Clinical Science (1981) 5s-10s
5s
STATE OF THE A R T REVIEW
Membranes, ions and hypertension
D. C. TOSTESON, N. A D R A G N A , I. BIZE, H. S O L O M O N
M. C A N E S S A
AND
Harvard Medical School, Boston, Massachusetts. U S A .
The theme of this lecture is developed around the
question, ‘What can we learn about the pathogenesis, diagnosis and treatment of essential
hypertension from investigations of the movements of ions, particularly sodium ions, across
cell membranes?’. This theme is not new. The
connection between salt intake and hypertension
has been recognized for at least 40 years and
investigated vigorously by many scientists, most
of whom are well known to the members of the
International Society of Hypertension. We think
especially of the pioneering contributions of L. K.
Dahl and his collaborators [ 1, 21 and ofL. Tobian
[31. Rather than attempt to review the works of
these and other investigators in detail, we will
summarize our current understanding of the
theme.
Abnormal transport of sodium and/or
potassium ions across membranes of various cells
in patients with essential hypertension and in
animals with different forms of experimental
hypertension has been reported by several
laboratories [3-131. The specific transport
pathway affected and the type of cell studied vary
considerably.
In human subjects, measurements have been
reported on erythrocytes [3-61, polymorphonuclear leucocytes 181 and lymphocytes [91.
Abnormal sodium transport reported in
erythrocytes of individuals with essential hypertension was observed in the presence of ouabain
and, therefore, probably does not involve the
adenosine triphosphate (ATP)-dependent Na+K+ pump that is specifically inhibited by cardiac
Key words: erythrocytes, leucocytes, lithiumsodium cotransport, ouabain-sensitive sodiumpotassium pump, sodium-potassium cotransport.
Correspondence: Dr D. C. Tosteson, Harvard
Medical School, 25 Shattuck Street, Boston,
Massachusetts 02 115, U.S.A.
glycosides. Some investigators have reported
reduced Na+ extrusion by a pathway interpreted
to be Na+K+ cotransport [6, 101. Others have
noted increased Li+ extrusion via a Li+-Na+
countertransport system in the erythrocytes of
patients with essential hypertension [71. Some 14,
81 have observed increased intracellular concentrations of Na+ in hypertensive subjects while
others have not [71. In leucocytes, reduced Na+
extrusion via the ouabain-sensitive Na+-K+
pump has been reported [81. de Wardener and his
collaborators have suggested that reduced Na+K+ pump activity in the cells of patients with
essential hypertension may be due to a circulating
inhibitor present in increased concentration in the
plasma of such individuals [ 111. The connection
between these various observations is not, at
present, clear, but the available evidence leads to
the conclusion that abnormal Na+ transport
across cell membranes is somehow involved in at
least some forms of essential hypertension.
This conclusion is supported by a substantially
larger body of information gathered on cells
taken from animals with various forms of
experimental hypertension. For example, Postnov
and his colleagues have shown increased
ouabain-resistant Na+ transport in the
erythrocytes of spontaneously hypertensive rats
[ 121. Haddy and his group have reported reduced
ouabain-sensitive Na+-K+ pump activity in the
blood vessel walls of rats and dogs with several
different forms of ‘volume overload’ hypertension
[131.
Several different hypotheses have been put
forward to account for the relation between
abnormal sodium and potassium transport and
essential hypertension. These hypotheses can be
thought of in two groups, aetiological and
physiological. The aetiological hypotheses deal
with the relative roles of genetic and environmental factors in determining abnormal transport and
hypertension. The physiological hypotheses ad-
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D. C. Tosteson
dress the ways in which abnormal transport of
ions could increase vascular peripheral resistance,
leading to hypertension.
Considering first aetiological arguments, both
genetic and environmental factors seem to be
involved in the pathogenesis of essential hypertension [14, 151. Both factors could also be
involved in the development of abnormal Na+
transport across membranes. The membrane
lesion could be either a necessary step in the
sequence of events leading to high blood pressure
or a separate and independent consequence of the
same factors that produce hypertension. Thus
one can imagine both direct and indirect genetic
and environmental relationships between the
causes of hypertension and abnormal transport.
