- Email: [email protected]
Comments on review by G.E. Gibson and Ch. Peterson ‘Calcium and the aging nervous system’
Neurobiolo.ey ofAgin~e, Vol.
8, pp. 370-372. '~!~P e r g a m o n J o u r n a l s Ltd., 1987. Printed in the U . S . A .
8. Segal, J. Studies on the age-related decline in the response of lymphoid cells to mitogens: measurements of concanavalin A binding and stimulation of calcium and sugar uptake in thymocytes from rats of varying ages. Mech Ageing Dev 33: 295-303. 1986. 9. Staiano-Coico, L., Z. Darzynkiewicz, J. M. Hefton, R. Dub kowski, G. J. Darlington and M. E. Weksler. Increased sensitivity of lymphocytes from people over 65 to cell cycle arrest and chromosomal damage. Science 219: 1335-1337, 1983. 10. Thoman, M. L. and W. O. Weigle. Cell-mediated immunity in aged mice: an underlying lesion in 1L 2 synthesis. J lmmunol 128: 2358-2361, 1982.
iii~7-4580/87 $3.00 ~ ~
1I. Tsien, R. Y., T. Pozzan and T. J. Rink. I-cell mitogens cause early changes in cytoplasmic free Ca z+ and membrane potential in lymphocytes. Nature 295: 68-70, 1982. 12. Vie, H. and R. A. Miller. Decline, with age, in the proportion of mouse T cells that express IL-2 receptors after mitogen stimulation. Mech Ageing Dev 33: 313-322, 1986. 13. Waiters, C. S. and H. N. Claman. Age-related changes in cellmediated immunity in BALB/c mice. J lmmunol 115: 14381443, 1975.
Comments on Review by G. E. Gibson and Ch. Peterson 'Calcium and the Aging Nervous System' IMRE ZS.-NAGY
F. Verzdr International Laboratory for Experimental Gerontology (VILEG) Hungarian Section, University Medical School, H-4012 Debrecen, Hungary
Although the working hypothesis of the involvement of an altered Ca 2+permeation across membranes and Caz+ transport in the aging of the brain is attractive, this review suffers from several serious weaknesses indicating that such a concept actually remains only a speculation without any safe experimental basis.
T H E review in the title has to be criticized at least from 2 main points of view. (1) The p a p e r per se suffers from some weaknesses rendering t h e general conclusions of the authors shaky. (2) The available knowledge has arbitrarily been selected in the review and relevant information on calcium effects which may be of importance even for the working hypothesis of the authors have been omitted.
tion of the neuronal networks, presynaptic elements are post-synaptic in other connections and vice versa. A n interneuron represents a typical illustration o f this situation. This implies that there is no safe basis to claim that aging causes a decrease or increase o f the cytosolic free calcium, especially not in the nerve cells where " . . . direct e x a m i n a t i o n . . , has not been d o n e . " It seems to be rather risky to create an aging theory for the nervous system based on results obtained in mitochondria, fibroblasts, lymphocytes and parotid glands, meanwhile the only structure o f the nervous system studied so far has been the synaptosomal preparation of the brain. Even if there is no doubt on the significance of studying synaptosomes for certain purposes, one cannot accept the working hypothesis [2] before serious investigations will be carried out on various types of nerve cells both in the central and the peripheric nervous system. In the Summary the authors admit the "largely speculative" character o f their working hypothesis as regards the brain. Nevertheless, they force the reader to consider " s h o r t t e r m " and "long term" effects o f the altered calcium homeostasis. Under the latter possibility they claim that it " . . may lead to changes in D N A and R N A synthesis, as well as changes in protein turnover and lysosomal e n z y m e metabolism." Although this possibility, as I will explain below. would be of the greatest importance in my opinion, nothing at all is mentioned in the review which woukl indicate even a most indirect interrelationship o f the calcium homeostasis and the D N A and R N A synthesis. What is then the basis for making such kind o f declarations?
