Nig. J. Pure & App. Sci. Vol. 2, pp.

6 – 13, 1987
INTERACTION OF SOME LOCAL ANAESTHETIC AND ANTI-SICKLING DRUGS
WITH CALCIUM-CARRIER PROTEINS IN BIOMEMBRANES

by

C. O. BEWAJI
Biomembrane Research Laboratories
Department of Biochemistry
University of Ibadan
Ibadan, Nigeria

AND

A. A. ODUTUGA
Department of Biochemistry
University of Ilorin, Ilorin
Nigeria

Abstract
The effects of p-butyl-amino-benzoyl-dimethylamino-ethanol (tetracaine), a local anaes-thetic,
3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-6-butyric acid (DBA), an antisickling agent; and p-
amino-benzoyl-diethylamino-ethanol (procaine), a local anaesthetic with antisickling properties, on
the calcium-carrier protein (Ca2+, Mg2+ - ATPase) and the modulator protein calmodulin, were
studied in mitochondria and plasma membranes isolated from rat liver, kidney and brain, and in
human erythrocyte ghost membranes.
The Ca2+, Mg2+ - ATPase activities in these membranes were markedly inhibited by pro-caine
and tetracaine in a concentration-dependent fashion, while DBA enhanced ATPase activity. The
concentration-effect studies were conducted at drug concentrations between 0.15 and 1.0 mM at pH
7.4. It is suggested that procaine and tetracaine, which have been reported to interefere with
calcium transport by displacing membrane-bound calcium, may also cause the dissociation of the
ATPase-calmodulin complex, hence a reduction in ATPase acticity. DBA probably produces a
perturbation in the local environment of the membrane-bound enzyme, thereby inducing a
conformational change in the ATPase which is optimal for ATP hydrolysis.

Introduction inhibition of the ruthenium red-insensitive Ca2+ efflux

The concentration of calcium within the red cell is
controlled not only by its extrusion by a specific calcium
pump or membrane-bound calcium-activated adenosine
triphosphatase, but also by the energy-dependent calcium
accumulation in the mitochondria and some other
organelles (Bygrave, 1977; Carafoli and Crompton, 1978;
Wikstrom et al., 1975). Lehninger et al. (1978) have shown
that respiring mitochondria of animal tissues rapidly take
up Ca2+ and retain it so long as the pyridine nucleotides are
kept in the reduced state. These workers suggested that the
active uptake of Ca2+ by mitochondria may be important in
the regulation of many Ca2+-dependent cellular activities.
Dawson et al. (1979) have also found, using
mitochondria from rat liver and brain, that the local
anaesthetics nupercaine and tetracaine, at concentrations
which do not inhibit Ca2+ uptake, cause considerable
observed under energized conditions. Local anaesthetics
have also been shown to affect membrane transport and the
permeability of membranes to calcium (Blaustein and
Goldman, 1966). In particular, procaine and tetracaine have
been shown to adsorb to membrane-bound Ca2+ from the
fixed negative sites (Bondani and Karter, 1970).
In this paper, we have shown that procaine and
tetracaine also inhibit the Ca2+, Mg2+ - ATPase in the
plasma membrane-rich fraction and mitochondria isolated
from rat liver, kidney and brain. Because of the potential
use and efficacy of DBA as an anti-sickling agent (Ekong
et al., 1975; Dean and Schechter, 1978), the effect of this
compound on the human erythrocyte ghost membrane
Ca2+, Mg2+ - ATPase was also investigated. Wee report that
DBA has a stimulating effect on the activity of the
membrane-bound enzyme in all the tissues studied.