Particular genes coding for products involved in
Na+ transport across membranes may have
alleles that express an abnormal transport system
which directly promotes increased vascular
resistance. In contrast to this direct genetic
relationship between the two phenomena, it is
also possible that two sets of genes, coding
respectively for products involved in ion transport and vascular resistance, segregate together
and lead to an indirect association between
abnormal transport and hypertension. Comparable direct and indirect relations between the two
types of dysfunction and primary environmental
factors can also be formulated. Indeed, mixed
aetiological theories in which certain environmental factors like high salt intake are necessary to
reveal the functional inadequacy of gene products
that lead directly to or are indirectly associated
with hypertension may also be imagined.
The physiological hypotheses address the
mechanisms that relate ion transport and vascular resistance. Again, both direct and indirect
relationships deserve consideration. Abnormal
ion transport in the membranes of vascular
smooth muscle cells may lead directly to
increased resistance by activating contraction
161 and/or stimulating hypertrophy and hyperplasia [ 171. Conversely, abnormal ion transport
in cells that influence vascular smooth muscle
may, indirectly, produce increased vascular
resistance. For example, such effects could be
mediated through the autonomic nervous system
or through the cells and hormones that regulate
fluid volume.
At present the data available do not permit a
clear decision between these and other conceivable hypotheses. To quote the comment by
Hippocrates inscribed in the wall of a building at
the Harvard Medical School, ... “the art is long,
decision difficult, experiment perilous”. However
perilous, more experiments are required to form a
clear picture of the relation between membrane
transport of ions and essential hypertension.
Several directions of future research seem worth
following.
Important methodological issues deserve address. For example, the design and implementation of the epidemiological studies necessary to
establish the roles of environmental and genetic
factors in the aetiology of hypertension and
abnormal transport are difficult and unsolved
problems. Further, the assays of blood pressure
and of transport that are currently being used
leave much to be desired. In many studies,
measures of blood pressure continue to be
indirect and episodic. The need for a non-invasive
method for continuously monitoring arterial
blood pressure has not yet been finally met.
The assays of membrane transport abnormalities are also inadequate. They are too
complicated for routine use in mass screening
studies. Furthermore, they do not measure
directly the amounts and properties of specific
transport proteins, but rather the operation of
transport systems that are mediated by membrane components that have not yet been
identified. The membrane proteins that comprise
these systems must be identified, isolated and
characterized. Only then will it be possible to
recognize the molecular basis of the abnormal
transport. Such knowledge is necessary for
devising assays that are more specific than those
with which we now struggle. It also is essential
for understanding the relation between membrane
events and the regulation of blood pressure.
The remainder of this paper describes recent
work in our laboratories at the Harvard Medical
School on Na+ transport in the erythrocytes of
patients with essential hypertension and in those
of normal control subjects. The results that have
emerged from these investigations express both
the hopes and the problems that are the ‘state of
the art’ of research on the relations between ion
transport across cell membranes and essential
hypertension.
Two types of abnormalities in erythrocyte
sodium transport have been reported to occur in
the erythrocytes of hypertensive patients. One is
increased Li+-Na+ countertransport first reported by Canessa et al. [71. The other is outward
reduced Na+-K+ cotransport first reported by
Garay et al. [lo]. We are currently measuring
simultaneously both of these modalities of transport in the erythrocytes of patients with essential
hypertension and of normal control subjects
without family history of hypertension. These two
pathways of Na+ transport, the methods that we
are using to assay them, and the preliminary
Membranes, ions and hypertension
1
7s
Na'
INSIDE
3
FIG. 1. Two state, four-site ion-exchange or countertransport system postulated for erythrocytes:
the system normally carries out a 1: 1 exchange of Na+ for Na+, but it can also exchange Li+ (a)
for Na+ (B). In the open state (1) the two sites can exchange Nat or Lit present in the solutions
bathing the inner and outer surfaces of the membrane. In the closed state (2) the two sites can
exchange ions with each other but not with the two solutions. Restoration of the initial state
completes the cycle (3). The selectivity of the sites is the same in the two states. In the body "at]
is lower inside the cells than it is outside; the countertransport system produces a net outward
movement of Li+ when the cell/plasma concentration ratio is greater for Li+ than it is for Na+.