WEAKNESSES OF THE REVIEW Although in the Abstract the authors correctly apply expressions like " m a y b e , " " p r o b a b l y , " " m a y , " " a p p e a r s , " etc., when summarizing the ideas about the role of calcium in the aging process, reflecting the real situation, i.e., the lack of knowledge about the majority of the related problems, their Summary at the end of the paper uses strict affirmations, like " f o r m a common mechanism," " o c c u r s , " " a r e critical," etc. The trouble is that the content of the review does not convince anybody that the firm statements: of the Summary are justified. This ambiguity is characteristic for the whole review as illustrated below by several examples. We learn from the Introduction section that according to biochemical evidence, aging decreases cytosotic free calcium concentration, whereas electrophysiology indicates an elevated level of cytosolic free calcium concentration. The statement of the authors that "This difference may simply be that calcium homeostasis is presynaptic and post-synaptic regions do not respond similarly to the effects o f aging although they may be interrelated" cannot be considered as a suitable explanation, since due to t h e well known organiza-
370
COMMENTARY
371
THE OMITTEDRELEVANTINFORMATION Although the review contains quite a large number (265) of references, it is by far not covering the field sufficiently. For example, the review contains a section entitled "Calcium binding to membrane components," however, it simply disregards important data. Namely, it has been demonstrated that numerous effects of calcium are Amiloridesensitive, i.e., they are realized through some changes in conformation and functional ability of the membrane-bound sodium-channel protein [6, 10-12]. These data might even be of greater importance in an eventual hypothesis regarding the regulatory role of calcium in aging of the nervous system than those listed by the authors of the review. The silence about these data is all the more strange since the authors mention the function of the sodium-dependent calcium transport. In addition, the review contains a chapter "Interaction of calcium with other ions." It remains obscure for the reader, why the authors restrict this only to Mg and AI. In some other places the review mentions the role of calcium in the alterations of the potassium currents in the membrane, however, this fact remains uninterpreted in the final conclusion. The inducer role of calcium in cell proliferation [1, 3, 13] remains also out of the review. If one speculates on the role of calcium in aging of nerve cells, one has to consider also the possibility of the age-dependent reduction of this inducer role in the nervous system. The eventual interrelationships between the calcium homeostasis and the monovalent ion transports deserve interest, since the role of the monovalent ion contents in the cellular aging process has extensively been studied. The present comment does not permit a sufficient explanation of the relevant data, however, a very brief reference to the ideas called membrane hypothesis of aging (MHA) seems to be appropriate 114, 15, 17-21], since it may point out to the importance of learning more about the regulatory role of calcium on the monovalent ion transports. The MHA represents a cell physiological mechanism which can explain the aging process with general validity. Essential point of this hypothesis is that the general physicochemical background of the aging process is the
cross-linking effect of the oxygen free radicals, especially of the OH. radicals. The OH. free radical reactions are basically density-dependent, therefore, they result in more intermolecular damage where the molecules are nearer to each other. Since the most compact structures of the cells are the cell membranes, the rate of their damage is of higher rate than that of the cytosolic components. The cell plasma membrane is exposed to an additional damage caused by the so-called residual heat deriving from the action potentials (see [15]). As a consequence, the leading point of cellular aging is a gradual loss of permeability of the cell membrane for potassium (and probably for water) which, on the other hand, results in a gradual increase of the intracellular density. It is well established that all enzyme activities show an inverse exponential correlation with the density of their molecular environment (see [18]). The increase of the intracellular density up to a certain level is an inherent phenomenon of the cell maturation and differentiation, however, this process does not stop at a certain age. Therefore, the continuous "maturation" becomes a suicide process for the cell, since the increasing intracellular density will cause a decrease of all enzyme activities. Among others, enzymes participating in the direct radical defense (e.g., superoxide dismutase, catalase, glutathione peroxidase) become less efficient, and furthermore, the enzymes participating in the elimination and replacement of the damaged components will also be insufficient. The result of this situation is a dehydration of the body, an increase of the cross-linked proteins [4, 5, 16, 22, 23], as well as of the waste-products (lipofuscin) in the cells accompanied by a slowing down of the RNA synthesis rate (and protein turnover) [7-9]. The age-dependent loss of the intracellular water content and a concomitant increase of the intracellular density in itself would be sufficient to explain all the observed age-dependent alterations of the calcium homeostasis without assuming any special causal role of calcium in this process. On the other hand, it would be extremely interesting to know more about the eventual role of calcium in the regulation of these processes at various levels of its metabolic activity. If the very speculative working hypothesis of Gibson and Peterson [2] will stimulate further studies on the role of calcium, it was worth while to publish it in spite of the defects listed above.
