Barka and Anderson (1962; I. Zs.-Nagy, unpublished observations).


Electron microscope lead salt methods showed that about half the cytosomes in the ganglia of A. cygnea are inactive for acid phosphatase in the normal state (Zs.-Nagy and Borovyagin, 1972). Others show a definite activity always localized on the periphery of the cytosomes and never within the lipid droplets (Fig. 7a).

During prolonged anoxia acid phosphatase activity increases considerably. Almost all the cytosomes show a more-or-less intense reaction (Fig. 7b). The activity is again localized in the membranous zones, and in many cases one can find precipitate in the intermem- brane space. Control experiments confirmed the reaction to be specific for acid phosphatase (Zs.-Nagy and Borovyagin, 1972).

F. ELECTRON MICROSCOPE VISUALIZATION OF ENERGY PRODUCTION IN CYTOSOMES

The energy-dependent accumulation of divalent cations can be used for electron microscope visualization of energy production, since Greenawalt and Carafoli (1966) showed that isolated mitochondria are able to accumulate, for example, strontium ions and inorganic phosphate at the expense of the energy yield of respiration to such an extent that they are precipitated in the form of insoluble crystals within the mitochondria. The same principle has been applied by Kerpel- Fronius and Haj6s (1970) to in situ mitochondria of mammalian tissues. This method reveals the function of the electron transport chain and the coupling of the ATP-generating pathways, since it can be blocked by potassium cyanide and dinitrophenol (DNP).

The method of strontium accumulation was applied to the nervous tissue of molluscs (Zs.-Nagy and Kerpel-Fronius, 1970b; Zs.-Nagy, 1971a; Kerpel-Fronius and Zs.-Nagy, 1973). It has been established that in the nerve cells of normal animals, using succinate as a respiratory substrate, an intense accumulation of strontium ions takes place in the internal compartment of the mitochondria, resulting in large, dense granules on the electron micrographs (Fig. 8). According to Greenawalt and Carafoli (1966), the form of precipitation also indicates the intensity of energy production, i.e., the appearance of needlelike crystals corresponds to a high level of energy production, whereas granular precipitation indicates a lower level. Several cytosomes also showed a fine granular precipitation in normal animals (Fig. 9), however, the great majority of cytosomes remained inactive. Needlelike crystals have not been found either in mitochondria or in cytosomes, therefore one can conclude that there is a lower level of energy production than in mammalian tissues (Kerpel-Fronius and Hajbs, 1970; Hajos and Kerpel-Fronius, 1971). The blocking effect of potassium

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FIG. 8. Energy-dependent strontium accumulation in the nerve cells ofM. gallopro- oincialis under normal conditions. Pedal ganglion. Arrows indicate mitochondria of strong activity. CY, Cytosomes displaying no activity. From Zs.-Nagy (1971a).

cyanide and DNP confirmed the specificity of the reaction both in mitochondria and cytosomes.

When the respiratory substrate was omitted from the incubation mixture, some strontium accumulation was also observed, which proved to be equally sensitive to the inhibitors discussed above. This reaction thus can be considered to be maintained by endogenous sub- strates. The relatively high concentrations of succinate (51-400 mg%) found in the tissues of some marine bivalves (Aoki, 1932) allows us to

FIG. 7. Electron microscope demonstration of acid phosphatase activity without staining of the sections. Incubation for 120 minutes at room temperature. (a) Cytosome from the cerebral ganglion ofA. cygneo under normal respiration. Arrows show weak reaction at the periphery of the lipid droplet. (b) Cytosome from the cerebral ganglion ofA. cygnea on the sixth day of anoxia. Note the striking difference in the intensity and localization of the reaction product (P). L, Lipid droplets. From Zs.-Nagy and Borov- yagin (1972), reproduced in Tissue 6 Cell, by permission of Longman Group Ltd. London.

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FIG. 9. Energy-dependent strontium accumulation in the nerve cells of A. cygnea under normal conditions. Cerebral ganglion. CY, Active cytosome in which the fine, granulated strontium phosphate deposits are indicated by arrows; M, active mitochon- drion. From Zs.-Nagy (1971a).

assume the presence of a sufficiently high concentration of endogenous succinate, even in A. cygnea.

Investigating the localization of energy-producing mechanisms during prolonged anoxia, one observes a characteristic reversion. The mitochondria1 reaction is restricted to a great extent, however, not completely. At the same time, cytosomal strontium accumulation increases significantly (Fig. 10). Almost all cytosomes show intense accumulation, and the anaerobic nature of this process is shown by the fact that it takes place with the same intensity even if incubation is carried out in a nitrogen atmosphere. However, it is very important to observe that even this cytosomal reaction can be blocked by DNP and potassium cyanide (Zs.-Nagy, 1971a).

These results indicate that during anoxia the main site of energy production is the cytosomes, and that the cytosomal mechanisms involve some pathways similar to aerobic ones in so far as succinate is


FIG. 10. Energy-dependent strontium accumulation in the cytosomes of A. cygnea on the fifth day of anoxia. Pedal ganglion. Arrows indicate very strong accumulation within the membranous part of the cytosome. From Zs.-Nagy (1971a).

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the substrate, the electron transport takes place to CyO (potassium cyanide), and coupled oxidative phosphorylation takes part in ATP generation (DNP sensitivity).

VI. Electron Acceptor Properties of the Cytosomal Lipochrome Pigment

The observation of Karnaukhov (1970) that the yellow autofluorescence of the pigmented area in isolated neurons of L. stagnalis decreases during anoxia indicates that electron distribution in these regions changes during anoxia. Since the lipochrome pigment of cytosomes contains carotenoids, the electron-transporting capacity of which is well known (Platt, 1959; Pullman and Pullman, 1963), the possibility has arisen that the pigment complex may represent some kind of electron acceptor.

Based on these considerations, experiments were carried out (Zs.- Nagy, 1971b) with isolated ganglia of A. cygnea and L. stagnalis in physiological solution (Marczynsky, 1959). The oxygen consumption was continuously measured polarographically after the surface was covered with paraffin oil. The redox potential of the solution was also determined (Jacob, 1970). Frog brain was used as control poikilo- thennic nervous tissue obviously devoid of lipochrome pigment (for technical details, see Zs.-Nagy, 1971b). The results are shown in Figs. 11 and 12. The behavior of the redox potential during these experiments suggests that the nervous tissue of molluscs contains a certain electron acceptor protecting the tissue components from reduction even during anoxia.

When the experiments were performed with physiological solutions containing no oxygen initially the redox potential decreased rapidly even in the case of Anodonta ganglia and only during the second day stopped for about 24 hours (Fig. 13). This result confirms the observation mentioned before (Section V,B), that animals are able to tolerate only gradually developed anoxia and not immediate anoxia.

