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339
Braz J Med Biol Res 34(3) 2001
Carbonic anhydrase and catecholamine activation
Brazilian Journal of Medical and Biological Research (2001) 34: 339-345
ISSN 0100-879X
Catecholamine-induced vasoconstriction
is sensitive to carbonic anhydrase I
activation
1
Romanian Medical Academy, Center for Research and Medical Assistance,
Simleu Silvaniei, Salaj, Romania
2
Faculty of Medicine, Oradea, Romania
3
Klinik Wilkenberg, Wilkenberg, Germany
4
Medical Care Unit, Staten Island, New York, NY, USA
I. Puscas
1
, M. Coltau
1
,
L. Gilau
2
, R. Pasca
1
,
G. Domuta
1
, M. Baican
3
and A. Hecht
4
Abstract
We studied the relationship between alpha- and beta-adrenergic ago-
nists and the activity of carbonic anhydrase I and II in erythrocyte,
clinical and vessel studies. Kinetic studies were performed. Adrener-
gic agonists increased erythrocyte carbonic anhydrase as follows:
adrenaline by 75%, noradrenaline by 68%, isoprenaline by 55%, and
orciprenaline by 62%. The kinetic data indicated a non-competitive
mechanism of action. In clinical studies carbonic anhydrase I from
erythrocytes increased by 87% after noradrenaline administration, by
71% after orciprenaline and by 82% after isoprenaline. The increase in
carbonic anhydrase I paralleled the increase in blood pressure. Similar
results were obtained in vessel studies on piglet vascular smooth
muscle. We believe that adrenergic agonists may have a dual mechan-
ism of action: the first one consists of a catecholamine action on its
receptor with the formation of a stimulus-receptor complex. The
second mechanism proposed completes the first one. By this second
component of the mechanism, the same stimulus directly acts on the
carbonic anhydrase I isozyme (that might be functionally coupled with
adrenergic receptors), so that its activation ensures an adequate pH for
stimulus-receptor coupling for signal transduction into the cell, result-
ing in vasoconstriction.
Correspondence
I. Puscas
Center for Research and
Medical Assistance
37 Dunarii Street
4775 Simleu Silvaniei, Salaj
Romania
Fax: +40-60-67-8320
E-mail: ccam@netcompsj.ro
Received August 11, 1999
Accepted December 12, 2000
Key words
Adrenergic agonists
Carbonic anhydrase
Arterial blood pressure
Introduction
Catecholamines released by the sympa-
thetic nervous system and adrenal medulla
are involved in regulating a host of physi-
ological functions, particularly the integra-
tion of responses to a range of stresses (1).
Norepinephrine is the major neurotransmit-
ter in the peripheral sympathetic nervous
system, whereas epinephrine is the primary
hormone secreted by the adrenal medulla in
mammals (2).
Important factors in the response of any
cell or organ to sympathomimetic amines are
the density and proportion of alpha- and
beta-adrenergic receptors (3,4). Studies on
DNA cloning have demonstrated the exist-
ence of at least nine types of adrenergic
receptors (5). Other studies have proved that
binding of agonists to these receptors occurs
when the catecholamine molecule is in the
protonated state (6), all of these receptors
being coupled with G proteins.
Carbonic anhydrase (CA) is a zinc-enzyme
discovered by Meldrum and Roughton in 1932
which catalyzes the reversible hydration reac-
340
Braz J Med Biol Res 34(3) 2001
I. Puscas et al.
tion of CO
2
, having a main role in the mainte-
nance of acid-basic equilibrium (7).
CA
CA
H
2
O + CO
2
H
2
CO
3
H
+
+ HCO
3
-
Eight isozymes have been described so
far, located in the membranes, cytoplasm
and mitochondria of all organs. CA I is
present both in erythrocytes and in vascular
walls and its physiologic role has been in-
completely elucidated (8). CA II is to be
found both in erythrocytes and in the cyto-
plasm. By its presence in the parietal cells of
the gastric mucosa, it has a central role in
HCl production, while in the kidney CA II is
involved in the maintenance of urinary pH
along with CA IV.
Regarding the physiological role of CA
isozymes, our studies have shown that CA I
is involved in the vascular changes (9) while
CA II and CA IV are isozymes involved in
the secretory processes (10).
The same studies showed that CA I and
CA II are activated by nonsteroidal anti-
inflammatory drugs (11) and vasodilating
prostaglandins and diuretic agents (12,13)
inhibit CA while vasoconstrictive prosta-
glandins activate the enzyme (12). These
studies have also demonstrated the involve-
ment of carbonic anhydrase in the regulation
of vascular and secretory processes in the
organism (14).
Our previous work has shown that alpha-
and beta-adrenergic agonists activated puri-
fied and red cell CA while adrenergic antago-
nists inhibited CA and reduced the activating
effect of agonists on this enzyme (15,16).
