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Clinical Science and Molecular Medicine (1977) 52,443-448. EDITORIAL REVIEW Claude Bernard’s (extended) milieu interieur revisited : autoregulation of cell and subcell integrity E. D. ROBIN Stanford University School of Medicine, Stanford, California, U.S.A. Next year will mark the hundredth anniversary of the death of the great French physiologist, Claude Bernard (10 February 1878). Perhaps it is not inappropriate to dedicate this Editorial to the memory of this remarkable scientist. One of his most powerful contributions was the concept of the milieu interieur, the internal environment. He regarded the plasma (extracellular fluid) as the internal environment of cells and viewed regulatory biology as a series of processes which operated to maintain the constancy of this environment (Bernard, 1878). Sick patients represent integrated whole-body or wholeorgan systems. Thus a focus on the alterations which occur in plasma as a result of disease provided a unifying theme which has remained a pillar of conceptual approaches to disease. For some years, we have been involved in extending this concept, and have suggestedthat the true internal environment is the watery protein-containing fluid within the plasma membranes of individual cells. Regulatory biology can be therefore viewed as those processes which maintain an optimum composition of intracellular fluid. As the internal environment differs from cell type to cell type, studies of extracellular fluid alone frequently fail to provide clear insight into the nature of regulatory processes in health and disease (Robin & Bromberg, 1959). During the past 10 years there has been a proliferation of knowledge concerning both normal cell function (cell physiology) and abnormal cell function (cell pathophysiology). These developments would Seem to confirm the validity of approaches based on the view that Correspondence: Professor Eugene D. Robin, Stanford University School of Medicine, Stanford, California 94305, U.S.A. intracellular fluid is the ‘true’ milieu interieur. Indeed, as subcellular organelles also possess inherent regulatory mechanisms, the concept of the internal environment can be extended to include the microenvironment of various subcellular organelles. In this Editorial I describe four examples of the ability of individual cellular or subcellular units to autoregulate their function. Each example describes processes which can be demonstrated in isolated cell or subcell systems, with a pertubation in extracellular composition evoking an autoregulatory response, detined as that intrinsic cellular response which permits more effective cell function, despite wide swings in extracellular composition. These examples thus focus on the primacy of the intracellular and subcellular milieu in regulatory biology. Regulation of energy metabolism enzymes by molecular oxygen Oxygen lack occurs in many lung diseases, as a consequence of abnormal pulmonary gas exchange. In turn, oxygen depletion has adverse effects on cell function, with a number of adaptive mechanisms operating at a cellular level permitting more or less normal cell function, despite the reduction of extracellular oxygen activity (Jobsis & LaManna, 1977; Robin, 1977). One such mechanism has recently been demonstrated in our laboratory in isolated alveolar macrophages, maintained in tissue culture. The pattern of energy metabolism in alveolar macrophages differs sharply from that in macrophages which originate in other tissues, e.g. peritoneal macrophages. Their rate of basal oxygen consumption is high, whereas the rates of both aerobic and anaerobic glycolysis are low, as compared with that in other macro443 444 E. D . Robin phages (Oren, Farnham, Saito, Milofsky & Karnovsky, 1963; Simon, Robin, Phillips, Acevedo, Axline & Theodore, 1977). These metabolic differences appear to depend on different activities of key enzymes in the pathways of oxidative phosphorylation and glycolysis. The activity of cytochrome oxidase, a key enzyme in oxidative phosphorylation, is high and the activities of three rate-limiting enzymes in glycolysis, pyruvate kinase, phosphofructokinase and hexokinase are low in alveolar macrophages compared with peritoneal macrophages (Table l). It is reasonable to suggest that these differences somehow reflect the high oxygen environment in which alveolar macrophages function. When isolated mouse alveolar macrophages are maintained in a low, rather than a