6
 C. O. BEWAJI and A. A. ODUTUGA
MATERIALS AND METHODS
Isolation of mitochondria
Female albino rats (Wistar strain) weighing between
100-200 g were used. The animals had a free access to food
and water, except for a 16 hour fasting period just before
they were sacrificed. The rats were killed by cervical
dislocation. The brain, kidney and liver were removed
immediately and put into separate beakers containing an
ice-cold solution of 0.25 M sucrose, buffered with 10 mM
Tris, pH 7.4. After the removal of connective tissue, each
organ was weighed, cut into small pieces and washed with
sucrose solution until free from blood. The pieces were
then homogenized in 5 volumes of the buffered sucrose,
suing a 50 ml Potter-Elvehjem glass homogenizer. The
Teflon pestle was driven mechanically at approximately
1,000 rpm. The homogenate was centrifuged at 1,500 x g
for 10 min. The supernatant therefrom was centrifuged at
10,000 x g for 10 min to obtain the mitochondrial pellet.
The supernatant from this step was treated as the plasma
membrane-rich fraction. The pellet was washed twice by
re-suspending it in the homogenizing medium and
centrifuging at 10,000 x g for 10 min. All the
centrifugation steps were carried out at 4oC in an MSE
angle 13 refrigerated centrifuge. Rates of oxygen uptake by
the mitochondria were measured polarographically with a
Clarke-type oxygen electrode in a thermostated reaction
vessel equipped with a stirring device and maintained at
37oC. Respiratory control ratios were calculated by
measuring the respiratory rates in the presence of added
ADP and the rates following ADP expenditure as described
by Chance and Williams (1955).
Preparation of erythrocyte ghost membranes
Haemoglobin-free ghost membranes deficient in
calmodulin were prepared from recently outdated normal
human blood, obtained from the Blood Bank of the
University College Hospital, Ibadan, Nigeria. The blood
was collected in acid citrate dextrose buffer (USP), stored
at 4oC and processed within two days. All the steps of the
membrane preparation were carried out at 4oC.
The whole blood was centrifuged at 5,800 x g for 10
min. The plasma and buffy layers were removed by
aspiration to obtain packed erythrocytes. The erythrocytes
were washed twice in 5 volumes of 130 mM KCl and 20
mM Tris, pH 7.4. The cells were then haemolysed in 5
volumes of 1 mM EDTA, 10 mM Tris, pH 7.4, and
centrifuged at 18,000g for 20 min. This step was repeated
three times. The membranes were then washed twice in the
haemolyzing buffer, and four additional times in 10 mM
HEPES, pH 7.4, without EDTA. The haemoglobin-free
ghosts were finally resuspended in a medium containing
7
130 mM KCl, 20 mM HEPES, pH 7.4, 500 M MgCl2 and
50 M CaCl2 and stored at 20oC till required for ATPase
assay.
Determination of protein
Protein concentrations were determined according to
the procedure of Lowry et al. (1955) using bovine serum
albumin as standard.
Assay of Ca2+, Mg2+ - ATPase
The Ca2+, Mg2+ - ATPase was measured
spectrophotometrically using a slight modification of the
method of Bond and Hudgins (1976). The assay medium
contained 25 mM Tris, pH 7.4, 3 mM MgCl2, 0.1 mM
EGTA, 0.2 mM CaCl2, 1 mM ouabain, 2 mM ATP and 10
g of rat tissue protein or 100 – 200 g erythrocyte
membrane protein, in a volume of 0.5 ml. The incubation
was carried out at 37oC for 30 min with constant shaking.
The reaction was then terminated by the addition of 0.2 ml
of a 5% solution of sodium dodecyl sulphate. The
inorganic phosphate liberated was measured by the method
of Fiske and Subbarow (1925). The blue colour that
developed after 30 minutes was read at 820 nm in a Unical
SP 600 spectrophotometer, using a red filter. Blanks were
run to correct for the non-enzymatic hydrolysis of ATP. All
assays were run in triplicates, and individual experiments
were repeated two to four times.
RESULTS
Table 1 shows the respiratory control and ADP:O ratios
of rat liver and kidney mitochondria in the presence of
succinate or pyruvate/malate as substrates. These values
agree well with those already established in the literature,
and demonstrate that electron transport in the mitochondria
is tightly coupled to ATP formation. The parameters could
not be measured in rat brain mitochondria probably
because of heavy contamination with synaptosomes.
Fig. 1 shows that the activity of the Ca2+, Mg2+ -
ATPase, in all membrane types tested, is inhibited by
procaine in a concentration dependent fashion. 0.15 mM
procaine inhibited the enzyme from rat liver, kidney and
brain mitochondria by 17.4 and 15 per cent respectively.
Increasing the concentration of procaine to 0.75 mM
similarly increased the degree of inhibition to 73, 68 and
78 per cent respectively. Corresponding values for the
degree of inhibition at a procaine concentration of 0.75
mM were 58, 49 and 81 per cent.
 Nig. J. Pure & App. Sci. Vol. 2, pp. 6 – 13, 1987
The effect of tetracaine on the Ca2+, Mg2+ - ATPase
activity in the various membranes are shown in Fig. 2. 0.2
mM tetracaine inhibited the enzyme by between 13 and 81
per cent. However, the degree of inhibition decreased as
the concentration of tetracaine was increased.
The activity of the enzyme was stimulated by the
addition of DBA (Fig. 3) The stimulating effect of DBA
gradually increased as its concentration was increased from
0.2 mM through 1.0 mM.
DISCUSSION
It is now generally accepted that the Ca2+, Mg2+ -
ATPase is the biochemical expression of the plasma
membrane calcium pump which is responsible for
extruding Ca2+ from the cytoplasm (Schatzmann, 1982;
Carafoli, 1984). An increase in intracellular Ca2+ activates
the enzyme and increases Ca2+ efflux. This action thus
constitutes a self-regulating device for maintaining a low
steady-state level of intracellular Ca2+ (Lynch and Cheung,
1979).
Although the mechanism of action of Ca2+ in regulating
a variety of cellular processes is still largely unknown,
available evidence suggests that the different effects of
Ca2+ are mediated through a homologous class of Ca2+-
binding proteins, particularly calmodulin, which serve as
intracellular Ca2+ receptors (Cheung, 1980). The present
concept of calmodulin-mediated Ca2+ efflux is that an
increase in intracellular Ca2+ concentrations (above 1 M)
promotes the formation of a Ca2+-calmodulin complex,
which in turn shifts the Ca2+, Mg2+ - ATPase from a low
Ca2+-affinity to a high Ca2+-affinity state (Cheung, 1980).
The present results demonstrate that procaine and
tetracaine inhibit the Ca2+, Mg2+ - ATPase in the plasma
membrane-rich fraction and mitochondria isolated from rat
tissues. These local anaesthetics carry positive charges and
have been shown to compete with and displace Ca2+ bound
to fixed negative sites in membranes (Bondani and Karter,
1970). It appears, therefore, that procaine and tetracaine
could prevent the binding of the Ca2+, Mg2+ - ATPase to
Ca2+ or the Ca2+-calmodulin complex.
However, there are differences in the pattern of the
enzymes by the two local anaesthetics even though they are
structurally similar. Whereas the inhibition of the enzyme
by procaine increases as the anaesthetic concentration is
increased, increasing the concentration of tetracaine leads
to a reduction in the degree of inhibition. Bond and
Hudgins (1976), working on the Na+, K+ -ATPase, have
shown that the non-ionized form of tetracaine is more
inhibitory on the enzyme than the ionized. These workers
showed that the non-ionized, lipid-soluble form of
tetracaine can distribute freely within the membrane,
whereas the ionized form is largely confined to the aqueous
phase by polar interactions with the charged heads of
membrane phospholipids and with water.