Another aspect of the electron acceptor capacity of the lipochrome pigment was also studied (Zs.-Nagy and Ermini, 1972a). It has been shown that ethanol or 2-propanol extracts obtained from the whole body or the isolated ganglia of the marine bivalve MytiZus gaZZopr-o- uinciaZis are able to oxidize NADH in uitro, whereas similar extracts of mouse brain tested under the same conditions do not have this property. Furthermore, it has been revealed that the oxidative capacity of the pigment extracts significantly decreased if the animals had previously been kept under conditions of prolonged (1- to 3-day) an-


ON

--------

I 2 3 4 5 6 - - - - - - - - - 3 O 34 30 42 46 50 54

hr

FIG. 11. Oxygen consumption of isolated ganglia of A. cygnea (curve 1) and frog brain (curve 2) in physiological solutions covered with paraffin oil. The curves are plotted on the basis of polarographic measurements. From Zs.-Nagy (1971b), reproduced in Comp. Biochem. Physiol. A, by permission of Pergamon Press Ltd. Oxford.

2 00 I

> E -

-350 h, \ - 5 5 0 -600

L L

I 1 1 1 1 1 1 1 1 I 1 ! I I 1 1

I 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5

Days

FIG. 12. Redox potential values of solutions containing 10 mg/ml Anodonta ganglia (curve l), 10 mg/ml Lymnaea ganglia (curve 2), and 5 mg/ml frog brain. The values are from the same experiment as the oxygen consumption values shown in Fig. 11. From Zs.-Nagy (1971b), reproduced in Comp. Biochem. Physiol. A, by permission of Pergamon Press Ltd. Oxford.

354 IMRE ZS.-NAGY

200 I50

100 50

-50 - -100 > E -150

h” -250 -300

- 350 -400 -450 -500 - 5 5 0

-600

v

5 -200

1 2 3 4 5 6 7

Days

FIG. 13. Redox potential values of the physiological solutions when oxygen was replaced by nitrogen before the experiment. Curve 1,lO mglml Anodonta ganglia; curve, 2 ,5 mg/ml frog brain. From Zs.-Nagy (1971b), reproduced in Comp. Biochem. Physiol. A, by permission of Pergamon Press Ltd. Oxford.

oxia. However, similar experiments gave negative results in the case ofA. cygnea. Neither whole-body nor ganglion extracts of this species showed any direct oxidative effect against NADH in uitro (I. Zs.- Nagy, unpublished results). This indicates that the lipochrome pigments of marine and freshwater species may have different composi- tions. As a matter of fact, the marine species contain taurine (2- aminoethanesulfonic acid) in a rather high concentration (Allen and Garrett, 1971; De Zwaan and Van Marrewijk, 1973), which is extract- able with 2-propanol (Pettit et al., 1973). However, in freshwater bivalves and snails taurine is undetectable (Simpson et d., 1959). The role of taurine in anaerobiosis is obscure; it decreases to a great extent in anoxia (De Zwaan and Van Marrewijk, 1973), however, it obviously is not involved in the anoxic metabolism of freshwater species.

VII. Adenosine Phosphate Concentrations of Tissues during Anoxia

The energetic state of tissues is characterized by the actual ATP content, reflecting the equilibrium of the energy-yielding and -con- suming mechanisms. Unfortunately, “facultatively anaerobic ani-


mals are neither widely known to biochemists, nor widely studied by hem” (Hochachka et al., 1973). Nevertheless, some biochemical data are at our disposal showing that the ATP level of numerous facultative anaerobes is surprisingly high (60% or more of the normal value) even during day- to week-long periods of anoxia (Saz, 1971; Zs.-Nagy and Ermini, 1972b; Zs.-Nagy, 1973b). Since this article considers only molluscs, this section does not include data obtained from other phyla.

The method used for the analysis of adenosine phosphates in M. galloprovincialis (Zs.-Nagy and Ermini, 1972b) and A. cygnea (Zs.- Nagy, 1973b) was that of Deutsch and Nilsson (1953). The main steps of preparation are homogenization in cold perchloric acid, separation of AMP, ADP, and ATP on an ion-exchange column, and spectropho- tometric measurement of the absorptions at 260 nm.

Mytilus galloprovincialis has a rather variable anoxic tolerance (24-110 hours). For this reason, the results obtained during anoxia should have been evaluated individually. Normal animals contained 133 & 9 nmoles ATP/gm wet weight (mean of three animals ? S.E.M.). Within 24-72 hours of gradually developed anoxia 6.8-60.9% of the normal value was found, whereas immediate anoxia gave 11.9% of the normal level 8 hours subsequent to the start of complete anoxia (Zs.- Nagy and Ermini, 1972b).

In the case of A. cygnea, survival is secure for 7 days at room temperature during gradually developed anoxia. Groups of three animals were analyzed after 1-7 days of anoxia, as well as during normal respiration (Zs.-Nagy, 1973b). The results are shown in Table 11. During the first 6 days of anoxia 5244% of the normal ATP level can be measured, and on the seventh day this value decreases to 11%. The animals start to die at this time, and none of them survives the tenth day if anoxia is maintained.

One can calculate several parameters of bioenergetic significance such as ATP/ADP and ATP/AMP ratios indicating the phosphorylation potential (Klingenberg and Pfaff, 1968), that is, the energetic state of the tissues (Newsholme and Gevers, 1967), as well as the “energy charge” parameter [(ATP + ?hADP)/ATP + ADP + AMP] (Atkinson and Walton, 1967; Atkinson, 1968). The values of these parameters are known for different aerobic tissues, and hypoxia or anoxia causes a decrease in them without exception (Racker, 1965; Ballard, 1970; Philip- pidis and Ballard, 1970; Williamson, 1970; Hems and Gaja, 1972; Ridge, 1972; Hearse and Chain, 1972). As opposed to this expectation, these parameters showed an increase during the first days of anoxia in A. cygnea (Table 11). This fact indicates that the energetic state of the molluscan tissues may be kept at a rather high level for a considerable

TABLE I1 CONCENTRATIONS OF ADENOSINE PHOSPHATES AND THE CALCULATED PARAMETERS OF THE

ADENYLATE POOL IN NORMAL AND ANOXIC ANIMALS

Normal First Second Third Fourth Fifth Sixth Seventh animals day day day day day day day

AMP Mean Range

ADP Mean Range

ATP Mean Range

Total adenosine phosphates in average

ATP as percent of normal level in average

ATP ADP -

117 61 74-159 24-87 106 55 87-140 34-88 73 38 56-85 26-53 296 154

100 52

38 21-52 49 34-66 60 45-76 147

82

29 23-38 46 37-60 49 43-54 124

67

44

48

59

151

3949

37-56

41-85

81

51 35-67 44 39-53 43 34-50 138

59

96

68 52-90 69

64-77 233

35-128

94

56 34-82 47 19-94 8 2-14

111

11

0.69 0.69 1.22 1.07 1.23 0.98 1.01 0.17

0.62 0.62 1.58 1.69 1.34 0.84 0.71 0.14

ATP + 35 ADP 0.43 0.43 0.57 0.58 0.55 0.47 0.44 0.28 ATP + ADP + AMP

a From Zs.-Nagy (1973b), reproduced in Acto Bi0chi.m. Biophys. Acad. Sci. Hung., by permission of Akadhmiai Kiad6, Budapest. * Concentrations are given in nanomoles per gram wet weight. Apart from the range values, each figure represents the average of three

measurements in different animals.


time, even with complete anoxia, and the animals start to die only when these parameters decrease below their normal values.