(From 'Lithium and mania' by D. C. Tosteson. Copyright 1981 by Scientific American Inc.; all
rights reserved [ 191.)
results that we are obtaining in the study will be
described. We will close with some reflections
on the meaning of these observations for
our understanding of the pathogenesis of
hypertension.
Li+-Na+ exchange or countertransport is a
one-for-one exchange of Na+ for Na+, or Li+ for
Na+, across the plasma membrane of human
erythrocytes [for reviews see 18, 191. The
membrane protein that mediates this pathway has
not yet been identified but can be imagined to
operate as described in Fig. 1. The four-site,
two-state model (Fig. 1) is one but not the only
scheme that is sufficient to account for the known
properties of Li+-Na+ exchange. In the open
state, both inward- and outward-facing sites can
exchange with the cytosol and extracellular fluid
respectively. In the closed state, the binding
sites can exchange with each other but not with
the external solutions. The system can open and
close only when all ion binding sites are
occupied. Such a system can perform exchange
but no net transport of ions. The Na+-Li+
exchange or countertransport system in the
human erythrocyte membrane requires either
Na+ or Li+ on both sides of the membrane. The
concentrations of internal Li+ and Na+ required
to produce half-maximal exchange are about 0.5
mmolll and 9.0 mmol/l respectively, indicating
that the affinity of the system is about 20 times
greater for Li+ than for Na+[201. In order to
produce maximal Li+-Na+ countertransport, it is
necessary to maintain saturating concentrations
of the exchange partners at both membrane
surfaces.
The procedure for assay of the maximum rate
of Li+-Na+ countertransport meets these conditions [71. Erythrocytes are loaded to contain
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D.C. Tosteson
about 10 mmol of Li+/l by preincubation in a
medium containing LiCl (150 mmol/l). This
concentration of internal Li+ is about 20 times
greater than that necessary to half-activate
exchange. The loss of Li+ from these Li+-loaded
cells is then measured both into an external
medium containing neither Li+ nor Na+ (Mg2+
substitution) and into a medium containing no
Li+ but Na+ (150 mmol/l). This concentration of
external Na+ is more than seven times greater
than the concentration required to half-activate
exchange. Thus, in the presence of 150 mmol/l
external Na+ and 10 mmol/l internal Li+,
Li+-Na+ countertransport is maximal, whereas,
in the absence of external Na+ and Li+, Li+ efflux
occurs only by pathways other than the countertransport system. The difference between Li+ loss
into the Na+ medium and the Mg2+ medium is
thus a good measure of the maximum rate of
Li+-Na+ countertransport.
Substantial differences between the maximal
rates of erythrocyte Li+-Na+ countertransport
in different individuals have been reported 120,
211. One individual with mania and some
members of his family were found to have
erythrocytes with absent or much reduced Li+Na+ countertransport [22]. Some but not all
patients with mania or bipolar mood disorders
have reduced Li+-Na+ countertransport that is,
at least in part, genetically determined [23]. On
the basis of this and other information, we
decided to explore the relation of Li+-Na+
countertransport to essential hypertension. We
have found that many but not all individuals with
essential hypertension have increased Lit-Na+
countertransport [71. Similar observations have
been made in several other laboratories and
reported to us in personal communications that
will soon be published (Roger Williams and his
group in Salt Lake City, J. Funder and his
colleagues in Copenhagen and Cusi and Bianchi
in Milan).
Na+-K+ cotransport is a coupled movement of
Nat and K+ in the same direction across the
membrane. The transport protein(s) involved in
this process have also not been isolated, but may
be imagined to function in a four-site, two-state
model (Fig. 2), one of several sufficient to
account for the characteristics of the system. In
the open state, two sites can exchange ions with
the cytosol and two with the extracellular fluid. In
the closed state, the outwardly and inwardly
facing sites can exchange ions with one another.
