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Parameters to monitor aging with a possible perspective for intervention — an immunological approach
Arch. Gerontol. Geriatr., 12 (1991)231-238
231
© 1991 Elsevier Science Publishers B.V. 0167-4943/91/$03.50
A G G 00390
Parameters to m o n i t o r aging with a possible perspective for intervention an i m m u n o l o g i c a l approach * Carlo Pieri, R i n a R e c c h i o n i , F a u s t o M o r o n i , M a r c o F a l a s c a
and Sandor Damjanovich 1 and
1
Cytology Center, Gerontological Research Department of INRCA, Ancona, Italy," Department of Bwphyslcs, University Medical School of Debrecen, H-4012, Debrecen, Hungary .
.
(Received 1 October 1990; accepted 13 December 1990)
Summary Reliable aging markers are very rare, which are better than the chronological age or those symptoms which have such great individual variability that their scientific value is questionable. The effect of aging on immunological behavior of h u m a n (and animal) individuals is reasonably well established. In this communication an attempt is made to find an immunological marker of aging at the level of cell surface phenomena. It was observed that ion-channel activities, having a complex regulation, loose their flexible responsiveness in lymphocyte membranes during aging. A recently discovered voltage regulation of the calcium-activated potassium channels showed a distinct change with aging of h u m a n lymphocytes. A possibility to find a better marker system in complex regulatory processes is also discussed. Lymphocyte membranes; Ca2+-activated K + channel; Voltage regulation; Aging
Introduction
Aging, apart from the chronological one is a very loosely defined parameter, describing the gradual loss or changing of abilities either inherited or acquired throughout the progression of the time of life. The great individual variations make a definition of a clearly worded indicator parameter extremely difficult. Since the effect of increasing age on the human (and also animal) immune system has been clearly documented, the present brief communication will concentrate on lympho-
* Paper presented by S. Damjanovich, and discussed in the program of the International Symposium 'Fritz Verzfir Project - 2000', held from 30 September to 3 October 1990 at Ancona, Italy. Correspondence to: S. Damjanovich, Department of Biophysics, University Medical School of Debrecen, H-4012 Debrecen, Hungary.
232
cytes (Thoman and Wiegle, 1989). The parameters to be analyzed are mostly regulatory ones and are connected to the lymphocyte plasma membrane. The reason for this aspect is that this is the level where the extracellular world encounters with the cells for the first time. Furthermore the plasma membrane is the main theater of the signal transmission between the outside and inside of cellular systems. Thus the altered functions of the plasma membrane could significantly contribute to the aging processes. A number of cell surface parameters investigated so far in connection with the immunological decline during aging showed rather vaguely observable changes, if any. Nevertheless, it is well documented that proliferation to mitogenic lectins and alloantigens, the generation of cytolytic effector cells, delayed type hypersensitivity, and primary and secondary antibody responses are diminished in the aged (Thoman and Weigle, 1989). The advent of refined technics in lymphocyte membrane physiology like patch clamp and the flow cytometric membrane potential measurements made possible the objective monitoring of individual channel activities and changes in the membrane potential for such cells with great accuracy (for review see Matko et al., 1988). Our attention has been directed towards this field, since hypothetically the fine regulatory processes of the immune response at this level are more likely to be impaired either by stochastically occurring injuries caused by extracellular agents like free radicals or any preprogrammed processes which are generally supposed to contribute to aging (Zs.-Nagy, 1978; O'Leary et al., 1985; Damjanovich et al., 1989; Varga et al., 1989). In the first part of this communication we intend to concentrate on some basic but complicated processes in the cell membrane and the means how to study them. In the second part, a different responsiveness of the calcium-activated potassium channels will be discussed with special reference to its age-dependent nature and its newly discovered voltage regulation (Matyus et al., 1990).