The energy demand of the tissues during anoxia in A. cygnea is not known quantitatively. It is probable that the functional energy consumption of the tissues decreases, since certain organ functions slow down (Salinki, 1965; P6csi and Salanki, 1964). At the same time, the amount of energy expended for the maintenance of homeostasis obviously cannot be reduced, on the contrary, assuming that the elimination of certain metabolites requires an extra energy demand in anoxia, it can even be higher than the normal consumption.

VIII. Carbohydrate Consumption of Tissues during Anoxia

The basic data regarding the carbohydrate metabolism of molluscs had been summarized by Martin (1961), as well as by Goddard and Martin (1966). From the point of view of this article, the most important question is whether or not the anaerobiosis of molluscs is accompanied by a Pasteur effect, i.e., whether or not the carbohydrate consumption increases during anoxia (Martin, 1961). The Pasteur effect has been observed in certain species, however, to a small extent (Brand, 1946; Brand et al., 1950), whereas in other species it is missing (Chapheau, 1932; Meenakshi, 1956, 1958). Since anaerobic glycolysis may realize only about 7% of the total energy content of the carbohydrates (Lehninger, 1965), the nearly normal energy level of tissues cannot be maintained during prolonged anoxia without a very significant Pasteur effect if the only source of energy is anaerobic glyco- or glycogenolysis. Even a 50% decrease in energy consumption would involve anaerobic carbohydrate consumption about six to seven times higher than normal.

In order to determine the anoxic carbohydrate consumption, experiments were performed with A. cygnea (Zs.-Nagy, 197313). The glycogen content of this species is rather high (Table 111); the average value varies between 4 and 5% of the fresh weight, however, the statistical scatter is high. Other analyses revealed similar data. In Ostrea a 4.38% glycogen content was found (Mitchell, 1915), and in Mytilus edulis it amounted to 10-35% of the dry weight (De Zwaan and Zandee, 1972a).

Because of the high scatter of the data, carbohydrate consumption was analyzed on the basis of the regression coefficient of the glycogen content (Zs.-Nagy, 197313). In normal animals this coefficient was - 156.98 mg% per day, while in anoxia it amounted to - 119.59 mg%

358 IMRE ZS.-NAGY

TABLE 111 GLYCOGEN CONTENT O F ANIMALS UNDER NORMAL AND

ANOXIC CONDITIONS AT 15.0 k 0.5"c"

Glycogen content (mg/100 pm)

Days Normal Anoxia

1 1 1 1 1 1 2 2 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7 7 7 8 8 8

5191.0 1515.0 4080.6 3408.2 4383.9 5814.9 4585.5 6323.4 2916.9 - - - - -

3150.0 5699.9 4221.9 3750.0 6283.8 5111.1 6780.9 4725.0 3049.7 2814.3 1901.2 1482.8 3904.0 5225.2 5224.5

4012.5 4713.3 4326.9 6834.8 3589.6

7138.8 2176.2 6935.4 4608.1 5238.0 3827.7 6129.9 6387.3 6078.6 1631.2 4944.6 4137.3 4449.9 5826.6 2801.7 7641.9 5549.4 3612.0 4243.3 4190.4 3801.6 2814.1 5141.1

-

From Zs.-Nagy (1973b), reproduced in Actu Biochtm. Btophys. Acad. Sct. Hung., by permission of Akadkmiai Kiad6, Budapest.

per day (Fig. 14). The difference is not significant. One can calculate that, even at the given level of statistical scattering of the data, a ratio of anaerobic to aerobic carbohydrate consumption of 2 results in a significant difference. One can conclude that in A. cygnea there is no Pasteur effect or, if there is, it is at a low level.

Measurements carried out on the whole body may mask some dif-


6000 I

2 4 5 6

Time (days)

FIG. 14. Normal (solid line) and anoxic (broken line) glycogen consumption of A. cygnea calculated on the basis of regression coefficients 2S.D. From Zs.-Nagy (1973b), reproduced in Acta Biochim. Biophys. Acad. Sci. Hung., by permission of Akadkmiai Kiad6, Budapest.

ferences between the organs. Data have been published regarding the organs of M . edulis (De Zwaan and Zandee, 1972b), in which only the adductors and the hepatopancreas displayed a slight Pasteur effect. Therefore the glycogen content of the whole body is an acceptable in- dicator of the total carbohydrate metabolism.

Anaerobic degradation of labeled ( I4C) glucose was followed in M. edulis (De Zwaan and Marrewijk, 1973). Eight animals received 5 pCi of activity each in form of 0.36 mg of glucose. Four animals were killed after 24 hours of anoxia, and the others after 48 hours of anoxia. Twenty-five percent of the total radioactivity was not found at all, 38% of it was present in the water, and only 37% remained in the tissue ho- mogenates. The last-mentioned activity further decreased when the homogenate was freeze-dried. These results indicate that a significant part of the radiolabel left the system, most probably in the form of volatile products such as carbon dioxide, or perhaps short-chain fatty acids, during the anoxia or the preparation. Unfortunately, the investigators did not identify the labeled components of the water, thus the possibility of hydrocarbonate formation cannot be excluded. About half the radioactivity found in the tissues was bound to the amino acid fraction, mostly to alanine, and a further one-third was found in organic acids, mainly as succinate. All these results indicate that the anaerobic carbohydrate metabolism of molluscs cannot be regarded as being resolved by alanine and succinate production (Stokes and Awa- para, 1968; Chen and Awapara, 1969; Bueding, 1962; Hochachka et

360 IMRE 2s.-NAGY

al., 1973), and the production of volatile substances deserves further study.