TABLE 1

Respiratory control and ADP:O ratios of mitochondria isolated from rat, liver and kidney

Substrate (mM) RCR ADP:O

A. Rat Liver
Succinate (5) 4.8 ± 0.6 2.1 ± 0.4
Pyruvate(5)/malate (5) 5.1 ± 0.4 2.6 ±0.5

B. Rat Kidney

Succinate (5) 4.1 ± 0.8 1.9 ± 0.2

Pyruvate(5)/malate (5) 4.5 ± 0.6 2.2 ± 0.3

Values represent the means ± standard deviations of three replicate

assays on four mitochondria preparations.

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C. O. BEWAJI & A. A. ODUTUGA

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Nig. J. Pure & App. Sci. Vol. 2, pp. 6 – 13, 1987

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C. O. BEWAJI & A. A. ODUTUGA

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 Nig. J. Pure & App. Sci. Vol. 2, pp. 6 – 13, 1987
Bond and Hudgins (1977) have also shown that at pH
7.4, the non-ionized form of tetracaine (pKa = 8.24) is 13
per cent of the total amount of tetracaine in solution. Since
tetracaine is further ionized on dilution, it is expected that
there will be a reduction in its inhibitory effect as the
concentration decreases. At pH 7.4, procaine is almost
completely ionized and its partition coefficient is about two
orders of magnitude lower than that of tetracaine (Bond
and Hudgins, 1977). This probably accounts for the
differences in the dose-response curves of the two local
anaesthetics.
The inhibition of the Ca2+, Mg2+ - ATPase, and
presumably the active transport of Ca2+, by procaine and
tetracaine suggests that the mechanism of action of these
local anaesthetics may be connected with their interference
with active cation transport.
The anti-sickling effect of DBA was first reported by
Ekong et al. (1975) who suggested that this effect may be
due to non-covalent binding of DBA to haemoglobin or the
red cell membrane. We have shown here that DBA, at
concentrations between 0.2 and 1.0 mM, stimulated the
activity of Ca2+, Mg2+ - ATPase in mitochondria and
plasma membranes isolated from various tissues (Fig. 3).
A number of substances have been shown to activate
plasma membrane Ca2+, Mg2+ - ATPase. These include
calmodulin, acidic phospholipids (including
polyphosphoinositides), fatty acids and proteases (Taverna
and Hanahan, 1980; Niggli et al., 1981; Penniston, 1982).
It is pertinent to know whether the mechanism of activation
by these substances are the same or not. This can only be
conclusively answered when the molecular architecture of
the active site of the enzyme is known. These agents
probably alter the conformation of the enzyme in such a
way as to make the active site more accessible.
It is not possible to conclude, from the present results,
whether DBA activates the enzyme by the same
mechanism as these alternative treatments. We cannot also
confirm whether it exerts its effects directly on the
pumping protein or via membrane modifications. These
questions will only be resolved when we are able to test its
effects on the purified protein.
ACKNOWLEDGEMENT
This work was supported by the Senate Research Grant
from the University of Ilorin, Nigeria.
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