Cell surface phenomena and biological information transfer in lymphocytes Transmembrane signalling is known to involve a number of physical and biochemical steps, which finally lead to cell activation, i.e., transcription of newly activated genes and in case of particular subsets of lymphocytes proliferation and differentiation (Berridge and Irvine, 1989). The signal-transducing pathways a r e dependent on the activation signal (Kozumbo et al., 1987; Mills et al., 1988; Valge et al., 1988; Tigges et al., 1989). Alloantigens or mitogenic lectins activate lymphocytes through a well studied pathway. The binding of the signal transducer to the appropriate cell surface receptor induces a receptor aggregation, sometimes translocation or assembly of different subunits (Berridge and Irvine, 1989). Changes in ion channel activities and membrane potential level are also well known and frequently observed concomitant early signs of signal transduction. However, their exact role in the transduction mechanism is not well understood. An increase in the intracellular C a 2+ level, phospholipase-C digested phosphatidylinositol breakdown products and finally the
233 activation of protein kinase C enzyme represent the key elements of a complicated biochemical cascade (Berridge and Irvine, 1989). This is a very frequently occurring chain of events, but it is far from being the only possible pathway. Activation of tyrosine kinases, cAMP and cGMP activated proteins (G-proteins), belong also to the possible alternative pathways depending on the kind of the activation signal (Tigges et al., 1989). The only common denominator is that all these pathways seems to phosphorylate proteins and these are supposedly the final messengers initiating gene transcription and cell proliferation. A high number of different gene products are among the early (a few minutes) and late (hours, days) markers of cell activation processes, but for only a few of them we can assign a function yet (Crabtree, 1989). The best studied early markers are the protooncogenes c-myc and c-fos and from among the late markers the c-myb. In case of the best studied T lymphocytes the interleukin-2 synthesis and the interleukin-2 and transferrin receptor expression in the plasma membrane are important factors of the cell activation. The interleukin-2, a lymphokine induced cell activation does not demand protein kinase C activation or an increase in the intracellular Ca 2+ level (Mills et al., 1988, Valge et al., 1988; Tigges et al., 1989). Recently we observed that a specific ion channel opener, bretylium tosylate, that opens otherwise silent sodium channels, influences cell activation (Pieri et al., 1989, 1990). At low doses of BT a dose dependent enhancement, at higher doses an inhibition of T lymphocyte proliferation was observed. The BT, which also requires a slight partial depolarization of lymphocytes prior to be able to open sodium channels, could also differentiate between alloantigenic (or lectin), and IL-2 induced cell activation processes. The activators using the protein kinase C activating stimulatory pathway for signal transduction, were negatively influenced, i.e., inhibited in the presence of BT concentrations which significantly enhanced the cell proliferation of IL-2 induced activation of so-called IL-2 primed, i.e., IL-2 receptor expressing human peripheral blood lymphocytes. The activation was influenced by the BT not solely at the level of 3H-thymidine incorporation. Northern blot analysis revealed that BT was able to enhance the expression of the IL-2 receptor gene, c-myc and c-myb oncogenes at the RNA synthesis level, when added together with IL-2. The same doses of BT (0.25-1 mM) added together with PHA to human peripheral blood lymphocytes inhibited the 3H-thymidine incorporation and blocked the cell proliferation mostly in G 1 and S phases. At the same time neither the IL-2 production nor the IL-2 and transferrin receptor expression was altered. Our earlier investigations indicated that BT causes a sustained hyperpolarization of the cells, due to the initial Na + influx which initiated an increased Na+-K + pump activity. Since toxic or irreversible effects of the BT could not be found either on the known metabolic or membrane events of the cell activation, the following model was suggested for the mode of action: the membrane potential regulates intermolecular interactions in the membrane, which have functional importance. The maintenance of only a relatively narrow 'window' of the membrane potential is suitable for the early molecular events of the transmembrane signalling. Any sustained deviation from this potential 'window' may result in an impairment of the
234 signal-transducing pathways. The changes in the membrane potential level caused by the signalling ligands themselves are far from being sustained ones. Generally, after the ligands bind their respective receptors, an internalization process removes the complexes from the cell surface and the 'normal' membrane potential is restored. There are a number of indications in our hands that aging may influence these delicate mechanisms (Damjanovich and Pieri, 1990).