REFERENCES
1. Epel, D. Ionic triggers in the fertilization of sea urchin eggs. Ann N Y Acad Sci 339: 74-85, 1980. 2. Gibson, G. E. and Ch. Peterson. Calcium and the aging nervous system. Neurobiol Aging 8: 32%343, 1987. 3. Jaffe, L. F. Calcium explosions as triggers of development. Ann N Y Acad Sci 339: 86-101, 1980. 4. Lustyik, Gy. and I. Zs.-Nagy. Alterations of the intracellular water and ion concentrations in brain and liver cells during aging as revealed by energy dispersive X-ray microanalysis of bulk specimens. Scan Electron Microsc 1985 h 323-337, 1985. 5. Nagy, K. and I. Zs.-Nagy. Alterations in the molecular weight distribution of proteins in rat brain synaptosomes during aging and centrophenoxine treatment. Mech Ageing Dev 28: 171-176, 1984. 6. Owen, N. E. and M. L. Villereal. Evidence for a role of calmodulin in serum stimulation of Na+-influx in human fibroblasts. Proc Natl Acad Sci USA 79: 3537-3541, 1982. 7. Richardson, A. and I. Semsei. Effect of aging on translation and transcription. In: Review o f Biological Research in Aging, Vol 3, edited by M. Rothstein. New York: Alan R. Liss, Inc., 1986, in press.
8. Richardson, A., M. S. Roberts and M. S. Rutherford. Aging and gene expression. In: Review o f Biological Research in Aging. vol 2, edited by M. Rothstein. New York: Alan R. Liss, Inc., 1985, pp. 395-419. 9. Semsei, I., F. Szeszfik and I. Zs.-Nagy. In vivo studies on the age-dependent decrease of the rates of total and mRNA synthesis in the brain cortex of rats. Arch Gerontol Geriatr 1: 29-42, 1982. 10. Vicentini, L. M. and M. L. Villereal. Activation of Na+/H+ exchange in cultured fibroblasts: Synergism and antagonism between phorbol ester, Caz+ ionophore, and growth factors. Proc Natl Acad Sci USA 82: 8053-8056, 1985. 11. Villereal, M. L. Sodium fluxes in human fibroblasts: effect of serum, Ca2+, and amiloride. J Cell Physiol 107: 35%369, 1981. 12. Villereal, M. L. Inhibition of the serum-dependent, amiloridesensitive sodium transport pathway in human fibroblasts by extracellular divalent cations. J Cell Physiol 111: 163-170, 1982. 13. Whitefield, J. F., A. L. Boynton, J. P. Macmanus, R. H. Rixon, M. Sikorska, B. Tsang and P. R. Walker. The roles of calcium and cyclic AMP in cell proliferation. Ann N Y Acad Sci 339: 216-240, 1980.
Neurobiology of Aging,
Vol. 8, pp. 372-375. " P e r g a m o n J o u r n a l s Ltd., 1987. Printed in the U . S . A .
14. Zs.-Nagy, I. A membrane hypothesis of aging. J Theor Biol 75: 189-195, 1978. 15. Zs.-Nagy, I. The role of membrane structure and function in cellular ageing: a review. Mech Ageing Dev 9: 237-246, 1979. 16. Zs.-Nagy, I. Energy dispersive X-ray microanalysis of biological bulk specimens: a review on the method and its application to experimental gerontology and cancer research. Scann Electron Microsc 1983 IH: 1255--1268, 1983. 17. Zs.-Nagy, I. Aging of the cellular membrane: Basic principles and pharmacological interventions. Geriatrika h 102-1 i 1, 1985. 18. Zs.-Nagy, I. Common mechanisms of cellular aging in brain and liver in the light of the membrane hypothesis of aging. In: Liver and Aging--1986, Liver and Brain, edited by K. Kitani. Amsterdam: Elsevier Science Publishers, 1986, pp. 373-387. 19. Zs.-Nagy, I. An attempt to answer the questions of theoretical gerontology on the basis of the membrane hypothesis of aging. In: Age Pigments: Biological Markers of" Aging and Environmental Stress?, edited by E. Aloj Totaro, F. A. Pisanti and P. Glees. London: Pergamon Press, in press, 1987.
