Evidence relating Na+,K +- ATPase inhibition in erythrocytes to the toxie effects of
cardiac glycosides on another tissue , the retina, comes from studies relating colour
vision disturbances to erythrocyte receptor occupancy and cation transport (Aronson
& Ford, 1978). Colour vision (which is impaired in patients with digoxin toxicity) has
been quantified in toxic and non-toxic subjects (Aronson & Ford, 1980). Some ofthe
results are shown in Figure 6. There are strong correlations between colour vision
score and each of th e three erythrocyte measurements. The effects of digoxin on
colour vision ma y be due to inhibition of retinal Na+,K+-ATPase (Bonting,
Caravaggio & Canady, 1964) and these data therefore strengthen the link between
erythrocyte Na+, K+- ATPase inhibition and inhibition in other tissues. Note,
howe ver, that the erythrocyte dat a from toxic patients discussed above suggest that
some of the man ifestations of digitalis toxicity ma y occur through mech anisms other
than inhibition ofNat. K+- ATPase.
Assuming that the apparent pharmacological 'tolerance' found in erythrocytes
during long-term therapy reflects simil ar 'tolerance' in the heart, one must now
consider the clinical implications ofthese pharmacological data. Ifsuch 'tolerance' to
the effects ofdigoxin in the hcart does occur, at least in some patients, then long-term
expect tha t withdrawal of digital is would not lead to an y subsequent deterioration in
clin ical state. That this ma y be so is suggested by the results of several studies
(Aronson , 1980) in which digital is has been withdrawn from patients on long-term
therapy. In most pati ents supposed to be taking adequ ate doses of digitali s but whose
plasma digital is concentrations are below the accept ed therapeutic range , digitali s
failure secondary to treatable cau ses such as anaemia or thyrotoxicosis). These
results are not unexpected. However in a variable percentage of cases (it is not
possible to quote an exact figure) in whom long-term treatment seem s to have been
justified and whose plasma digitalis concentrations are weil within the accepted
therapeutic range during chronic therapy, withdrawal does not result in clinical
Thus, during digoxin therap y, changes occur in the digitali s receptors of patients'
erythrocytes, those changes being in con cordance with the clinical effects ofthe drug
observed during both short-term and long-term therapy. During short-term
treatment there is evidence of Na+,K+-ATPase inhibition, the degre e of presumed
inhibition correlating weil with some mea sure s of the clinical result s of treatment,
results which are thought to be secondary to Na+,K+-ATPase inhibition. During
long-term treatment there is an apparent loss of inhibition , an observation which is
consistent with the observations of others on th e absence of clinical detriment in
some patients on withdrawal oftherapy after long-term use.
The next logical que stion to ask is whether any changes in erythrocytic digitalis
receptor function which occur during withdrawal of digox in after long-term therapy
reflect concomitant clin ical changes after withdrawal. A study designed to an swer
that que stion is currcntly being carried out.
Aron son, J. K. (1980). Digitalis and the anti-dysrhythmic dru gs. In Meyler's Side Effects 0/
Drugs Annual lV. Chapter 18, ed. Duk es, M. N. G. Amsterdam: Excerpta Medica.
Aronson, J. K. & Ford , A. R. (1978). The use ofquantitative colour vision testing in diagnosing
digitalis toxicity. 7th International Pharmacology Congress, Paris. Abstract No. 2894 .
Aronson, J. K. & Ford, A. R. (1980). The use of colour vision measurement in the diagnosis of
digoxin toxicity. Quart . J. Med., 49, 273-282.
Aronson, J. K., Graharne-Smith , D. G., Hallis, K. F., Hibble, A. & Wigley, F. (1977).
Monitoring digoxin therapy: 1. Plasma concentration s and an in vitro assay of tissue
response. Brit. J. clin. Pharmac.. 4,213-221.
Astrup , J. (1974). The elfect of hypokalacmia and of digoxin therap y on red cell sodium and
potassium content. Some c1 inical aspects. Scand. J. clin. lab. In vest.. 33, 11-16.
rhodopsin. Exp. Eye Res.. 3,47-56.
Erdmann, E. & Hasse, W. (1975). Quantitative aspects of ouabain binding to erythrocyte and
cardiac membranes. J. Physiol., 251,671-682.
Ford, A. R., Aronson , 1. K., Grahame-Smith, D. G. & Rose, J. A. (l979a). The characteristics of
the binding of 12-a-pH]-digoxin to the membrane s of intact human erythrocytes:
relevance to digoxin therapy. Brit. J. clin. Pharmac., 8,115-124 .