In the present investigation we studied
the relationship between alpha- and beta-
adrenergic agonists and CA activity in vaso-
constriction mechanism.
Material and Methods
Material
Purified human erythrocyte CA I and
CA II, adrenaline, noradrenaline, isoprena-
line, orciprenaline, HEPES buffer, p-
nitrophenol, and Na
2
SO
4
were obtained
from Sigma Chemical Co. (Deisenhofen,
Germany), orciprenaline (Alupent) was
purchased from Boehringer (Ingelheim,
Germany), and noradrenaline and isopre-
naline (vials) were obtained from Sicomed
(Bucharest, Romania).
Experimental designs
Erythrocyte studies. We studied the ef-
fects of adrenaline, noradrenaline, isoprena-
line and orciprenaline on CA I and CA II
purified from human erythrocytes. Kinetic
determinations were performed at concen-
trations between 10 nM and 100 µM.
Kinetic studies were carried out in order
to identify the mechanism of action of adre-
nergic agonists on CA. Maximum reaction
rate (V
max
) and the Michaelis constant (K
m
)
were determined.
Clinical studies. The study was conducted
according to the Declaration of Helsinki as
modified by the 21st World Medical Assem-
bly, Venice, Italy, 1983 and later by the 41st
World Medical Assembly, Hong Kong, 1989.
All patients gave informed consent for a
protocol approved by the Ethics Committee
of the Center for Research and Medical As-
sistance in Simleu Silvaniei.
We selected 42 healthy male volunteers
aged 30 to 50 years and weighing 60-74 kg,
who were randomly divided into three groups.
All subjects resided in the community and
were in good general health. Subjects were
screened before participation by being sub-
mitted to physical examination, a complete
blood count, fasting serum glucose, and rou-
tine chemistry, urinalysis, and electrocardio-
gram and their medical history was taken.
Patients were excluded from participation if
they exceeded 135% of ideal body weight,
had a past history of hypertension, diabetes
mellitus, had a fasting serum glucose of >6.7
mM, were taking any medications, had ortho-
341
Braz J Med Biol Res 34(3) 2001
Carbonic anhydrase and catecholamine activation
static hypotension, or had evidence from the
screening tests of underlying illness or sig-
nificant laboratory or electrocardiogram ab-
normalities. In the acute experiment, group I
patients (N = 12) received noradrenaline iv
at the dose of 4 mg/1000 ml isotonic solution
(4 µg/min over 30-min periods), group II
patients (N = 14) received orciprenaline
(Alupent) iv at the dose of 0.5 mg, and group
III patients (N = 16) received isoprenaline
(Isoprenalin) sc at the dose of 0.2 mg.
Red cell CA I and CA II activity and
arterial blood pressure values were deter-
mined before and 30 min after drug adminis-
tration and the blood count, routine chemis-
try, urinalysis and electrocardiogram were
repeated.
Vessel studies. In the animal experiments,
20 piglets weighing 25-30 kg were housed in
air-conditioned quarters and had free access
to tap water and standard food. Animals
were divided into 4 groups of 5 piglets each
and treated as follows in the acute experi-
ment: group 1 - noradrenaline, iv doses of 2
µg/min for 30 min; group 2 - orciprenaline
(Alupent), iv doses of 0.25 mg; group 3 -
isoprenaline (Isoprenalin), sc doses of 0.1
mg, and group 4 (control group) - placebo.
Arterial blood pressure was determined
30 min after drug administration and all
piglets were sacrificed for isolation of vas-
cular smooth muscle CA I. CA I activity was
determined and compared to that obtained
for the control group.
Experimental procedure
Differentiation of red cell CA I from CA
II activity was performed by the nicotinate
test (17), which relies on selective inhibition
of CA I activity.
Vascular smooth muscle CA I was iso-
lated from the small mesenteric arteries of
the animals according to the technique of
Lonnerholm et al. (8).
CA I and CA II activity was assessed
using the stopped-flow method (18), which
consists of measuring the enzymatic activity
of CO
2
hydration and is based on a colori-
metric method which measures changing pH.
The time needed for the pH of the reagent
mixture to decrease from its initial value of
7.5 to its final value of 6.5 was measured.
The reaction was monitored spectrophoto-
metrically at 400 nm using a rapid kinetic
Hi-Tech SF-51MX spectrophotometer (Hi-
Tech Scientific Ltd., Salisbury, England)
equipped with a mixing unit and a system of
two syringes which supply the reagents. The
signal transmitted by the photomultiplier from
the mixing chamber is received and visual-
ized by a computer equipped with a math-
ematical coprocessor and the kinetic soft-
ware package RKBIN IS1.
We used p-nitrophenol (0.2 mM) as color
indicator and HEPES (20 mM) as buffer.
Na
2
SO
4
(0.1 M) was used to keep a constant
ionic strength. The CO
2
solution at a concen-
tration of 15 mM (as substrate) was obtained
by bubbling twice-distilled water with CO
2
to
saturation. All reagents were maintained at pH
7.5 and at room temperature (22-25
o
C).