tt197-4580/87 $3.00 ~ .I~1
20. Zs.-Nagy, I. Functional consequences of free radical damage to cell membrane. In: CRC Handbook of Biomedicine of Free Radicals and Antioxidants, edited by J. Micluel, H. Weber and A. Quintanilha. Boca Raton, FL: CRC Press Inc., 1987, submitted. 21. Zs.-Nagy, I. and I. Semsei. Centrophenoxine increases the rates of total and mRNA synthesis in the brain cortex of old rats~ an explanation of its action in terms of the membrane hypothesis of aging. Exp Gerontol 19: 171-178, 1984. 22. Zs.-Nagy, I., K. Nagy, V. Zs.-Nagy, A. Kalmfir and E. Nagy. Alterations in total content and solubility characteristics of proteins in rat brain and liver during aging and centrophenoxine treatment. Exp Gerontol 16: 229-240, 1981. 23. Zs.-Nagy, I., Gy. Lustyik and C. Bertoni-Freddari. Intracellular water and dry mass content as measured in bulk specimens by energy-dispersive X-ray microanalysis. Tissue Cell 14: 47-60, 1982.
Authors' Response to Commentaries GARY E. GIBSON* A N D CHRISTINE PETERSONt *Department o f Neurology, Cornell University Medical College at the Burke Rehabilitation Center, White Plains, N Y 10605 and t D e p a r t m e n t o f Psychobiology University o f California, Irvine, CA 92717
THE goal o f the Review [8] was to examine the literature on calcium homeostasis during aging and to relate these findings to current knowledge of calcium homeostasis in a cohesive manner. Each reviewer expands on at least one aspect of cellular calcium homeostasis that may be critical to the aging process. F o r example, Trabucchi and Govoni [28], as well as Leslie [15], extend our limited treatment of calcium channels, while Zs.-Nagy [30] emphasizes the importance of amelioride sensitive calcium movements and calcium's interaction with potassium. The combination of our Review and the commentaries delineate the current status of research on calcium homeostasis during aging and identify questions that are unanswered due to technical limitations or experiments that have just not been done. In the Review, we tried to synthesize all o f the present results that relate calcium and aging into an hypothesis that reconciles the apparent discrepancy in neurochemical and electrophysiological findings and that would suggest future experimental directions. Both direct and indirect studies suggest that aging leads to altered calcium distributions and diminished movement o f calcium across membranes; both o f these changes appear to be physiologically important. As Michaelis stresses [17], these are vague generalities, however, these alterations appear to be a common denominator in most published studies of aging. Both the cause and the extent o f the alterations in calcium homeostasis, as well as their relation to normal cellular function, need to be established. As pointed out, particularly by Lust [16], Barritt [1] and Zs.-Nagy [30], this is exceedingly difficult due to the
complexity of the nervous system. Even in various muscle types [26], aging may change calcium homeostasis differently. Because of these complexities, some recent studies have concentrated on more homogeneous cell populations (i.e. lymphocytes [18] and fibroblasts [21]) in order to understand aging. The Review was not intended to provide an all inclusive theory of aging. The membrane hypothesis of aging [30] attempts to do this, but it is unclear how it relates to changes in brain calcium homeostasis, which occur without measurable changes in water content. Etienne and Baudry [4] extend the theory on calcium activated proteases to encompass a more general theory of aging. Although the ealpain hypothesis may account for some age-related brain pathology, it is not clear how brain calpain may account for differences in maximal life span across species. Man often dies o f non-brain related disorders and this presumably haPl~ns in o t h e r species, as well. Furthermore, the method for s~lecting a brain region to do cross species analysis is unclear; the distribution of calpain varies considerably between brain regions and cell types. Some of the reviewers strongly suggest that aging research should move away from studying the ~ r d t i e s of calcium homeostasis. Lust suggests that calcium is too complicated to study, especially in vivo. This criticism should create caution in intmlm~ting results, but n o t encourage abandonment o f the area. " S i m p l e " and intact systems are attractive for more precisely d e t e ~ w h i c h steps are altered, so that a result can be placed in a physiological
372
8, pp. 370-372. '~!~P e r g a m o n J o u r n a l s Ltd., 1987. Printed in the U . S . A .