Ford, A. R., Aronson , J. K., Grahame-Smith, D. G. & Carver, J. G. (l979b). Changes in cardiac
glycoside receptor sites, 86rubidium uptake and intracellular sodium concentrations in the
erythrocytes of patients receiving digoxin during the early phases of treatment of cardiac
failure in regular rhythm and ofatrial fibrillation , Brit. J. clin. Pharma c..8, 125-134.
Ford, A. R., Aronson , J. K., Grahame-Smith, D. G. &Carver, J. G. (l979c). The acute changes
seen in cardiac glycoside receptor sites, 86 rubidium uptake and intracellular sodium
concentrations in the erythrocytes of patients during the early phases of digoxin therapy
are not found during chronic therapy: pharmacological and therapeutic imp1ications for
chronic digoxin therapy. Brit. J. clin. Pharmac.. 8, 135-142.
Funder, J. & Wieth, J. O. (1974). Combined elfects of digitalis therapy and of plasma
bicarbonate on red cell sodium and potassium. Scand. J. clin.lab. Invest.. 34, 153-160.
Kettlewell, M., Nowers, A. & White , R. (1972). Elfect of digoxin on human red blood cell
electrolytes. Brit. J. Pharmac.. 44,165-167.
Schwartz , A., Lindenmayer, G. E. & Allen, J. C. (1975). The sodium-potassium, adenosine
triphosphatase: pharmacological, physiological and biochemical aspects. Pharm ac. Rev..
Weissler, A. M., Lewis, R. P. & Leighton, R. F. (1972). The systolic time intervals as a measure
of left ventricular performance in man. In Progress in Cardiology, pp. 155-183.
Withering, W. (1785). An Account ofthe Fox glove and some ofits Medical Uses: with Practical
Remarks on the Dropsy and other Diseases. Birmingham: G. G. J. &J. Robinson.
ß-ADRENERGIC RECEPTOR FUNCTION
Howard Hu ghes M edical In stitute Laboratory,
Departments 0/ Medicin e (Cardiovascular) and Biochemistry,
Duke University Medical Cent er,
Durham, North Carolin a 27710,
The advent ofdirect radiol igand bind ing studies ofa variety ofreceptors over the past
few years has made it possible to directly investigate the role ofreceptor alterations in
mediating a variety ofphysiological changes in drug respons iveness. Among the more
interesting properties of many biologically active agonist drugs is the fact that they
not only stimulate target tissues but they also desensitize them . Th is means that after
aperiod of agonist stimulation the response of the target tissue often wanes even in
the continued presence of the agonist. Many terms have been used to describe these
phenomena includ ing desensitization, tolerance, tachyphylaxis and refractoriness.
Desensitization is of interest to basic as weil as c1inical pharmacologists because it
may be a major mechanism which limits the therapeutic etTectiveness of various
drugs. If one could understand the molecular basis for desensitization it might be
possible to design therapeutic strategies to alter it. However, since only agonists
desensitize tissues (antagonists do not), it seems likely that the activating and
desensitizing properties of various agonists are very intimately linked . Thus, a very
detailed understanding of these processes is likely to be necesary if they are to be
Since there appear to be a variety oftypes of desensitization which are likely to be
mediated by various distinct biochemical processes, it is useful to try to c1assify these
phenomena. Su, Cubeddu & Perkins (1976) have suggested that there are two broad
c1asses of desensitization which they have termed homologous and heterologous
desensitization respectively . Homologous desensitization refers to a situation where
incubation of a responsive tissue with an agonist leads to a very restricted loss of
further responsiveness only to that agonist or to closely related drugs. Other agonists
which stimulate the system through distinct receptors evoke normal responses. By
contrast, heterologous desensitization refers to a situation where exposure ofa system
to any agonist leads to subsequent loss of responsiveness to other agonists and
stimulators as weil. There is mounting evidence that, in general , homologous forms
of desensitization appear to involve alterations in drug receptors whereas
heterologous forms of desensitization may involve alterations in more distal
components ofthe system (Lefkowitz, Wesseis & Stadel, 1980). In this presentation,
recent studies will be reviewed which have been focused on a model of homologous
desensitization; the ß-adrenergic receptors coupled to adenylate cyc1ase in frog
erythrocyte plasma membranes. It is believed that this system shows many of the
typical features ofhomologous desensitization and that insights which can be gleaned
from it may be generally applicable to other systems demonstrating homologous
o ' I I I I I 'F O~_L---:!-----+---7-----+- 8'7 6 5 4 3 6 0 I 2 3 4 5
(-) Isoproterenol concentration 1: (-) (3H] Dihydroalprenolol cancentratian
Figure la Isoproterenol (isoprenaline) stimulation of adenylate cyclase in frog erythrocyte
membranes from cells preincubated with and without isoproterenol. 'Maximum stimulation'
refers to the highest activity observed in the control preparations (that in the presence ofO.l to
1.0 mM isoproterenol) and was 396 ± 82 pmol cAMP generated mg protein! min-I
(Mean ± s.e.mean for 10experiments). (Mickey et al., 1975).