IX. Fatty Acid Composition of Total Lipids during Anoxia

Recent biochemical analyses using radioisotope techniques have revealed that, surprisingly enough, fatty acid synthesis in the land snail Cepaea nemoralis L., which has high anoxic tolerance, persists even during prolonged anoxia and, what is more, synthesis of saturated fatty acids is considerably stimulated. This involves saturation processes which may act as an electron acceptor mechanism for the reoxidation of NADH or NADPH (Van der Horst, 1974; Oudejans and Van der Horst, 1974). Furthermore, the production of some volatile fatty acids has also been demonstrated as a possible contribution to anoxic ATP production (for reference, see Hochachka et al., 1973).

Two types of experiments have been performed with A. cggnea (Zs.-Nagy and Galli, 1976). In the first type the fatty acid composition of total lipids was analyzed in the ganglia of 10 animals on the sixth day of anoxia by gas chromatography and compared to that of 10 normal animals. The anoxic energy-producing mechanism still functions rather well at this time (Section VII). The main findings were as follows. More fatty acids were present ( + 29%) in the total lipids of anoxic ganglia than in the controls. This increase was much higher in saturated fatty acids (55%) than in unsaturated ones (17%). The ratio of saturated to unsaturated fatty acids increased from 0.428 to 0.566 at the same time in anoxia. The changes were manifested in the fatty acids below 20:4; especially, 16: 0 and 16: 1 acids increased (65 and 47%, respectively).

In the second type of experiment, the fatty acid composition of total lipids was studied in the whole body of animals on the eighth and ninth days of anoxia, also by gas chromatography; and the saponifiable and nonsaponifiable fractions of the total lipids (Van der Horst, 1970) were measured, too. By the time the latter experiments took place the anoxic energy-producing mechanism had been heavily damaged, and the animals were near death. These analyses showed a strong decrease in fatty acid content of the total lipids; about 77 and 87% of the normal value was lost by the eighth and ninth day of anoxia, respectively. Fatty acids below 20:4 showed a more intense decrease. The saponifiable fraction of the total lipids amounted to 55-60% in normal animals during different experiments. After 8 or 9 days of anoxia these values were in the range 15-30%. At the same time, the nonsaponifiable fraction increased.


On the basis of these experiments (Zs.-Nagy and Galli, 1976) the following conclusions can be drawn: (1) Until the anoxic energy- producing mechanism begins to function well, fatty acid synthesis persists also in A. cygnea; the synthesis of saturated fatty acids is more intense than that of unsaturated ones. This result agrees well with those of others (Van der Horst, 1974; Oudejans and Van der Horst, 1974). (2) Parallel with the exhaustion of the anoxic energy-producing mechanism, drastic changes can be observed in the fatty acid metabolism of the whole body, and seemingly this is the reason for the limita- tion of anoxic tolerance. This indicates that fatty acids play an important role in the anaerobic metabolism of molluscs.

X. General Discussion

A. CYTOSOMES AS CELL ORGANELLES

It is evident that cytosomes are of general occurrence in the pigmented tissues of molluscs (Sections I1 and 111). Structures similar to cytosomes were also described in the nerve cells of worms (Rohlich et al., 1962; Coggeshall and Fawcett, 1964) and in the corpus cardiacum of insects (Scharrer, 1963). However, the classification of cytosomes into known cell organelle categories encounters some difficulties. For example, they show certain relations to lysosomes (Section V,E), since some of them contain acid phosphatase. However, the morphology of cytosomes, as well as their high lipid and pigment content, represent remarkable differences as compared to lysosomes, even considering the enormous morphological variability of the latter (Novikoff, 1961, 1973; De Duve and Wattiaux, 1966; Nehemiah and Novikoff, 1974). A further difference between cytosomes and lysosomes is that respiratory enzymes have never been found in the latter (Gahan, 1967). The acid phosphatase activity of cytosomes, which increases especially during prolonged anoxia, can be interpreted as a sign of lytic processes occurring within the cytosomes, however, just the temporary character of this enzyme activity, together with the other distinctive features mentioned above, indicate some significant differences between 1 ysosomes and cytosomes. Further problems of cytosome- lysosome relationships are discussed by Nicaise (1973).

The functional significance of cytosomes is indicated by the fact that molluscs having a central nervous system rich in cytosomes display considerable anoxic tolerance, whereas those devoid of cytosomes cannot tolerate anoxia at all (Section V,B). Respiratory enzyme activity (Section IV) must play an important role in cytosomal functions, un- derlining the possibility of some connection between mitochondria

362 IMRE 2s.-NAGY

and cytosomes (Lacy and Home, 1956; Fahrmann, 1961). However, the morphological changes in cytosomes during anoxia and those in mitochondria during reoxygenation suggest a possible mitochondrial transformation into cytosomes, thus explaining the presence of mitochondrial enzymes in the cytosomes, but the autophagic origin of these enzymes cannot be excluded (Section V,C).

A further experimental result suggesting a connection between cytosomes and mitochondria is the energy-dependent accumulation of divalent cations in cytosomes (Section V,F). The only essential difference between mitochondrial and cytosomal ion accumulation is that under normal conditions the mitochondrial mechanism plays the main role, whereas the cytosomal one becomes activated only during prolonged anoxia. It seems to be significant that cytosomal ion accumulation remains sensitive to potassium cyanide and DNP even in the complete absence of molecular oxygen.

The properties of cytosomes suggest a functional analogy between the polymelanosomes of higher animals (Van Woert et al., 1967) and cytosomes. Polymelanosomes have been studied mostly in the liver of amphibia. They consist of melanin granules, a matrix, and some internal membranes enclosed by an outer limiting membrane; morphologically they are very similar to cytosomes. Mitochondria1 enzymes have been found in polymelanosomes, and accumulation of divalent cations was also observed (Prasad et al., 1965; Van Woertet al., 1967). Melanin is an extremely good electron acceptor, because of its semi- quinone free radicals (Pullman and Pullman, 1961). The isolated polymelanosomes of amphibia showed no phosphorylating activity in the presence of oxygen (Van Woert et al., 1967), whereas those obtained from mouse melanomas phosphorylated as strongly as the well- prepared mitochondria (Dorner and Reich, 1961). Although one cannot exclude the possibility of mitochondrial contamination in these melanosome preparations, this observation deserves attention. However, polymelanosomes also show certain relationships to lysosomes as regards their enzyme activity and morphogenesis (Novikoff et al., 1968). With the histochemical method of Lillie (Pearse, 1972), melanin could not be detected in the cytosomes of molluscs (own unpublished observation), however, the electron acceptor role of melanin can also be performed by the lipochrome pigment of cytosomes.

The changes in cytosomes during anoxia (Section V) can be interpreted by assuming that the cytosomes are in a certain state of rest during aerobic respiration of the cells and only during anoxia become activated. This activation of cytosomal mechanisms needs a certain latency time and is probably induced by decreasing oxygen tension

CYTOSOMES AND ANAEROBIOSIS O F MOLLUSCS 363

directly or through its functional consequences. One can assume that this latency is connected with the observation that only gradual anoxia is tolerated well, while immediate anoxia causes death. The consumption of cytosomal lipids during anoxia (Section V,D) indicates in itself that some peculiar process takes place, since under anaerobic conditions one would not expect fatty acid oxidation (Fairbairn, 1970).