8. Segal, J. Studies on the age-related decline in the response of lymphoid cells to mitogens: measurements of concanavalin A binding and stimulation of calcium and sugar uptake in thymocytes from rats of varying ages. Mech Ageing Dev 33: 295-303. 1986. 9. Staiano-Coico, L., Z. Darzynkiewicz, J. M. Hefton, R. Dub kowski, G. J. Darlington and M. E. Weksler. Increased sensitivity of lymphocytes from people over 65 to cell cycle arrest and chromosomal damage. Science 219: 1335-1337, 1983. 10. Thoman, M. L. and W. O. Weigle. Cell-mediated immunity in aged mice: an underlying lesion in 1L 2 synthesis. J lmmunol 128: 2358-2361, 1982.
iii~7-4580/87 $3.00 ~ ~
1I. Tsien, R. Y., T. Pozzan and T. J. Rink. I-cell mitogens cause early changes in cytoplasmic free Ca z+ and membrane potential in lymphocytes. Nature 295: 68-70, 1982. 12. Vie, H. and R. A. Miller. Decline, with age, in the proportion of mouse T cells that express IL-2 receptors after mitogen stimulation. Mech Ageing Dev 33: 313-322, 1986. 13. Waiters, C. S. and H. N. Claman. Age-related changes in cellmediated immunity in BALB/c mice. J lmmunol 115: 14381443, 1975.
Comments on Review by G. E. Gibson and Ch. Peterson 'Calcium and the Aging Nervous System' IMRE ZS.-NAGY
F. Verzdr International Laboratory for Experimental Gerontology (VILEG) Hungarian Section, University Medical School, H-4012 Debrecen, Hungary
Although the working hypothesis of the involvement of an altered Ca 2+permeation across membranes and Caz+ transport in the aging of the brain is attractive, this review suffers from several serious weaknesses indicating that such a concept actually remains only a speculation without any safe experimental basis.
T H E review in the title has to be criticized at least from 2 main points of view. (1) The p a p e r per se suffers from some weaknesses rendering t h e general conclusions of the authors shaky. (2) The available knowledge has arbitrarily been selected in the review and relevant information on calcium effects which may be of importance even for the working hypothesis of the authors have been omitted.
tion of the neuronal networks, presynaptic elements are post-synaptic in other connections and vice versa. A n interneuron represents a typical illustration o f this situation. This implies that there is no safe basis to claim that aging causes a decrease or increase o f the cytosolic free calcium, especially not in the nerve cells where " . . . direct e x a m i n a t i o n . . , has not been d o n e . " It seems to be rather risky to create an aging theory for the nervous system based on results obtained in mitochondria, fibroblasts, lymphocytes and parotid glands, meanwhile the only structure o f the nervous system studied so far has been the synaptosomal preparation of the brain. Even if there is no doubt on the significance of studying synaptosomes for certain purposes, one cannot accept the working hypothesis [2] before serious investigations will be carried out on various types of nerve cells both in the central and the peripheric nervous system. In the Summary the authors admit the "largely speculative" character o f their working hypothesis as regards the brain. Nevertheless, they force the reader to consider " s h o r t t e r m " and "long term" effects o f the altered calcium homeostasis. Under the latter possibility they claim that it " . . may lead to changes in D N A and R N A synthesis, as well as changes in protein turnover and lysosomal e n z y m e metabolism." Although this possibility, as I will explain below. would be of the greatest importance in my opinion, nothing at all is mentioned in the review which woukl indicate even a most indirect interrelationship o f the calcium homeostasis and the D N A and R N A synthesis. What is then the basis for making such kind o f declarations?