normal, oxygen environment, there is a striking change. TABLE1. Comparison of key energy metabolism enzymes in rabbit alveolar and peritoneal macrophages Activities are expressed as mean enzyme units/mg of protein (k I SD). Cytochrome oxidase Pyruvate kinase Phosphofructokinase Hexokinase Alveolar macrophages Peritoneal macrophages 5 . l k 1.5 30.3+ 13 9.7+ 1.2 1.6+ 0.37 3.5+ 1.2 327+ 128 32.7+ 2.6 2.6+ 0.74 Cytochrome oxidase activity decreases and pyruvate kinase and phosphofructokinase activities increase. The rate of anaerobic lactate production increases. Indeed, the alveolar macrophage is converted into a cell whose pattern of energy metabolism enzymes resembles that of peritoneal macrophages (Table 2). The cell is changed from a cell that is well adapted to a highly aerobic environment to a cell which is better adapted to a relatively anaerobic environment. The mechanism of these alterations could involve alterations of the rate of biosynthesis/biodegradation of specific enzymes. In turn, this decreases the oxygen consumption rate and increases the glycolysis rate. The sequence of the changes may be: a fall in ambient molecular oxygen leads to a changed biosynthesis/biodegradation of enzymes of energy metabolism, leading to a more suitable pattern of energy metabolism for a relatively anaerobic environment (Simon et a/., 1977). This differs from a feedback loop in that the primary disturbance, reduction of ambient oxygen, persists. The precise molecular basis of these adaptations is not known. However, the physiological implications are clear. Alveolar and peritoneal macrophages originate from common precursor cells in the bone marrow (Volkman & Gowans, 1965) and have similar functions. The differences in bioenergetics could theoretically arise as a result of inherent differences TABLE2. Effect of hypoxic exposure on energy metabolism enzymes and lactate production in cultivated mouse alveolar macrophages The differences between the normoxic alveolar macrophages and hypoxic alveolar macrophages are statistically significant for each variable. All values are not statistically different between the hypoxic alveolar macrophages and the freshly harvested peritoneal macrophages except for pyruvate kinase, which is actually higher in the hypoxic alveolar macrophages as compared with the peritoneal macrophages. Enzyme activities are expressed as mean enzyme units h -* mg of protein (f1 SD). Lactate production is expressed as pmol of lactate h mg -' of protein. -' -' Alveolar macrophages Phosphofructokinase Pyruvate kinase Cytochrome oxidase Lactate production Po2 = 20 kPa (150 mmHg) Po2 = 2.0 kPa (15 mmHg) Peritoneal macrophages 7.5f 3.0 59.5+23 C 4.5 + 4.4 0.17+ 0.07 21.7k8.6 299+ 65 11.05 3.0 0.35k 0.08 15.7+ 6.0 158+ 16 9 . 8 5 2.6 0.25 f0.06 Cell and subcell autoregulation in the two cell types or as a result of the differences in oxygen environment (high oxygen in the alveolar macrophage and low oxygen in the peritoneal macrophage). The present studies suggest that changes in the availability of oxygen in the external environment evoke autoregulatory changes involving alterations in the biosynthesis or biodegradation of key enzymes, which in turn equip the cell for function in its specific environment. In addition to serving this function, the ability to change the pattern of bioenergetics presumably is useful under conditions of alveolar hypoxia, permitting alveolar macrophages to withstand severe oxygen depletion. Osmotic autoregulation Alveolar macrophages suspended in a hypotonic medium undergo rapid swelling, as do most cells. In 200 mosmol/l fluid, within 5 min, mean cell volume increases from about 1625 to about 1950 ,um3. If the cells remain in this medium without any additional manipulation, they show a slow decrease in cell volume and, by 45 min, mean cell volume has returned to control values. The cells are thus able to autoregulate cell volume in the face of mild osmotic perturbation. We have called this phenomenon ‘osmotic autoregulation’. The process has a specific requirement for K+ and occurs only when external [K+] is maintained between 2 and 45 mmol/l. At external [K+] of less than 2 and greater than 45 mmol/l cell swelling persists even after 45 min (O’Brien, Theodore & Robin, 1977). The ability of cells to autoregulate volume or tonicity is by no means confined to macrophages. Autoregulatory processes after hypotonic and hypertonic alterations have been previously described in a number of different cell systems. The