Ion-channel activity changes in lymphocytes Lymphocytes have practically all those ion channels which used to be a prerequisite of excitable cells. Ca 2+, Na +, K + and a number of anion channels of many different types have been detected in lymphoid cells (Grinstein and Dixon, 1989). The ion channels localized in lymphocyte membranes have basically the same forms of gatings, i.e., regulatory control, like the excitable cells: namely the ligand, voltage and G-protein gating. A ligand gating means an all or none type opening or closing of a channel, upon binding a ligand to or in the immediate vicinity of an ion channel. A number of hormones, drugs, peptides, alloantigens or lectins can be classified to this group. Voltage gating is a very fast and versatile gating of a channel, when a decrease in the membrane potential induces such a conformational change in the membrane bound channel structure that ions can pass through it. An increase in the potential level generally closes channel activities. G-protein gating is a mechanism whereby a usually 3-component protein in the membrane is regulating ion channels, and which can activate specific enzymes. The G-proteins are usually activated by binding guanosin triphosphates, (this is the origin of their name) and are deactivated by the dephosphorylation of the GTP into GDP. The considerable changes in the charge distribution of the G protein during GTP or G D P binding is the explanation for their intricate regulatory role. Of course, mixed types of regulation may also occur. Recently we described a new type of voltage and ligand gated Na + channel and detected an additional voltage regulation of the Ca 2+ activated potassium channels. The ligand and voltage gated Na + channels, discovered by the aid of bretylium tosylate (a quaternary ammonium ion that opens otherwise silent sodium channels of slightly depolarized lymphocytes), may have an even higher importance than the already described other sodium channels. The voltage regulated Na + channels have been detected only in about 3% of the lymphocyte population. The ligand and voltage gated ones seem to be ubiquitous both in T and B lymphocytes regardless of their human or murine origin as was observed by flow cytometric membrane potential as well as patch clamp experiments (Matyus et al., 1990; Pieri et al., 1989, 1990; Tron et al., 1990). These channels are per se influenced by membrane potential changes. The increasing rigidity of membrane structure and the decreasing responsiveness of membrane ion channels and also of other cell membrane functions are likely and yet less investigated targets of age-related processes. The observation by Gardos (1958) of calcium flux regulated K + channels in
235 erythrocytes initiated a rich harvest of data on channel regulations. The ion-regulated channel activities were found ubiquitous and prominently among them the Ca z÷ activated K ÷ channels. However, until recently the measurements studying the responses of these channels were carried out either by isotopes or fluorescence dyes, and only to a lesser extent by electrophysiological methods like patch clamp. It is generally accepted that these channels belong to the more sophisticated ones. A voltage regulation of the calcium-channel-side of the calcium activated potassium channels was suggested recently by Sarkady at al. (1990). The voltage regulation of the potassium-channel-side of the Ca 2 + activated potassium channels was described by us (Matyus et al., 1990). The same effect was found in human and murine T and B cells. However, there was a significant difference in the sensitivity and responsiveness of these channels depending on the cell lineage. T-lymphocytes are likely to have more calcium activated potassium channels, and also their responsiveness is greater than in B-cells. A one day incubation of fresh cells in RPMI-1640 usually produces a down-regulation of this type of channel activity. Again, the B-cells were more sensitive to this decay of regulation. The membrane potential was monitored using bisoxonol, a negatively charged dye that is excluded from normally polarized lymphocytes. Freshly prepared human lymphocytes were treated with 1 mM extracellular calcium in the presence of 2 /~M specific calcium ionophore (inomycin). The response was in most cases a significant depolarization. When the calcium concentration was dropped by an order of magnitude (100/xM) the hyperpolarization of the cells, due to the activated potassium channels and the resulting diffusion potential was mandatory. Theoretical calculations, using a simple model, showed that the capacity to change the potential values exists in those conditions where the depolarizing effect of the calcium is observed. On the other hand the higher calcium concentrations themselves can inhibit the potassium channels. However, when the same (1 mM) calcium concentrations were applied to partially depolarized cells which caused depolarization in the normally polarized cells, a strong hyperpolarization was observed. Potassium channel blockers like 4-amino-pyridine or quinine, did not influence the depolarization. However, the quinine, which is known to inhibit the calcium activated potassium channels, inhibited the hyperpolarizing effects (Matyus et al., 1990). Based on the above briefly described experimental evidence, we suggested a voltage gating (or rather modulating) of the calcium activated potassium channels (Matyus et al., 1990). This phenomenon seemed to be a very fine tuning of the channels in the plasma membrane. Thus it was tested, in the hope to find some differences, in lymphocyte populations of healthy volunteers divided into two groups of significantly different age. One group consisted of volunteers in their twenties or thirties and the other group consisted of elderly, 85 + 6 years of age. According to our expectations, the lymphocytes of the elderly reacted very differently from those of the young. The voltage modulation of the lymphocytes from elderly was less expressed and one could always find those conditions where they were depolarized with doses of calcium, which invariably hyperpolarized the lymphocytes from the young. The
236 striking consistency of the results in case of 8 elderly and a similar n u m b e r of y o u n g patients suggests a possibility for opening a new trend in the exploration of reliable aging markers.