WEAKNESSES OF THE REVIEW Although in the Abstract the authors correctly apply expressions like " m a y b e , " " p r o b a b l y , " " m a y , " " a p p e a r s , " etc., when summarizing the ideas about the role of calcium in the aging process, reflecting the real situation, i.e., the lack of knowledge about the majority of the related problems, their Summary at the end of the paper uses strict affirmations, like " f o r m a common mechanism," " o c c u r s , " " a r e critical," etc. The trouble is that the content of the review does not convince anybody that the firm statements: of the Summary are justified. This ambiguity is characteristic for the whole review as illustrated below by several examples. We learn from the Introduction section that according to biochemical evidence, aging decreases cytosotic free calcium concentration, whereas electrophysiology indicates an elevated level of cytosolic free calcium concentration. The statement of the authors that "This difference may simply be that calcium homeostasis is presynaptic and post-synaptic regions do not respond similarly to the effects o f aging although they may be interrelated" cannot be considered as a suitable explanation, since due to t h e well known organiza-
370
COMMENTARY
371
THE OMITTEDRELEVANTINFORMATION Although the review contains quite a large number (265) of references, it is by far not covering the field sufficiently. For example, the review contains a section entitled "Calcium binding to membrane components," however, it simply disregards important data. Namely, it has been demonstrated that numerous effects of calcium are Amiloridesensitive, i.e., they are realized through some changes in conformation and functional ability of the membrane-bound sodium-channel protein [6, 10-12]. These data might even be of greater importance in an eventual hypothesis regarding the regulatory role of calcium in aging of the nervous system than those listed by the authors of the review. The silence about these data is all the more strange since the authors mention the function of the sodium-dependent calcium transport. In addition, the review contains a chapter "Interaction of calcium with other ions." It remains obscure for the reader, why the authors restrict this only to Mg and AI. In some other places the review mentions the role of calcium in the alterations of the potassium currents in the membrane, however, this fact remains uninterpreted in the final conclusion. The inducer role of calcium in cell proliferation [1, 3, 13] remains also out of the review. If one speculates on the role of calcium in aging of nerve cells, one has to consider also the possibility of the age-dependent reduction of this inducer role in the nervous system. The eventual interrelationships between the calcium homeostasis and the monovalent ion transports deserve interest, since the role of the monovalent ion contents in the cellular aging process has extensively been studied. The present comment does not permit a sufficient explanation of the relevant data, however, a very brief reference to the ideas called membrane hypothesis of aging (MHA) seems to be appropriate 114, 15, 17-21], since it may point out to the importance of learning more about the regulatory role of calcium on the monovalent ion transports. The MHA represents a cell physiological mechanism which can explain the aging process with general validity. Essential point of this hypothesis is that the general physicochemical background of the aging process is the
cross-linking effect of the oxygen free radicals, especially of the OH. radicals. The OH. free radical reactions are basically density-dependent, therefore, they result in more intermolecular damage where the molecules are nearer to each other. Since the most compact structures of the cells are the cell membranes, the rate of their damage is of higher rate than that of the cytosolic components. The cell plasma membrane is exposed to an additional damage caused by the so-called residual heat deriving from the action potentials (see [15]). As a consequence, the leading point of cellular aging is a gradual loss of permeability of the cell membrane for potassium (and probably for water) which, on the other hand, results in a gradual increase of the intracellular density. It is well established that all enzyme activities show an inverse exponential correlation with the density of their molecular environment (see [18]). The increase of the intracellular density up to a certain level is an inherent phenomenon of the cell maturation and differentiation, however, this process does not stop at a certain age. Therefore, the continuous "maturation" becomes a suicide process for the cell, since the increasing intracellular density will cause a decrease of all enzyme activities. Among others, enzymes participating in the direct radical defense (e.g., superoxide dismutase, catalase, glutathione peroxidase) become less efficient, and furthermore, the enzymes participating in the elimination and replacement of the damaged components will also be insufficient. The result of this situation is a dehydration of the body, an increase of the cross-linked proteins [4, 5, 16, 22, 23], as well as of the waste-products (lipofuscin) in the cells accompanied by a slowing down of the RNA synthesis rate (and protein turnover) [7-9]. The age-dependent loss of the intracellular water content and a concomitant increase of the intracellular density in itself would be sufficient to explain all the observed age-dependent alterations of the calcium homeostasis without assuming any special causal role of calcium in this process. On the other hand, it would be extremely interesting to know more about the eventual role of calcium in the regulation of these processes at various levels of its metabolic activity. If the very speculative working hypothesis of Gibson and Peterson [2] will stimulate further studies on the role of calcium, it was worth while to publish it in spite of the defects listed above.