Figure Ib Specific (-)[3H]dihydroalprenolol binding as a function of ligand concentration in
frog erythrocyte membranes from cells preincubated with and without isoproterenol
(isoprenaline). (Mickey et al., 1975).
The data shown in Figures la and Ib, demonstrate the basic phenomenology of
catecholamine induced hornologous desensitization in the frog erythrocyte model
system (Mickey, Tate & Lefkowitz, 1975). When these cells are exposed to a
ß-adrenergic catecholamines for minutes to several hours there is a progressive loss
of catecholamine responsive ß-adrenergic receptor-coupled adenylate cyc1ase
activity (Figure la). This is apparent, primarily, as a decrease in the maximum
level of catecholamine stimulated adenylate cyc1ase activity. The abil ity of other
hormone activators, such as prostagiandin EI or ofthe nonspecific activator fluoride
to stimulate the system remains unatTected (Mickey et al.. 1975). Figure Ib
demonstrates that, as assessed by saturation analysis with the specific ß-adrenergic
antagonist radioligand (3H]-dihydroalprenolol, there is approximately a 50%
decrease in the number of assayable ß-adrenergic receptor binding sites in the
membranes from the desensitized cells. These data suggest that at least one
mechanism for the ß-adrenoceptor agonist induced desensitization of catecholamine
responsive adenylate cyc1ase is the agonist induced loss of functional receptor
binding·capacity. Interestingly, antagonists do not desensitize the system and do not
lead to the loss in receptor capac ity. At the present time the details of the molecular
mechanisms by which agonists lead to the loss of receptors is unc1ear. In the frog
erythrocyte the process appears to be slowly reversible, seemingly without the need
DESENSITIZATION ANDß-ADRENERGIC RECEPTOR FUNCTION 147
for new protein synthesis (Mickey, Tate, Mullikin & Lefkowitz, 1976; Mukkerjee,
Caron & Lefkowitz, 1976). However, in other cell types wh ich have been studied new
protein synthesis appears to be required for resensitization (Terasaki, Brooker, de
Vellis, English, Hsu & Moylon, 1978). Recent data suggest that the lost ß-adrenergic
receptors may be internalized in analogy with the ability of peptide horrnones to
induce the internalization oftheir receptors (Chuang & Costa, 1979; Harden, Cotton,
Waldo, Lutton & Perkins, 1980). It should be pointed out, however, that this has not
been proven for the ß-adrenergic systerns. The evidence published thus far seems to
indicate that after agonist exposure ß-adrenoceptors can be found in small
membrane vesicles which may represent an internalized form of the receptor or at
least asequestered form of the receptor not available for stimulation of adenylate
Obviously, agonist-induced loss of plasma membrane receptors is an attractive
mechanism for the mediation of homologous forms of desensitization, since it is the
receptor binding sites which convey the specificity of hormone action. Loss of
receptors induced by the specific agonist would be expected to lead to a loss only of
the specific response to that class of hormones producing the homologous
phenomenology. Recent data, however, have suggested that in addition to actual
receptor loss , other alterations of the receptors may be contributing to the overall
loss of homone responsiveness. These studies are important because they indicate
that the simple 'counting' of receptor binding sites with antagonist ligands, although
giving important new insights into the regulation ofreceptors, does not tell the whole
story. It seems almost self-evident to state that there must be fundamental differences
in the binding of agonists and antagonists to their receptors. In fact, recent binding
studies confirrn this and indicate in a quantitative fashion the nature ofsome ofthese
differences. In order to understand how agonist interactions with their receptors may
be perturbed after desensitization it is necessary to review briefly some material
concerning these important differences between agonist and antagonist binding to the
receptors. Figures 2a and 2b depict competition curves of an agonist, isoproterenol
4 Ol..-_-'---~--'-----'----'--_--J'-----'
(-) Alprenolol concentration (-l09Io Molar)
Figure 2a Computerized curve fitring of binding data from displacement of (-)[3H)-
dihydroalprenolol by (-)alprenolol in frog erythrocyte membranes. Ligand concentration
was 1.5nM. The solid line is a computer generated curve fitting the observed data points.
(isoprenal ine) (lP), a nd an antagonist, propranolol, with ß-adren oceptors in frog
erythrocyte membran es as assessed by their ability to co m pe te for th e sites with the
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