term ‘isosmotic volume regulation’ was proposed by Jeuniaux to define the cellular adaptation that reverts the hydration and size of a cell to its original value by adjustment of intracellular osmotic particles when the osmolality of the bathing medium is altered (Jeuniaux, Bricteux-Gregoirie & Florkin, 1961). Osmotic autoregulation has been described in duck and human erythrocytes (Kregenow, 1971; Poznansky & Solomon, 1972) and mouse lymphoma cells (Rosenberg, Shank & Gregg, 1972). In these cell types it has been 445 suggested that autoregulation depends on increased K+ efflux, possibly mediated by a configurational change in the membrane protein which controls cation transport. A different mechanism for autoregulation was described by Fugelli (1967) in flounder erythrocytes and also in Ehrlich ascites tumour cells (Hendil & Hoffman, 1974). After exposure to dilute blood plasma, shrinkage of cells toward a normal volume was accompanied by a decreased intracellular concentration of free ninhydrin-positive substances. It was suggested that these substances were free amino acids, and that the regulatory mechanism consisted either of efflux of ninhydrin-positive substances out of the cell or conversion of the free amino acids into proteins. Either process would reduce intracellular concentrations of osmotically active particles, so accounting for volume regulation (Fugelli, 1967). Another mechanism has been described by Ripoche in the frog bladder. The instillation of hypotonic solutions leads to decreased water permeability, returning bladder cells toward the original volume. Conversely, the instillation of hypertonic solutions leads to increased water permeability, increasing cell volumes toward normal (Ripoche, Bourget & Parisi, 1973). It is clear that a wide variety of cell types have intrinsic mechanisms for autoregulating cell volume or cell tonicity despite changes in extracellular osmolality. Autoregulation of intracellularp H Recent studies permit a conceptual synthesis of the regulation of intracellular pH. Two different patterns are apparent. Some cell types (erythrocytes: Tosteson, 1959; macrophages: Robin, Smith, Tanser, Adamson, Millen & Packer, 1971) have free permeability to C1-. The resting transmembrane potential (TMP) may be calculated from the Nernst eqation: TMP = (RT/F) * ln(ClT/Cl;). In these cells TMP is about 10 mV, with the inside negative to the outside. Equilibrium for H* would dictate an internal pH 7.2 at a plasma pH 7.4. Direct measurements show this value (Bromberg, Theodore, Robin & Jensen, 1965; Laman, Theodore &Robin, 1976). Such cell types are quite permeable to carbon dioxide and also to H+/HCO;. Cells of this type are in diffusion equilbrium with respect 446 E. D. Robin to H + . As a consequence, changes in intracellular pH are largely dependent on changes in plasma pH. In other cell types with free permeability to K + (skeletal muscle, nerve), resting TMP, as determined by both the Nernst relationship [TMP = (RT/F) . In(K: /K:)] and direct measurement amounts to about 90 mV, with the inside negative to the outside. In these cells thermodynamic equilibrium for H + would dictate an internal pH of 5.9 at a plasma pH -7.4. Measurements of intracellular pH by either indicator methods (Waddell & Butler, 1959; Adler, 1970) or with pHsensitive glass microelectrodes (Paillard, 1972 ; Thomas, 1974) show an internal pH-7.4. Clearly H + is not in thermodynamic equilibrium and intracellular pH is actively regulated, Active regulation could involve H + , HCO-, or OHpumps (Thomas, 1974), or could involve selective intracellular binding of H + (Ling, 1965) or other as yet undefined mechanisms. In this kind of cell carbon dioxide is rapidly permeable, whereas H + and HCO, penetrate intracellular water slowly. Studies by Boron & De Weer (1976) in the giant axon of the squid, which has a high transmembrane potential, show that an autoregulatory mechanism exists for preventing changes in intracellular pH. The initial response to exposure of the axon to 5 % carbon dioxide is a rapid decrease in intracellular pH from 7.4 to 6.8. During the next 5 min, without further manipulation, intracellular pH increases slowly, by approximately 0.1 pH unit. If the cell is now restored suddenly to a carbon dioxidefree medium, intracellular pH rises to a value actually higher than the control pH. This overshoot reflects the additional HCO-, generated by autoregulation. These workers attribute autoregulation to activation of a