REFERENCES
1. Epel, D. Ionic triggers in the fertilization of sea urchin eggs. Ann N Y Acad Sci 339: 74-85, 1980. 2. Gibson, G. E. and Ch. Peterson. Calcium and the aging nervous system. Neurobiol Aging 8: 32%343, 1987. 3. Jaffe, L. F. Calcium explosions as triggers of development. Ann N Y Acad Sci 339: 86-101, 1980. 4. Lustyik, Gy. and I. Zs.-Nagy. Alterations of the intracellular water and ion concentrations in brain and liver cells during aging as revealed by energy dispersive X-ray microanalysis of bulk specimens. Scan Electron Microsc 1985 h 323-337, 1985. 5. Nagy, K. and I. Zs.-Nagy. Alterations in the molecular weight distribution of proteins in rat brain synaptosomes during aging and centrophenoxine treatment. Mech Ageing Dev 28: 171-176, 1984. 6. Owen, N. E. and M. L. Villereal. Evidence for a role of calmodulin in serum stimulation of Na+-influx in human fibroblasts. Proc Natl Acad Sci USA 79: 3537-3541, 1982. 7. Richardson, A. and I. Semsei. Effect of aging on translation and transcription. In: Review o f Biological Research in Aging, Vol 3, edited by M. Rothstein. New York: Alan R. Liss, Inc., 1986, in press.
8. Richardson, A., M. S. Roberts and M. S. Rutherford. Aging and gene expression. In: Review o f Biological Research in Aging. vol 2, edited by M. Rothstein. New York: Alan R. Liss, Inc., 1985, pp. 395-419. 9. Semsei, I., F. Szeszfik and I. Zs.-Nagy. In vivo studies on the age-dependent decrease of the rates of total and mRNA synthesis in the brain cortex of rats. Arch Gerontol Geriatr 1: 29-42, 1982. 10. Vicentini, L. M. and M. L. Villereal. Activation of Na+/H+ exchange in cultured fibroblasts: Synergism and antagonism between phorbol ester, Caz+ ionophore, and growth factors. Proc Natl Acad Sci USA 82: 8053-8056, 1985. 11. Villereal, M. L. Sodium fluxes in human fibroblasts: effect of serum, Ca2+, and amiloride. J Cell Physiol 107: 35%369, 1981. 12. Villereal, M. L. Inhibition of the serum-dependent, amiloridesensitive sodium transport pathway in human fibroblasts by extracellular divalent cations. J Cell Physiol 111: 163-170, 1982. 13. Whitefield, J. F., A. L. Boynton, J. P. Macmanus, R. H. Rixon, M. Sikorska, B. Tsang and P. R. Walker. The roles of calcium and cyclic AMP in cell proliferation. Ann N Y Acad Sci 339: 216-240, 1980.
Neurobiology of Aging,
Vol. 8, pp. 372-375. " P e r g a m o n J o u r n a l s Ltd., 1987. Printed in the U . S . A .
14. Zs.-Nagy, I. A membrane hypothesis of aging. J Theor Biol 75: 189-195, 1978. 15. Zs.-Nagy, I. The role of membrane structure and function in cellular ageing: a review. Mech Ageing Dev 9: 237-246, 1979. 16. Zs.-Nagy, I. Energy dispersive X-ray microanalysis of biological bulk specimens: a review on the method and its application to experimental gerontology and cancer research. Scann Electron Microsc 1983 IH: 1255--1268, 1983. 17. Zs.-Nagy, I. Aging of the cellular membrane: Basic principles and pharmacological interventions. Geriatrika h 102-1 i 1, 1985. 18. Zs.-Nagy, I. Common mechanisms of cellular aging in brain and liver in the light of the membrane hypothesis of aging. In: Liver and Aging--1986, Liver and Brain, edited by K. Kitani. Amsterdam: Elsevier Science Publishers, 1986, pp. 373-387. 19. Zs.-Nagy, I. An attempt to answer the questions of theoretical gerontology on the basis of the membrane hypothesis of aging. In: Age Pigments: Biological Markers of" Aging and Environmental Stress?, edited by E. Aloj Totaro, F. A. Pisanti and P. Glees. London: Pergamon Press, in press, 1987.