B. OXYGEN STORAGE AS A BASIS FOR ANOXIC ENERGY PRODUCTION

The conclusion that cytosomes take part in anoxic energy production was also reached by Karnaukhov (1970, 1971a,b,c). This inves- tigator studied the isolated neurons of L. stagnalis by means of mi- crospectrophotometry and interpreted the cytosomal functions assuming that the carotenoids of the lipochrome pigment may bind oxygen by means of their conjugated double bonds, in this way forming an oxygen store. This is an attractive hypothesis, however, there are some difficulties in connection with it; namely, there is no evidence showing that lipochrome pigments are able to bind oxygen (Pearse, 1972). Nevertheless, this hypothesis has been analyzed from a quantitative point of view (Zs.-Nagy, 1974). The main conclusion of this analysis was that oxygen storage can be realized according to the as- sumption of Karnaukhov, even supposing an overestimated oxygen saturation of carotenoids (5 moles oxygen/ 1 mole carotene), if 100 gm wet gangiion tissue contains 25.8 gm of P-carotene instead of the 10 mg/100 gm found by LBbos e t al. (1966).

Oxygen storage may be effected by heme pigments, too. Different concentrations of hemoglobin, myoglobin, and hemocyanin are present in the hemolymph and tissues of molluscs (Arvanitaki and Chalazonitis, 1952; Chalazonitis and Arvanitaki, 1963; Chalazonitis and Gola, 1964; Prosser and Brown, 1961; Read, 1966; Ghiretti, 1966; Kamaukhov, 1970), however, no quantitative data are at our disposal as regards A. cygnea. In spite of the lack of data, one can make some calculations starting from certain suppositions, in order to decide whether the oxygen stored by heme pigments can play a role in anoxic energy production. Without going into details (Zs.-Nagy, 1974), we refer to the results showing that, even supposing that the total dry mass of the ganglion tissue (20%) consists of hemoglobin displaying an oxygen-binding capacity similar to that of human hemoglobin, only less than 1% ofthe oxygen demand for 150 hours could be fixed by the ganglion tissue.

These calculations exclude stored oxygen from any serious consideration as the basis of anoxic tolerance.

364 IMRE ZS.-NAGY

c. ANAEROBIC GLYCO- OR GLYCOGENOLYSIS IN ANOXIA

The most evident form of biological energy production under anaerobiosis may be glyco- or glycogenolysis. If a living system ex- changes aerobic metabolism for anaerobic metabolism, the following phenomena can be observed jointly: (1) Carbohydrate consumption increases considerably (Pasteur effect); (2) the lactic acid production of the system increases in proportion to the carbohydrate consumption; (3) an oxygen debt is formed which is repaid during reoxygenation. Let us consider how these conditions are realized in molluscs during anoxia.

1. The Pasteur effect has been observed in certain molluscs, however, only in moderate form; the ratio of anaerobic to aerobic carbohydrate consumption was between 1.2 and 2.4. Values of4.1 and 4.5 were also found in two species as exceptions (Brand, 1946; Brand et al., 1950). In other species this ratio was even below unity (Chapheau, 1932; Meenakshi, 1956,1958). Also, recent studies on A. cygnea (Sec- tion VIII) showed no Pasteur effect during anoxia. 2. Lactic acid production was analyzed in 20 species of snails under

anaerobic conditions (Brand et al., 1950). However, the total amount of lactic acid produced could explain only 1-12% of the carbohydrates consumed in the majority of cases. Only three species showed higher values, namely, Planorbarius corneus (22%), L. stagnalis (49%), and Lymnaea natalensis (50%). It is noted here that only these species displayed a low anoxic tolerance, they survived 24, 6, and 6 hours, respectively, whereas the other species survived for much longer times, up to 64 hours. Dugal and Fortier (1941) as well as Wernstedt (1944), could not observe increased lactic acid production during anoxia in Ostrea and in Dressenia, respectively. However, a certain increase in lactic acid production was found in Venus (Dugal, 1939) and in the oyster (Humphrey, 1949). One has to take into consideration that a certain level of increased lactic acid production during anoxia may also indicate the glycolytic activity of certain organs for which this is the only means of energy production. For instance, the heart of L. stagnalis contains very few or no cytosomes, and so do the adductors of A. cygnea (Section 111). It has been observed in the heart of a snail (Haeser and De Jorge, 1971) that the glycogen content is almost completely used up during an anoxic period of 25 hours.

Indirect but remarkable evidence against anaerobic glyco- or glycogenolysis as the only source of energy during anoxia is the fact that in A. cygnea the tissues still contain a large amount of glycogen when


anoxic tolerance has ended (Section VIII). If we assume that the absence of lactic acid production is due to a peculiar mechanism eliminating the lactic acid during anoxia, the limit of anoxic tolerance should be exhaustion of the glycogen reserves; however, this is not the case.

3. A moderate oxygen debt has been observed during anoxia of certain species (Martin, 1961; Goddard and Martin, 1966). However, this is insufficient evidence in itself for the exclusiveness of the anaerobic glyco- or glycogenolysis. It is noted that these types of analyses also require consideration of the changes in oxygen consumption accom- panying the different phases of the periodic activity of bivalves (Gart- kiewicz, 1923; Hers, 1943; Saliinki and Lukacsovics, 1967).

After all one has to accept that glyco- or glycogenolysis cannot be the exclusive energy-yielding mechanism in molluscs during anoxia. This opinion is shared by others (Hochachka et al., 1973). Of course, the first step of carbohydrate metabolism is realized by these processes even in anoxia, however, one has to suppose further metabolic events which are able to explain the high energy yield and the maintenance of a redox balance in the tissues.

D. A POSSIBLE MECHANISM OF CYTOSOMAL ENERGY PRODUCTION

From the data presented in this article (Sections V, VI, and X,A) it is obvious that cytosomes play an important role in anoxic energy production. Since oxygen storage as a cytosomal function could be excluded (Section X,B), one had to search for an acceptable hypothesis which could give a more-or-less complete explanation of the known experimental results.