Future perspectives The observed slower responsiveness of the calcium and voltage regulated potassium channels might be positively influenced by a minor lowering of the m e m b r a n e potential of lymphocytes. This finding is in full accordance with those well k n o w n p h e n o m e n a that the so-called excitable cells (nerve, muscle) show a much higher responsiveness at lower m e m b r a n e potential levels. The resting plasma m e m b r a n e potential is described with a good approximation as a potassium potential by the Nernst equation. Thus, any sustained drug effect, which relaxes the lymphocyte m e m b r a n e potential by a few millivolts, m a y be advantageous for an improvement in the i m m u n e regulation. However, very complex interrelationships exist between different channel activities, cell activation processes of different organs, and the final positive or negative result in the whole organism. Thus, further detailed studies are required to clarify such questions. One important conclusion m a y be drawn: the complex regulatory processes are likely to be the first and very sensitive targets of aging processes, regardless of the ultimate, supposedly multiparametric causes. The possible points of an interference with the inconvenient consequences of aging probable can be found at these levels.
Acknowledgements One of the authors (S.D.) wishes to express his gratitude to Prof. Dr. Imre Zs.-Nagy, coordinator of the V.I.L.E.G. Hungarian Section, who organized his participation at the International Symposium ' F r i t z Verzfir Project - 2000'. The paper was presented at the Conference by S.D. The work described in the paper was partially supported by grants of the Hungarian A c a d e m y of Sciences to S.D. ( O T K A No. 112 and O K K F T 1.1.4.2).
Discussion of the lecture Cutler: Maybe I missed this point in your presentation, but I wonder what might be the physical basis of the changes in activation of cells you described. Damjanooich: The physical basis of the changes in responsiveness may come from the changes of the membrane structure, however, other factors can also be involved. A series of processes can be listed which have something to do with the modifications of the structure and function of the surface membrane, and resulting in the altered responsiveness. Zs.-Nagy: I would like to suggest to perform an experiment with the addition of the components of the Fenton reaction in order to see whether the changes you demonstrated will occur to an increasing
237 effect or not. In other words, it should be decided whether your model behaves as other aging models or not. Damjanouich: You are right. I have no doubt that such experiments will give similar alterations, but for me even such a result would not answer the question whether the alterations are generated in vivo by the same mechanism. Oxyradical induced damage is one of the possibihties, since the cross-linking of proteins will result in similar alterations as we observed.
Hofecker: You have mentioned the effect of calcium. Its role may be very important for the cell membrane. It is known that calcium is accumulated during aging in some tissues of animals and man. This accumulation influences in some way arterial smooth muscle cells, they start dividing, migrate into the intima, and in many aspects show altered behavior. Could you comment on the mechanism of these alterations from the point of view of membrane physiology? Damjanovich: I think the calcium stores are filled up during aging, but the really acting ionized calcium is not changing. It would be very bad for us. In my view the acting calcium level is regulated very strictly, therefore, I do not see the effect you mentioned.
References Berridge, M.J. and Irvine, R.F. (1989): Inositol phosphates and cell signalling. Nature, 341, 197-205. Crabtree, G.R. (1989): Contingent genetic regulatory events in T lymphocyte activation. Science, 243, 355-361. Damjanovich, S. and Pieri, C. (1990): Electro immunology: membrane potential, ion channel activities and stimulatory signal transduction in human T lymphocytes from young and elderly. Ann. N.Y. Acad. Sci. (in press). Damjanovich, S., Zs.-Nagy, I. and Somogyi, B. (1989): Application of a molecular enzyme kinetic model for aging cells and tissues. Arch. Gerontol. Geriatr., 8, 37-45. Gardos, G. (1958): The function of calcium in the potassium permeability of human erythrocytes. Biochim. Biophys. Acta, 30, 653-654. Grinstein, S. and Dixon, S.J. (1989): Ion transport, membrane potential and cytoplasmic pH in lymphocytes: changes during activation. Physiol. Rev. 69, 417-481. Kozumbo, W.J., Harris, D.T., Gromkowski, S., Cerottini, J.-C. and Cerutti. P. (1987): Molecular mechanism involved in T cell activation, I1. The phosphatidylinositol signal transduction mediates lymphokine production but not IL-2 induced proliferation in cloned cytotoxic T lymphocytes. J. Immunol., 138, 606-612. Matko, J., Szollosi, J., Tron, L. and Damjanovich, S. (1988): Luminescence spectroscopic approaches in studying cell surface dynamics. Q. Rev. Biophys., 21,479-544. Matyus, L., Pieri, C., Recchioni, R., Moroni, F., Bene, L., Tron, L. and Damjanovich, S. (1990): Voltage gating of Ca2+-activated potassium channels in human lymphocytes. Biochem. Biophys. Res. Comm., 171,325-329. Mills, G.B., Girard, P., Grinstein, S. and Gelfand, E.W. (1988): Interleukin-2 induces proliferation of T lymphocyte mutants lacking protein kinase C. Cell, 55, 91-100. O'Leary, J., Jackola, D.R., Mehta, C. and Hallgreen, H.M. (1985): Enhancement of mitogen response and surface marker analysis of lymphocytes from young and old donors after preliminary incubation in vitro. Mech. Aging Dev., 29, 239-253. Pieri, C., Recchioni, R., Moroni, F., Bene, L., Marian, T., Tron, L. and Damjanovich, S. (1989): Ligand and voltage gated sodium channels may regulate electrogenic pump activity in human, mouse and rat lymphocytes. Biochem. Biophys. Res. Comm., 160, 999-1002. Pieri, C., Recchioni, R., Moroni, F., Marcheselli, F., Falasca, M. and Damjanovich, S. (1990): A sodium channel opener inhibits stimulation of human peripheral T lymphocytes. (Submitted.) Sarkady, D., Tordai, A. and Gardos, G. (1990): Membrane depolarization selectively inhibits receptoroperated calcium channels in human T (Jurkat) lymphoblasts. Biochim. Biophys. Acta, 1027, 130-140.