proton-extruding pump, as this autoregulation was blocked by cyanide (Boron & De Weer, 1976), which at least indicates that autoregulation requires metabolic energy derived from oxidative phosphorylation. These studies are particularly relevant to the theme of this Editorial. Are regulatory mechanisms in acid-base disease primarily geared to changes in extracellular pH or to changes in intracellular pH (Robin, 1961)? Evidence has slowly accumulated that the latter is true. The role of spinal fluid pH in the ventilatory response during acid-base disturbances (Mitchell, 1966) implies that the cells which are the source of spinal fluid primarily regulate the ventilatory response. We have shown that muscle intracellular pH is more narrowly regulated than plasma pH in patients with chronic stable hypercapnia (Tushan, Bromberg, Shively & Robin, 1970). Similarly the renal regulation of acid-base equilibrium in chronic hypocapnia is not geared to the maintenance of a constant extracellular pH (Cohen, Madias, Wolf & Schwartz, 1976). If we assume that cells have autoregulatory mechanisms for defending their own internal pH, and also that intracellular pH in key cells (brain, kidney) ultimately determines the plasma pH changes in patients with acid-base disturbances, it is reasonable to consider that intracellular pH and not extracellular pH is the variable which should provide the major insight into the pathophysiological consequences of acid-base abnormalities. Having described autoregulation at a cellular level, let us now turn to autoregulation at a subcellular level. Autoregulation of brain mitochondrial energy metabolism with hyperosmotic exposure The brain shows a subcellular autoregulatory response to changes in osmolarity which is not present in other cells or organ systems. Both isolated cell and isolated mitochondrial preparations have shown that hypotonicity and hypertonicity produce important alterations in mitochondria1 oxygen utilization (Atsmon & Davis, 1967; Campbell, Raison & Brady, 1975; Lewiston, Theodore & Robin, 1976). In turn, these alterations of bioenergetics must account for some of the pathophysiological consequences of cellular oedema and dehydration. In recent studies of the coupling of oxygen consumption to ATP generation in isolated brain mitochondria subjected to progressive external hyperosmolarity, there are decreases in P/O ratios and low respiratory control ratios (indices of the coupling between oxygen consumption and ATP generation), within 5 min of exposure to hypertonic solution (osmolalities 450-950 mosmol/l). These abnormalities increase progressively with increasing tonicity. If, however, the mitochondria are maintained for 20 min in the same medium, without further Cell and subcell autoregulation manipulation, values of both P/O ratios and of respiratory control index return toward normal. With moderate hypertonicity, autoregulation is adequate to restore normal respiratory control. With severe hypertonicity, biochemical respiratory control partially returns toward normal values. The molecular mechanism for the original changes of respiratory control are unknown, as are the mechanism(s) of autoregulation. However, it is clear that even at a mitochondria1 (subcellular) level mechanisms exist which defend the integrity of subcellular units, despite changes in the composition of extramitochondrial (intracellular) fluid (Holtzman, Lewiston, Herman, Desautel, Brewer & Robin, 1977). The above examples all demonstrate two common features of autoregulation. One is that the range of perturbations which can be compensated for by autoregulation has circumscribed limits. Maintaining alveolar macrophages at a Poi of zero will kill the cell rather than evoke altered enzyme activities. When macrophages are exposed to osmolalities below 200 mosmol/l the cell becomes irreversibly stretched (O’Brien et al. 1977). If ambient carbon dioxide is increased sufficiently, we presume that a point will be reached when intracellular pH no longer returns toward normal. Severe hypertonicity results in irreversible changes of respiratory control in isolated brain mitochondria. The other common feature is that autoregulation has an important time-dependent dimension. However acute the primary disturbance, a substantial period is required for the regulatory process to become effective. In the examples cited, periods ranging from minutes (osmotic autoregulation) to days (autoregulation of energy metabolism enzymes) are necessary. Thus studies