The supposed cytosomal mechanism was called “anoxic endogenous oxidation” (Zs.-Nagy and Ermini, 1972b; Zs.-Nagy, 1973b, 1974), the essential points of which are as follows. Carbohydrate metabolism may take place as during aerobic respiration, however, at the end of the terminal electron transport chain the molecular oxygen is replaced by another molecule having a sufficiently high positive redox potential for electron acceptance. If the terminal electron transport chain functions, the coupled oxidative phosphorylation mechanisms may produce ATP. The energy yield will be proportional to the redox potential difference between NADH and the terminal electron acceptor. This assumed electron acceptor may be present in cytosomes in the preformed state, however, it may also be reproduced continuously during anoxia. Anoxic endogenous oxidation may function until the capacity of this electron acceptor is not exhausted, i.e.,

366 IMRE ZS.-NAGY

the resynthesis of the electron acceptor is possible. Such a mechanism requires some isolated compartments whch may be at the disposal within the cytosomes.

A basically similar mechanism has been revealed in certain facultative anaerobes (for references, see Bueding, 1962; Hochachka et al., 1973). It has been shown in some bacteria and also in invertebrates that NADH can be oxidized by fumarate during anoxia, that is, SDH functions in the reverse direction; it acts as a fumarate reductase and in this way oxidizes the flavoproteins; meanwhile ATP is generated. Fumarate is reproduced during the preceding metabolic steps by carbon dioxide fixation. In this pathway the electron acceptor function of the molecular oxygen is replaced by that of an organic molecule (fumarate), and the electron transfer is accompanied by the production of biologically utilizable energy.

However, the anoxic energy-producing processes in molluscs differ in many respects from the above-mentioned succinate-producing pathway. The latter process is localized in the mitochondria and, for example, for Ascaris it offers the main energy-yielding possibility, since the environment of this species is almost always anoxic (Bued- ing, 1962; Chen and Awapara, 1969), whereas the former processes take place within special cell organelles (cytosomes) and may function only for a certain period of time. A further difference is that in the muscle ofAscaris there is no CyO (Bueding, 1962); it is replaced by a terminal flavin oxidase, therefore the aerobic process itself is not sensitive to potassium cyanide, whereas in molluscs this enzyme is present (Section V,F).

Some essential conditions of the functioning of anoxic endogenous oxidation are discussed below, suggesting that this mechanism may be a physiological reality in molluscs.

1. The Presence and Nature of the Electron Acceptor The behavior of the redox potential of physiological solutions con-

taining isolated ganglia during anoxia proved that the pigmented nervous tissue contains an electron acceptor that is different from the oxygen (Section VI). As the experiments showed, this property of the ganglia is attributed to the lipochrome pigment of the cytosomes, and this electron acceptor capacity is exhausted during prolonged anoxia. The redox potential of the physiological solution at the beginning was about Ecal = 100-150 mV, which corresponds to a hydrogen electrode value of EH = 350-400 mV. At the same time the oxygen content was already rather low. The redox potential of the lipochrome pigment may be even higher, since this value is the resultant of the redox bal-


ance between the pigment and the reduced metabolites of the tissue. The decrease in the redox potential during anoxia indicates the increasing reduction of the pigment components. The value EH = 350-400 mV is higher than the redox potential of the CyO (Lehninger, 1965), therefore electron transfer from the latter to the pigment is phys- icochemically possible.

As regards the carotenoid components of the lipochrome pigment, one can exclude their role as definitive electron acceptors on the same quantitative basis excluding their oxygen-storing function (Section X,B). However, it is known that carotenoids are able to transport electrons easily from donor molecules to acceptors (Platt, 1959; Pullman and Pullman, 1963), therefore one can attribute a certain “electron pump” function to them.

The definitive electron acceptor function may be performed by unsaturated fatty acids. The results presented in this article (Sec- tions V,D and IX) clearly show an intense participation of cytosomal lipids in anoxic events. Since fatty acid synthesis requires ATP, one would expect a decrease in this process or even a complete stop during anoxia, when the energy shortage may reach a considerable extent. However, in spite of this expectation, fatty acid synthesis not only persisted even during anoxia, but saturated fatty acids were produced to a greater extent (Van der Horst, 1974; Oudejans and Van der Horst, 1974). A way to explain this unexpected phenomenon is to assume that during anoxia mainly unsaturated fatty acids are synthesized at the expense of a certain amount of ATP, and that they become saturated in a second step during which the original energy expense is repaid with a higher energy yield. The saturation of fatty acids represents an oxidative step for the hydrogen donors.

The biohydrogenation of unsaturated fatty acids is quite possible in molluscan tissues also, since an enzyme (NADPH-enoyl-CoA reductase) has been found in Candida cells and in rat liver. This enzyme is able to function only if NADPH is present as a specific electron donor (Ishidate et al., 1973; Mizugaki and Uchiyama, 1973). Similar enzymes may function in molluscs as well. If a suitable coupling exists between the hydrogen transfer from NADPH to the fatty acids and the phosphorylating mechanisms, ATP may be synthesized. Since cytosomal cation accumulation proved to be sensitive to DNP (Section V,F), one can accept the existence of this coupling.

The drastic decrease in the fatty acid content of the total lipids at the end of anoxic tolerance (Section IX) supports the electron acceptor function of fatty acids, although indirectly. Anoxic tolerance is most probably limited by the failure of hrther synthesis of unsaturated fatty

368 IMRE ZS.-NAGY

acids, and the animals die in spite of the presence of rather high glycogen reserves (Section VIII).

The question arises, Why do saturated fatty acids show no higher accumulation during anoxia and during the terminal phase? It is known that many poikilothermic animals possess a regulatory mechanism eliminating excess saturated fatty acids from lipids, maintaining in this way a sufficiently low lipid melting point (Farkas and Herodek, 1964; Knipprath and Mead, 1966; 1968; Kemp and Smith, 1970). Although in C. nemoralis the fatty acid composition showed no varia- tions during an annual cycle (Van der Horst and Zandee, 1973), one cannot exclude the functioning of this regulatory mechanism in all molluscs, since in other species it was confirmed (Thiele, 1959,1960). There is direct experimental evidence at our disposal showing that molluscs are able to transform rather rapidly excessive palmitic adid (16 : 0) into saturated hydrocarbons (Van der Horst and Oudejans, 1972). Such a mechanism can explain the rather high total lipid content accompanied by a drastic decrease in fatty acids in the whole body at the end of anoxic tolerance.

2. Bioenergetics of Anoxic Endogenous Oxidation

Within certain limits, the actual ATP content of the tissues may indicate the total energy balance, too. Therefore one can compare the theoretically calculated energy yield of anoxic endogenous oxidation with the real ATP content. If the redox potential of the lipochrome pigment is placed in the scheme of the terminal electron transport chain (Fig. 15), one can see that, except for the last step of ATP production taking place between CyO and oxygen, all other quantities of ATP may be produced. In this case the electron transfer is finished at the redox potential level of the pigment.