238 Thoman, M.L. and Wiegle, W.O. (1989): The cellular and subcellular bases of immunosenescence. Adv. lmmunol., 46, 221-261. Tigges, M.A., Casey, L.S. and Koshland, M.E. (1989): Mechanism of interleukin-2 signalling: mediation of different outcomes by a single receptor and transduction pathway. Science, 243, 781-786. Tron, L., Pieri, C., Marian, T., Balkay, L., Emri, M. and Damjanovich, S. (1990): Bretylium causes activation of Na+-K + pump in slightly depolarized rat mouse and human lymphocytes which is independent of the Na+/H + exchange. Mol. Immunol., 27, 1307-1311. Valge, V.E., Vong, J.G.P., Datlof, B.M., Sinskey, A.J. and Rao, A. (1988): Protein kinase C is required for responses to T cell receptor ligands but not to interleukin-2 in T cells. Cell, 55, 101-112. Varga, Zs., Bressani, N., Zaia, A.M., Bene, L., Fulop, T. Leovey, A., Fabris, N. and Damjanovich, S. (1989/1990): Cell surface markers, inositol phosphate levels and membrane potential of lymphocytes from young and old human patients, lmmunol. Lett., 23, 275-280. Zs.-Nagy, 1. (1978): A membrane hypothesis of aging. J. Theor. Biol., 75, 189-195.
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© 1991 Elsevier Science Publishers B.V. 0167-4943/91/$03.50
A G G 00390
Parameters to m o n i t o r aging with a possible perspective for intervention an i m m u n o l o g i c a l approach * Carlo Pieri, R i n a R e c c h i o n i , F a u s t o M o r o n i , M a r c o F a l a s c a
and Sandor Damjanovich 1 and
1
Cytology Center, Gerontological Research Department of INRCA, Ancona, Italy," Department of Bwphyslcs, University Medical School of Debrecen, H-4012, Debrecen, Hungary .
.
(Received 1 October 1990; accepted 13 December 1990)
Summary Reliable aging markers are very rare, which are better than the chronological age or those symptoms which have such great individual variability that their scientific value is questionable. The effect of aging on immunological behavior of h u m a n (and animal) individuals is reasonably well established. In this communication an attempt is made to find an immunological marker of aging at the level of cell surface phenomena. It was observed that ion-channel activities, having a complex regulation, loose their flexible responsiveness in lymphocyte membranes during aging. A recently discovered voltage regulation of the calcium-activated potassium channels showed a distinct change with aging of h u m a n lymphocytes. A possibility to find a better marker system in complex regulatory processes is also discussed. Lymphocyte membranes; Ca2+-activated K + channel; Voltage regulation; Aging
Introduction
Aging, apart from the chronological one is a very loosely defined parameter, describing the gradual loss or changing of abilities either inherited or acquired throughout the progression of the time of life. The great individual variations make a definition of a clearly worded indicator parameter extremely difficult. Since the effect of increasing age on the human (and also animal) immune system has been clearly documented, the present brief communication will concentrate on lympho-
* Paper presented by S. Damjanovich, and discussed in the program of the International Symposium 'Fritz Verzfir Project - 2000', held from 30 September to 3 October 1990 at Ancona, Italy. Correspondence to: S. Damjanovich, Department of Biophysics, University Medical School of Debrecen, H-4012 Debrecen, Hungary.