of autoregulatory processes require sequential observations, for isolated measurements which look at cell function statically may mask the presence of autoregulation. Turning now from these specific examples to more general considerations,several concepts emerge. Early in evolution, all life forms were single cells which possessed the ability to defend their internal integrity despite wide swings in the ambient environment, i.e. the ability to autoregulate. With the development of multicellular forms, there was the parallel development of a 447 self-contained, watery environment, the extracellular fluid. This was not accompanied by loss of ability to autoregulate and, indeed, the composition of extracellular fluid is itself strongly influenced by the independent activities of various body cells. This suggests an interesting property of multicellular systems. One cell type does not have the same impact on the composition of extracellular fluid as another. Cells involved in general regulatory functions, such as brain and kidney, have a more decisive role in body economy than other cells. Multicellular systems thus do not operate ‘democratically’, so that each cell has an equal share in determining the composition of the extracellular fluid to which it is exposed. Rather, regulatory cells have a disproportionately large influence on the composition of extracellular fluid. Because they are responding to their own microenvironment, their influence may not provide optimum conditions for other cell types (Robin, 1961). Autoregulation, providing as it does some degree of independence from the tyranny of control cell types, may be as important in multicellular systems as it is in unicellular forms. (Anyone who has dealt with the administration of a modern American university will recognize the validity of this assertion.) Above all, the existence of cellular and subcellular autoregulation emphasizes the importance of clinical approaches based on measurement and therapeutic manipulations of the intracellular and subcellular environment. It was the genius of Claude Bernard which provided a unified approach to biological regulation. In turn, this unified approach had an enormous impact on clinical science. In similar fashion, the contributions of cell physiology are revolutionizing modern clinical science. Bernard was a cell physiologist as well as a multi-organ physiologist (Bernard, 1872). Thus he may well have welcomed the extension of the milieu interieur to the inside of cellular and subcellular units. References ADLER,S. (1970) Simultaneous measurement of intracellular pH with a weak acid and weak base. Clinical Research, 18,493. A ~ M O NR. , & DAVIS,R.P. (1967) Mitochondria1 respiration under conditions of varying osmolanty Biochimica et Biophysica Acta, 131, 221-223. . 448 E. D. Robin BERNARD, C. (1872) Physiologie Gkneralk. Baillitre, Paris. BERNARD, C. (1878) Lecons sur les Phenomknes de la Vie Communs et aux Vegetaux. Bailliere, Paris. BORON,W.F. & DE WEER,P. (1976) Active proton transport stimulated by COJHCOZ, blocked by cyanide. Nature (London). 259, 240-241. BROMBERG, P.A., THEODORE, J., ROBIN,E.D. & JENSEN, W. (1965) Anion and hydrogen ion distribution in human blood. Journal of Laboratory and Clinical Medicine, 66,464475. CAMPBELL, L.C., RAISON,J.K. & BRADY,C.J. (1975) Factors limiting mitochondria1 respiration in media of high solute content. 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(1961) Contribution des acides amines lib& a la regulation osmotique intracellulaire chez deux curstacks euryhalins Leander serratus F and Leander squilla. Cahiers de Biologie Marine, 2, 373-380. JOBSIS,F.F. & LA MANNA, J.C. (1977) Kinetic aspects of intracellular redox reactions. In vivo effects during and after hypoxia and ischemia. In: Extrapulmonary Manifestations of Respiratory Disease. Ed. Robin, E. D. Marcel Dekker, New York (In press). KREGENOW, F.M. (1971) The response of duck erythrocytes to nonhemolytic hypotonic media. Evidence for a volume-controlling mechanism. Journal of General Physiology, 58, 372-392. LAMAN,P.D., THEODORE, J. & ROBIN,E.D. (1976) Regulation of intracellular pH in rabbit alveolar macrophages. Clinical Research, 25, 386. LEWISTON, N.J., THEODORE, J. & ROBIN,E.D. (1976) Intracellular edema and dehydration. Effects on energy metabolism in alveolar macrophages. Science, 191,4034. LING,G.N. (1955) The physical state of water in living cell and model systems. 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