During carbohydrate metabolism, 93% of the total energy is realized by the terminal oxidation of NADH or NADPH (Lehninger, 1965). Electron transport to the pigment can yield about 56-57% of the total energy of the carbohydrate molecule. Therefore if the carbohydrate consumption is not increased, the anoxic endogenous oxidation may theoretically produce about two-thirds of the normal ATP level. The ATP contents of molluscs during anoxia (Section VII) agree rather well with these considerations, therefore one can assume anoxic endogenous oxidation to be a real possibility. It should be noted that the assumed biochemical mechanisms of anoxic energy production listed by Hochachka et al. (1973) may give maximally only 28% of the normal ATP yield of 1 mole of glucose 6-phosphate, and in addi- tion these mechanisms also require 2 moles of aspartate and 2 moles of glutamate per 1 mole of glucose.


Rcdox Pot rnx NADH

-300- \ } - 1 2 , 4 0 O c o l / m o l FP

0- cyt b p- } -4100 col/mol

c y t c c y t d / - I O , I O O cal/rnol

+ 30

+ 600 - - 24,400 eol/rnal I + 900 -

Total -52,000 cal/rnol

FIG. 15. Schematic drawing of the electron transport mechanism from NADH to oxygen with values of free-energy changes according to Lehninger (1965). The under- lined values of energy are used for ATP synthesis. The metabolization of 1 mole of glucose produces 12 pairs of electrons, that is, 624,000 cal representing about 93% of the total energy content of glucose. Anoxic endogenous oxidation functions at the expense of the oxidative capacity of the lipochrome pigment, therefore it can produce only 56-57% ofthe total energy realized in aerobiosis, i. e., it can yield roughly two-thirds of the normal ATP level. From Zs.-Nagy and Ermini (1972b), reproduced in Comp. Bio- chem. Physiol. B , by permission of Pergamon Press Ltd. Oxford.

3. Carbon Dioxide Production during Anoxia

Anoxic endogenous oxidation as explained above involves the functioning of the Krebs cycle, that is, carbon dioxide must be formed even during anoxia. It has been shown that in Mya arenaria (Collip, 1920, 1921) and Venus mercenaria (Dugal, 1939) the carbon dioxide content of the body fluid increases during anoxia, meanwhile the calcium content also increases. In Venus the carbon dioxide content increased from 6 to 130 vol% during 10 days of anoxia, and the increase in calcium content was even higher, however, the pH remained unchanged. In A. cygnea the calcium level increased by about four times after 8 days of anoxia at 15°C with a practically constant pH (Dotterweich and Elssner, 1935). Some carbon dioxide production was also observed during the anaerobic degradation of radiolabeled glucose in C. nemoralis, however, it was of much lesser extent than during aerobiosis (Oudejans and Van der Horst, 1974). The data of De Zwaan and Van Marrewijk (1973) (Section VIII) also suggest carbon dioxide production during anoxia, however, indirectly, since the water-soluble and volatile products originating in the labeled glucose were not identified.

Nevertheless, one has to accept that carbon dioxide production

370 IMRE ZS.-NAGY

occurs during anoxia in molluscs. This fact had been interpreted to indicate that carbon dioxide becomes liberated from the shells as an effect of lactic acid being produced by anaerobiosis. Since, however, investigations have not confirmed an increase in lactic acid production during anoxia (Section X,C), and because even the strongest organic acids are not able to liberate carbon dioxide from the carbonates of the shells, one has to reject this interpretation. It seems to be quite possible that the carbon dioxide is a product of the Krebs cycle even during anoxia, if anoxic endogenous oxidation really functions, and the mobilization of calcium should prevent acidosis of the tissues. At present nothing is known about the origin of this calcium, however, the possibility of its mobilization from the cation stores of the glioin- terstitial system (for details, see Nicaise, 1973) seems to be an attractive hypothesis.

The shells can hardly be responsible for this calcium mobilization, since experimental facts prove that in uivo or in uitro the shell and the mantle tissue of the oyster are able to incorporate very quickly labeled hydrocarbonate fiom the body fluid, and the isotope carbon atoms ap- pear in the carbonates or as components of the conchioline of the shell (Hammen and Wilbur, 1959). Under such circumstances the shells may rather represent a calcium-fixing site instead of a calcium- mobilizing one. These data show that in bivalves a carbon dioxide- fixing mechanism exists which, apart from the composition of the shells, may influence the carbon dioxide content of the body fluid under all circumstances. Thus acidosis of the tissues can be avoided even if carbon dioxide production is maintained in a closed system like the anoxia experiments. At the same time, these results call our attention to the necessity for careful interpretation of experimental results regarding anaerobic carbon dioxide production in molluscs.

Another possibility regarding the fixation of carbon dioxide during anoxia may be incorporation into organic acids, especially into succinate, as shown by Hammen and Wilbur (1959) and by Hammen (1966) in the mantle tissue of the oyster. This mechanism is rather similar to succinate production in other invertebrates (Bueding, 1962; Hochachka et al., 1973) and may be the reason for the high succinate content of certain bivalves (Aoki, 1932). Experimental data have shown that, for example, in the gills of two bivalves succinate (Ma- langa and Aiello, 1972), and in the mantle tissue of Rangia cuneata succinate and alanine, were produced, the latter in equimolar amounts, during anoxia (Stokes and Awapara, 1968). The latter phenomenon has been interpreted to indicate that half the carbohydrates consumed were transformed into succinate by means of oxidoreductive


steps, whereas the other half appeared as alanine through transamina- tion of pyruvate. However, these observations may be valid only for the mantle tissue, since in M. edulis succinate production increased only during anoxia, however, that of alanine did not (De Zwaan and Zandee, 1972b) and, what is more, the total amount of succinate, alanine, and D-lactate could explain only about half the carbohydrates consumed. The latter results suggest the possibility that succinate productive and cytosomal mechanisms may even function in parallel in different organs of the same animal.

Summarizing the considerations outlined in Section X,D, one can see that several essential conditions of anoxic endogenous oxidation exist in pigmented molluscan tissues. This mechanism of anoxic energy production may explain numerous phenomena of molluscan anaerobiosis. It should, however, be emphasized that numerous details are still unknown and, until biochemistry reveals more new facts, the theory of anoxic endogenous oxidation is considered a working hypothesis which offers a basis for further experiments.

XI. Summary and Conclusions

Many molluscan tissues contain yellow pigment granules called cytosomes. The greatest number of cytosomes occurs in nerve cell so- mata, but cytosomes can generally be found in all basic tissues. Cyto- soma1 pigments are lipochromes.

There are some species whose nervous systems are devoid of cytosomes, and these animals are unable to survive anoxic conditions, whereas those having a strongly pigmented central nervous system can survive complete anoxia for day- to week-long periods at room temperature without any essential damage.