232
cytes (Thoman and Wiegle, 1989). The parameters to be analyzed are mostly regulatory ones and are connected to the lymphocyte plasma membrane. The reason for this aspect is that this is the level where the extracellular world encounters with the cells for the first time. Furthermore the plasma membrane is the main theater of the signal transmission between the outside and inside of cellular systems. Thus the altered functions of the plasma membrane could significantly contribute to the aging processes. A number of cell surface parameters investigated so far in connection with the immunological decline during aging showed rather vaguely observable changes, if any. Nevertheless, it is well documented that proliferation to mitogenic lectins and alloantigens, the generation of cytolytic effector cells, delayed type hypersensitivity, and primary and secondary antibody responses are diminished in the aged (Thoman and Weigle, 1989). The advent of refined technics in lymphocyte membrane physiology like patch clamp and the flow cytometric membrane potential measurements made possible the objective monitoring of individual channel activities and changes in the membrane potential for such cells with great accuracy (for review see Matko et al., 1988). Our attention has been directed towards this field, since hypothetically the fine regulatory processes of the immune response at this level are more likely to be impaired either by stochastically occurring injuries caused by extracellular agents like free radicals or any preprogrammed processes which are generally supposed to contribute to aging (Zs.-Nagy, 1978; O'Leary et al., 1985; Damjanovich et al., 1989; Varga et al., 1989). In the first part of this communication we intend to concentrate on some basic but complicated processes in the cell membrane and the means how to study them. In the second part, a different responsiveness of the calcium-activated potassium channels will be discussed with special reference to its age-dependent nature and its newly discovered voltage regulation (Matyus et al., 1990).
Cell surface phenomena and biological information transfer in lymphocytes Transmembrane signalling is known to involve a number of physical and biochemical steps, which finally lead to cell activation, i.e., transcription of newly activated genes and in case of particular subsets of lymphocytes proliferation and differentiation (Berridge and Irvine, 1989). The signal-transducing pathways a r e dependent on the activation signal (Kozumbo et al., 1987; Mills et al., 1988; Valge et al., 1988; Tigges et al., 1989). Alloantigens or mitogenic lectins activate lymphocytes through a well studied pathway. The binding of the signal transducer to the appropriate cell surface receptor induces a receptor aggregation, sometimes translocation or assembly of different subunits (Berridge and Irvine, 1989). Changes in ion channel activities and membrane potential level are also well known and frequently observed concomitant early signs of signal transduction. However, their exact role in the transduction mechanism is not well understood. An increase in the intracellular C a 2+ level, phospholipase-C digested phosphatidylinositol breakdown products and finally the
233 activation of protein kinase C enzyme represent the key elements of a complicated biochemical cascade (Berridge and Irvine, 1989). This is a very frequently occurring chain of events, but it is far from being the only possible pathway. Activation of tyrosine kinases, cAMP and cGMP activated proteins (G-proteins), belong also to the possible alternative pathways depending on the kind of the activation signal (Tigges et al., 1989). The only common denominator is that all these pathways seems to phosphorylate proteins and these are supposedly the final messengers initiating gene transcription and cell proliferation. A high number of different gene products are among the early (a few minutes) and late (hours, days) markers of cell activation processes, but for only a few of them we can assign a function yet (Crabtree, 1989). The best studied early markers are the protooncogenes c-myc and c-fos and from among the late markers the c-myb. In case of the best studied T lymphocytes the interleukin-2 synthesis and the interleukin-2 and transferrin receptor expression in the plasma membrane are important factors of the cell activation. The interleukin-2, a lymphokine induced cell activation does not demand protein kinase C activation or an increase in the intracellular Ca 2+ level (Mills et al., 1988, Valge et al., 1988; Tigges et al., 1989). Recently we observed that a specific ion channel opener, bretylium tosylate, that opens otherwise silent sodium channels, influences cell activation (Pieri et al., 1989, 1990). At low doses of BT a dose dependent enhancement, at higher doses an inhibition of T lymphocyte proliferation was observed. The BT, which also requires a slight partial depolarization of lymphocytes prior to be able to open sodium channels, could also differentiate between alloantigenic (or lectin), and IL-2 induced cell activation processes. The activators using the protein kinase C activating stimulatory pathway for signal transduction, were negatively influenced, i.e., inhibited in the presence of BT concentrations which significantly enhanced the cell proliferation of IL-2 induced activation of so-called IL-2 primed, i.e., IL-2 receptor expressing human peripheral blood lymphocytes. The activation was influenced by the BT not solely at the level of 3H-thymidine incorporation. Northern blot analysis revealed that BT was able to enhance the expression of the IL-2 receptor gene, c-myc and c-myb oncogenes at the RNA synthesis level, when added together with IL-2. The same doses of BT (0.25-1 mM) added together with PHA to human peripheral blood lymphocytes inhibited the 3H-thymidine incorporation and blocked the cell proliferation mostly in G 1 and S phases. At the same time neither the IL-2 production nor the IL-2 and transferrin receptor expression was altered. Our earlier investigations indicated that BT causes a sustained hyperpolarization of the cells, due to the initial Na + influx which initiated an increased Na+-K + pump activity. Since toxic or irreversible effects of the BT could not be found either on the known metabolic or membrane events of the cell activation, the following model was suggested for the mode of action: the membrane potential regulates intermolecular interactions in the membrane, which have functional importance. The maintenance of only a relatively narrow 'window' of the membrane potential is suitable for the early molecular events of the transmembrane signalling. Any sustained deviation from this potential 'window' may result in an impairment of the
234 signal-transducing pathways. The changes in the membrane potential level caused by the signalling ligands themselves are far from being sustained ones. Generally, after the ligands bind their respective receptors, an internalization process removes the complexes from the cell surface and the 'normal' membrane potential is restored. There are a number of indications in our hands that aging may influence these delicate mechanisms (Damjanovich and Pieri, 1990).
