Download autoregulation of cell and subcell integrity

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.
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