The structure of cytosomes characteristically changes during anoxia. The homogeneous lipid droplets become transformed into membranes, and at the end of anoxic tolerance they are destroyed. Histo- chemical methods proved a significant decrease in the cytosomal lipid content during anoxia.

While cytosomes show very low or no acid phosphatase activity during normal respiration, this activity strongly increases during anoxia, indicating an important role of this enzyme and most probably also of other hydrolases in the realization of cytosomal functions.

Cytosomal membranes contain CyO and SDH activity. With the method of energy-dependent accumulation of divalent cations, an energy-producing mechanism can be revealed in cytosomes, which is

372 IMRE ZS.-NAGY

strongly activated during anoxia. This mechanism is inhibited completely be DNP and almost completely by potassium cyanide.

Ganglion tissue contains an electron acceptor which is able to main- tain a redox potential of E = 350-400 mV even during complete anoxia. The electron acceptor substance can be extracted together with the lipochrome pigment by ethanol or propanol.

In the whole body, one can find 52-94% of the normal ATP level up to the sixth day of anoxia in A. cygnea. At the end of anoxic tolerance, this value decreases to 11%. This high ATP yield cannot be explained by known anaerobic energy-producing mechanisms.

The carbohydrate consumption of A. cygnea at constant temperature proved to be identical under normal and anoxic conditions, that is, no Pasteur effect was observed.

Quantitative analyses of the possibilities for oxygen storage revealed that neither carotenoids nor heme pigments can store an amount of oxygen sufficient for the high energy yield during prolonged anoxia.

Intense fatty acid synthesis persists during anoxia, producing mainly saturated fatty acids, until anoxic tolerance is completely over. A drastic decrease in the fatty acid content of total lipids was observed when anoxic tolerance ended.

It is assumed that energy production in molluscs during prolonged anoxia is realized by means of a mechanism called anoxic endogenous oxidation. This mechanism is localized in the cytosomes, and its essential point is that the terminal electron acceptor function of the molecular oxygen is replaced by an internal electron acceptor. Nu- merous experimental results indicate that the assumed electron acceptor function during anoxia is performed by unsaturated fatty acids, the biohydrogenation of which represents an oxidative metabolic step for hydrogen donors like NADH and NADPH. The theoretically calculated energy yield of such a mechanism agrees with the experi- mentally measured ATP content of the tissues. This working hypothesis explains numerous phenomena of molluscan anaerobiosis.

ACKNOWLEDGMENT

The author thanks Dr. Christine M. Betts (London) for correcting the English in this paper.

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477.

Akadbmiai Kiad6, Budapest.

Grenoble,” Vol. 3, p. 155.

NOTE ADDED IN PROOF Since completion of this review (November, 1975) the following information has be-

come available: 1. Adenosine phosphate has been measured in crude TCA and PCA extracts of My-

tilus edulis by means of enzymic assay based on oxidation of NADH and calculation of concentrations from the change in absorbance at 340 nm, extrapolated to the starting point of the reaction [Wijsman, T. C. M. (1976),]. Comp. Physiol. 107, 1291. The ATP and ADP concentrations obtained with this method are much higher than those referred to in Section VII of this review, however, and are very different for TCA and PCA, whereas that of the AMP was in the same range. This discrepancy finds its explanation in the fact that crude TCA and PCA extracts of Mytilus gulloprooincialts show a considerable, although quantitatively different, oxidative effect on NADH in oitro even in the absence of the test solution enzymes used by Wijsman. This direct oxidation results in a pseudoreaction in the system [Zs.-Nagy, I. (1976), Comp. Biochem. Physiol., in press] which may greatly contribute to the ATP or ADP concentrations given by Wijsman. Therefore, the results obtained using crude extracts of marine bivalves for the enzymic


assay of adenosine phosphates cannot be regarded as reliable and, consequently, the conclusions and calculations based on these results by Wijsman are to be revised.

2. The anaerobic metabolism of bivalves has been reviewed [De Zwaan, A,, and Wijsman, T. C. M. (1976), Comp. Blochem. Physiol. 54B, 3131. These authors calculate, among other parameters, the metabolic rate for M . edulis under normal and anoxic conditions. Their ratio of aerob to anaerob ATP demand is almost 20. However, it should be pointed out that this calculation seems to be rather arbitrary. While the aerob ATP production was correctly calculated on the basis of average normal oxygen consumption, the ATP production in anaerobiosis is based only on the increase of the known anaerobic end products @lactate, alanine, succinate, and propionate) as well as on the decrease measured in ATP and the phosphoarginine pool. The sum of these ATP yields is taken as the total ATP demand in anoxia. The authors cited above therefore exclude a priori any possibility of production of ATP by other mechanisms that would result in end products different from the above-mentioned ones. The factor of 20 indicates that the known anaerobic energy-yielding mechanisms can offer only about 5% ofthe aerobic ATP demand in M . edulis in anoxia, which is most probably far below the values required for the maintenance of homeostasis. As a matter of fact, the ratio between active (aerobic) and standard (anaerobic) metabolism was found to be about 2.0 just in M . edulis widdows, J. (1973), Neth.]. Sea Res. 7,3871. Therefore, the argument against the existence of anoxic endogenous oxidation, based on the factor of 20 mentioned above [De Zwaan, A., Kluytmans, J. H. F. M., and Zandee, D. I. (1976), Biochem. Soc. Symp. 41, 1331, does not stand. On the contrary, the high value of this factor alone as calculated above suggests the existence of some anaerob energy-yielding mechanism apart from the known ones. On the other hand, if 5% of the aerob ATP production is the total anoxic ATP demand, this figure may easily be accounted for even by the classic lactic acid producing pathway without succinate productive and other mechanisms, but this is not the case.

3. The pyruvate kinase of the ganglia in M . edulis has recently been studied [De Zwaan, A., and Schoop, H. H. C. (1976), personal communication]. The characteristics of the ganglionic PyK proved to be different from those of the adductor muscle and mantle tissue by showing less pronounced allosteric behavior. At all examined pHs (6.2-7.5) the saturation curve for PEP was hyperbolic, with relatively small changes in K, values. At pH 7.5 alanine did not change the saturation curve in a sigmoid shape and there was only a minor increase in K, (0.77 to 1.5 mM/nPEP), whereas at low pH (6.5) there was a shift from hyperbolic to sigmoid shape when alanine was added. Therefore, it seems that this type of PyK is not as strongly controlled by pH and alanine as the muscle and mantle. At the same time PEP-carboxykinase activities are maintained at extremely low levels; therefore, even under anaerobic conditions it is quite possible that reasonable PyK activities persist, which is consistent with the hypothesis of anoxic endogenous oxidation. In this case all available PEP can be converted into pyruvate by PyK, i.e., the anaerobic function of the citrate cycle is quite possible.

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