Ion-channel activity changes in lymphocytes Lymphocytes have practically all those ion channels which used to be a prerequisite of excitable cells. Ca 2+, Na +, K + and a number of anion channels of many different types have been detected in lymphoid cells (Grinstein and Dixon, 1989). The ion channels localized in lymphocyte membranes have basically the same forms of gatings, i.e., regulatory control, like the excitable cells: namely the ligand, voltage and G-protein gating. A ligand gating means an all or none type opening or closing of a channel, upon binding a ligand to or in the immediate vicinity of an ion channel. A number of hormones, drugs, peptides, alloantigens or lectins can be classified to this group. Voltage gating is a very fast and versatile gating of a channel, when a decrease in the membrane potential induces such a conformational change in the membrane bound channel structure that ions can pass through it. An increase in the potential level generally closes channel activities. G-protein gating is a mechanism whereby a usually 3-component protein in the membrane is regulating ion channels, and which can activate specific enzymes. The G-proteins are usually activated by binding guanosin triphosphates, (this is the origin of their name) and are deactivated by the dephosphorylation of the GTP into GDP. The considerable changes in the charge distribution of the G protein during GTP or G D P binding is the explanation for their intricate regulatory role. Of course, mixed types of regulation may also occur. Recently we described a new type of voltage and ligand gated Na + channel and detected an additional voltage regulation of the Ca 2+ activated potassium channels. The ligand and voltage gated Na + channels, discovered by the aid of bretylium tosylate (a quaternary ammonium ion that opens otherwise silent sodium channels of slightly depolarized lymphocytes), may have an even higher importance than the already described other sodium channels. The voltage regulated Na + channels have been detected only in about 3% of the lymphocyte population. The ligand and voltage gated ones seem to be ubiquitous both in T and B lymphocytes regardless of their human or murine origin as was observed by flow cytometric membrane potential as well as patch clamp experiments (Matyus et al., 1990; Pieri et al., 1989, 1990; Tron et al., 1990). These channels are per se influenced by membrane potential changes. The increasing rigidity of membrane structure and the decreasing responsiveness of membrane ion channels and also of other cell membrane functions are likely and yet less investigated targets of age-related processes. The observation by Gardos (1958) of calcium flux regulated K + channels in
235 erythrocytes initiated a rich harvest of data on channel regulations. The ion-regulated channel activities were found ubiquitous and prominently among them the Ca z÷ activated K ÷ channels. However, until recently the measurements studying the responses of these channels were carried out either by isotopes or fluorescence dyes, and only to a lesser extent by electrophysiological methods like patch clamp. It is generally accepted that these channels belong to the more sophisticated ones. A voltage regulation of the calcium-channel-side of the calcium activated potassium channels was suggested recently by Sarkady at al. (1990). The voltage regulation of the potassium-channel-side of the Ca 2 + activated potassium channels was described by us (Matyus et al., 1990). The same effect was found in human and murine T and B cells. However, there was a significant difference in the sensitivity and responsiveness of these channels depending on the cell lineage. T-lymphocytes are likely to have more calcium activated potassium channels, and also their responsiveness is greater than in B-cells. A one day incubation of fresh cells in RPMI-1640 usually produces a down-regulation of this type of channel activity. Again, the B-cells were more sensitive to this decay of regulation. The membrane potential was monitored using bisoxonol, a negatively charged dye that is excluded from normally polarized lymphocytes. Freshly prepared human lymphocytes were treated with 1 mM extracellular calcium in the presence of 2 /~M specific calcium ionophore (inomycin). The response was in most cases a significant depolarization. When the calcium concentration was dropped by an order of magnitude (100/xM) the hyperpolarization of the cells, due to the activated potassium channels and the resulting diffusion potential was mandatory. Theoretical calculations, using a simple model, showed that the capacity to change the potential values exists in those conditions where the depolarizing effect of the calcium is observed. On the other hand the higher calcium concentrations themselves can inhibit the potassium channels. However, when the same (1 mM) calcium concentrations were applied to partially depolarized cells which caused depolarization in the normally polarized cells, a strong hyperpolarization was observed. Potassium channel blockers like 