Transitions between the open and closed states
can occur only when one of the two pairs of sites,
inwardly or outwardly facing, are occupied by
both Na+ and K+.
The Na+-K+ cotransport system is consider-
ably more complicated and less well characterized than Li+-Na+ countertransport. Most of
the observations on the kinetic properties of the
system have been made by measuring the
frusemide-inhibited efflux of Na+ and K + into a
medium containing either Mg2+ or choline but
neither Na+ or K + 1241. Under these conditions,
the ratio of frusemide-sensitive Na+ and K+
effluxes is 1 over the entire range of internal Na+
and K+ concentrations. The maximal rates of
outward Na+ and K+ frusemide-sensitive transport occur when both the internal Na+ and K+
concentrations are in the range 40-60 mmol/l.
When Na+ is substituted for K+, the concentration of internal Na+ required to produce
half-maximal activation of frusemide-sensitive
Na+ and K+ effluxes is about 13 mmol/l. The
shape of the curve relating effluxes to internal
Na+ concentrations is not hyperbolic but sigmoid, suggesting that more than one Na+ is
required to activate the system. External K + but
not external Na+ inhibits the frusemide-sensitive
effluxes of K + and Na+. There is evidence that
the cotransport system in human erythrocytes
also involves C1[25], as is the case in the better
characterized Na+-K+ cotransport system in
duck erythrocytes [261.
The assays for Na+-K+ cotransport that have
been used in studies of essential hypertension are
based on the assumption that the net frusemidesensitive movements of Na+ and K + into a
medium free of these ions is the operational
definition of Na+-K+ cotransport. Garay et al.
have reported observations with several different
assays of Na+-K+ cotransport [6, 10, 271. Most
recently [271, they have used erythrocytes loaded
by the p-chloromercuribenzene sulphonate
(PCMBS) method to contain about 20 mmol of
Na+/l and 20 mmol of K+/I (choline
substitution). Na+ and K+ effluxes were measured
from these cells into media containing MgCl, but
no Na+ or K+. Since the concentration of Na+
required to half-activate the outward cotransport
is about 13 mmol/l, this method detects both
variations in the maximal rate of cotransport and
changes in the affinity of the system for internal
Na+. We have recently undertaken measurements
of countertransport and cotransport in the
erythrocytes of normal subjects and in those of
patients with essential hypertension. To assay
cotransport we have used cells loaded by the
PCMBS method to contain about 40 mmol of
Na+/l and 60 mmol of K+/I suspended in a
medium containing Mg2+but no Na+ or K + [241.
This assay measures the maximal rate of cotransport and is relatively insensitive to changes in the
affinity of the system for internal Na+.
There is considerable variation in the rate of
3QISNI
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D.C.Tfisteson
coupled. How are these two modes of ouabaininsensitive Na+ transport related to the known
transport proteins of the erythrocytes membrane,
e.g. the Na+-K+ pump, the anion-exchange
protein (band 3) and the Ca2+ pump? Are the
differences in erythrocyte Na+ transport between hypertensive patients and control subjects
due to genetic and/or environmental factors? Is
abnormal Na+ transport across cell membranes a
necessary condition for the development of
hypertension? In what cells does abnormal Na+
transport lead to pathological function producing
increased vascular resistance: in vascular smooth
muscle cells or in cells regulating vascular smooth
muscle? Does abnormal Na+ transport exert its
effects on vascular smooth muscle by altering the
intracellular concentration andlor distribution of
CaZ+, as suggested by Blaustein [16]? Do
changes in ion transport and concentrations alter
vascular resistance by increasing the contraction
of vascular smooth muscle and/or producing
hypertrophy? There are many hopeful paths of
inquiry now open to students of the role(s) of
membrane transport of ions in the pathogenesis
of essential hypertension.
Acknowledgment
Supported by NIH Grants GM 25686 and HL
25064.
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