tt197-4580/87 $3.00 ~ .I~1
20. Zs.-Nagy, I. Functional consequences of free radical damage to cell membrane. In: CRC Handbook of Biomedicine of Free Radicals and Antioxidants, edited by J. Micluel, H. Weber and A. Quintanilha. Boca Raton, FL: CRC Press Inc., 1987, submitted. 21. Zs.-Nagy, I. and I. Semsei. Centrophenoxine increases the rates of total and mRNA synthesis in the brain cortex of old rats~ an explanation of its action in terms of the membrane hypothesis of aging. Exp Gerontol 19: 171-178, 1984. 22. Zs.-Nagy, I., K. Nagy, V. Zs.-Nagy, A. Kalmfir and E. Nagy. Alterations in total content and solubility characteristics of proteins in rat brain and liver during aging and centrophenoxine treatment. Exp Gerontol 16: 229-240, 1981. 23. Zs.-Nagy, I., Gy. Lustyik and C. Bertoni-Freddari. Intracellular water and dry mass content as measured in bulk specimens by energy-dispersive X-ray microanalysis. Tissue Cell 14: 47-60, 1982.
Authors' Response to Commentaries GARY E. GIBSON* A N D CHRISTINE PETERSONt *Department o f Neurology, Cornell University Medical College at the Burke Rehabilitation Center, White Plains, N Y 10605 and t D e p a r t m e n t o f Psychobiology University o f California, Irvine, CA 92717
THE goal o f the Review [8] was to examine the literature on calcium homeostasis during aging and to relate these findings to current knowledge of calcium homeostasis in a cohesive manner. Each reviewer expands on at least one aspect of cellular calcium homeostasis that may be critical to the aging process. F o r example, Trabucchi and Govoni [28], as well as Leslie [15], extend our limited treatment of calcium channels, while Zs.-Nagy [30] emphasizes the importance of amelioride sensitive calcium movements and calcium's interaction with potassium. The combination of our Review and the commentaries delineate the current status of research on calcium homeostasis during aging and identify questions that are unanswered due to technical limitations or experiments that have just not been done. In the Review, we tried to synthesize all o f the present results that relate calcium and aging into an hypothesis that reconciles the apparent discrepancy in neurochemical and electrophysiological findings and that would suggest future experimental directions. Both direct and indirect studies suggest that aging leads to altered calcium distributions and diminished movement o f calcium across membranes; both o f these changes appear to be physiologically important. As Michaelis stresses [17], these are vague generalities, however, these alterations appear to be a common denominator in most published studies of aging. Both the cause and the extent o f the alterations in calcium homeostasis, as well as their relation to normal cellular function, need to be established. As pointed out, particularly by Lust [16], Barritt [1] and Zs.-Nagy [30], this is exceedingly difficult due to the
complexity of the nervous system. Even in various muscle types [26], aging may change calcium homeostasis differently. Because of these complexities, some recent studies have concentrated on more homogeneous cell populations (i.e. lymphocytes [18] and fibroblasts [21]) in order to understand aging. The Review was not intended to provide an all inclusive theory of aging. The membrane hypothesis of aging [30] attempts to do this, but it is unclear how it relates to changes in brain calcium homeostasis, which occur without measurable changes in water content. Etienne and Baudry [4] extend the theory on calcium activated proteases to encompass a more general theory of aging. Although the ealpain hypothesis may account for some age-related brain pathology, it is not clear how brain calpain may account for differences in maximal life span across species. Man often dies o f non-brain related disorders and this presumably haPl~ns in o t h e r species, as well. Furthermore, the method for s~lecting a brain region to do cross species analysis is unclear; the distribution of calpain varies considerably between brain regions and cell types. Some of the reviewers strongly suggest that aging research should move away from studying the ~ r d t i e s of calcium homeostasis. Lust suggests that calcium is too complicated to study, especially in vivo. This criticism should create caution in intmlm~ting results, but n o t encourage abandonment o f the area. " S i m p l e " and intact systems are attractive for more precisely d e t e ~ w h i c h steps are altered, so that a result can be placed in a physiological
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