4-amino-pyridine or quinine, did not influence the depolarization. However, the quinine, which is known to inhibit the calcium activated potassium channels, inhibited the hyperpolarizing effects (Matyus et al., 1990). Based on the above briefly described experimental evidence, we suggested a voltage gating (or rather modulating) of the calcium activated potassium channels (Matyus et al., 1990). This phenomenon seemed to be a very fine tuning of the channels in the plasma membrane. Thus it was tested, in the hope to find some differences, in lymphocyte populations of healthy volunteers divided into two groups of significantly different age. One group consisted of volunteers in their twenties or thirties and the other group consisted of elderly, 85 + 6 years of age. According to our expectations, the lymphocytes of the elderly reacted very differently from those of the young. The voltage modulation of the lymphocytes from elderly was less expressed and one could always find those conditions where they were depolarized with doses of calcium, which invariably hyperpolarized the lymphocytes from the young. The
236 striking consistency of the results in case of 8 elderly and a similar n u m b e r of y o u n g patients suggests a possibility for opening a new trend in the exploration of reliable aging markers.
Future perspectives The observed slower responsiveness of the calcium and voltage regulated potassium channels might be positively influenced by a minor lowering of the m e m b r a n e potential of lymphocytes. This finding is in full accordance with those well k n o w n p h e n o m e n a that the so-called excitable cells (nerve, muscle) show a much higher responsiveness at lower m e m b r a n e potential levels. The resting plasma m e m b r a n e potential is described with a good approximation as a potassium potential by the Nernst equation. Thus, any sustained drug effect, which relaxes the lymphocyte m e m b r a n e potential by a few millivolts, m a y be advantageous for an improvement in the i m m u n e regulation. However, very complex interrelationships exist between different channel activities, cell activation processes of different organs, and the final positive or negative result in the whole organism. Thus, further detailed studies are required to clarify such questions. One important conclusion m a y be drawn: the complex regulatory processes are likely to be the first and very sensitive targets of aging processes, regardless of the ultimate, supposedly multiparametric causes. The possible points of an interference with the inconvenient consequences of aging probable can be found at these levels.
Acknowledgements One of the authors (S.D.) wishes to express his gratitude to Prof. Dr. Imre Zs.-Nagy, coordinator of the V.I.L.E.G. Hungarian Section, who organized his participation at the International Symposium ' F r i t z Verzfir Project - 2000'. The paper was presented at the Conference by S.D. The work described in the paper was partially supported by grants of the Hungarian A c a d e m y of Sciences to S.D. ( O T K A No. 112 and O K K F T 1.1.4.2).
Discussion of the lecture Cutler: Maybe I missed this point in your presentation, but I wonder what might be the physical basis of the changes in activation of cells you described. Damjanooich: The physical basis of the changes in responsiveness may come from the changes of the membrane structure, however, other factors can also be involved. A series of processes can be listed which have something to do with the modifications of the structure and function of the surface membrane, and resulting in the altered responsiveness. Zs.-Nagy: I would like to suggest to perform an experiment with the addition of the components of the Fenton reaction in order to see whether the changes you demonstrated will occur to an increasing
237 effect or not. In other words, it should be decided whether your model behaves as other aging models or not. Damjanouich: You are right. I have no doubt that such experiments will give similar alterations, but for me even such a result would not answer the question whether the alterations are generated in vivo by the same mechanism. Oxyradical induced damage is one of the possibihties, since the cross-linking of proteins will result in similar alterations as we observed.
Hofecker: You have mentioned the effect of calcium. Its role may be very important for the cell membrane. It is known that calcium is accumulated during aging in some tissues of animals and man. This accumulation influences in some way arterial smooth muscle cells, they start dividing, migrate into the intima, and in many aspects show altered behavior. Could you comment on the mechanism of these alterations from the point of view of membrane physiology? Damjanovich: I think the calcium stores are filled up during aging, but the really acting ionized calcium is not changing. It would be very bad for us. In my view the acting calcium level is regulated very strictly, therefore, I do not see the effect you mentioned.
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