Carbonic anhydrase activity was obtained
by the formula:
A =
T
0
-
T
[enzyme units/ml]
T
where T
0
represents the uncatalyzed reaction
time, and T the catalyzed reaction time (in
the presence of CA). Activity is reported as
enzyme units (EU) per ml.
In the CO
2
hydration reaction catalyzed
by CA one enzyme unit will cause the pH to
drop from 7.5 to 6.5 per minute, at 25
o
C.
In humans, blood pressure was measured
with a standard mercury sphygmomanom-
eter in the classical sitting position and is
reported as the mean of three measurements.
In piglets, blood pressure was measured
under anesthesia by intraperitoneal injection
of pentobarbital sodium (35 mg/kg body
weight) and catheterization of the femoral
artery.
342
Braz J Med Biol Res 34(3) 2001
I. Puscas et al.
Statistical analysis
When repeated measure ANOVA showed
significant differences between groups, the
Newman-Keuls multiple comparison test was
performed to determine which groups dif-
fered significantly. Probabilities of P<0.05
were considered significant.
Results
Erythrocyte studies
Adrenaline, noradrenaline, isoprenaline
and orciprenaline increased CA I and CA II
activity in a dose-dependent manner. The
effect started at 10 nM and reached a peak at
100 µM (Table 1).
Adrenaline increased CA I activity from
0.425 ± 0.01 to 0.743 ± 0.02 EU/ml (75%)
(P<0.001), and CA II activity from 1.00 ±
0.01 to 1.472 ± 0.02 EU/ml (47%) (P<0.001).
Noradrenaline increased CA I activity
from 0.425 ± 0.01 to 0.714 ± 0.01 EU/ml
(68%) (P<0.001), and CA II activity from
1.00 ± 0.01 to 1.436 ± 0.02 EU/ml (43%)
(P<0.001).
Isoprenaline increased CA I activity from
0.425 ± 0.01 to 0.658 ± 0.02 EU/ml (55%)
(P<0.001), and CA II activity from 1.00 ±
0.01 to 1.342 ± 0.01 EU/ml (34%) (P<0.001).
Orciprenaline increased CA I activity
from 0.425 ± 0.01 to 0.688 ± 0.02 EU/ml
(62%) (P<0.001), and CA II activity from
1.00 ± 0.01 to 1.488 ± 0.01 EU/ml (49%)
(P<0.001).
The kinetic data processed according to
the Michaelis-Menten equation showed a
non-competitive mechanism of action with
an increase in V
max
and a constant K
m
.
The
kinetic results show that adrenergic agonists
were bound to the active site of CA I in a
position different from that of the enzyme
substrate, CO
2
(Table 2).
Clinical studies
In group 1, noradrenaline increased red
cell CA I activity from 0.268 ± 0.026 to
0.501 ± 0.042 EU/ml (87%) (P<0.001)
and CA II activity from 1.081 ± 0.116 to
1.598 ± 0.134 EU/ml (48%) (P<0.05) (Fig-
ure 1). Arterial blood pressure rose from 130
± 10 to 175 ± 15 mmHg (P<0.05) (Figure 2).
In group 2, orciprenaline increased red
cell CA I activity from 0.251 ± 0.030 to
0.429 ± 0.027 EU/ml (71%) (P<0.001)
and CA II activity from 1.135 ± 0.110 to
1.288 ± 0.194 EU/ml (27%) (P<0.05) (Fig-
ure 1). Arterial blood pressure rose from 120
± 10 to 155 ± 5 mmHg (P<0.05) (Figure 2).
Table 1 - Effect of adrenergic agonists on isozyme I and II of carbonic anhydrase (CA).
The table shows the increase of the activity of CA isozymes induced by therapeutic
agents such as adrenaline, noradrenaline, isoprenaline and orciprenaline. Values are
reported as means ± SEM (N = 5 assessments) *P<0.05 compared with basal activity
for each isozyme (Newman-Keuls multiple comparison test).
Substance Concentration Purified CA I Purified CA II
(basal activity = (basal activity =
0.425 ± 0.01 EU/ml) 1.00 ± 0.01 EU/ml)
Adrenaline 10 nM 0.519 ± 0.01* 1.194 ± 0.01*
1 µM 0.638 ± 0.01* 1.341 ± 0.02*
100 µM 0.743 ± 0.02* 1.472 ± 0.02*
Noradrenaline 10 nM 0.472 ± 0.02* 1.168 ± 0.02*
1 µM 0.603 ± 0.01* 1.305 ± 0.03*
100 µM 0.714 ± 0.01* 1.436 ± 0.02*
Isoprenaline 10 nM 0.468 ± 0.01* 1.082 ± 0.01*
1 µM 0.595 ± 0.02* 1.258 ± 0.02*
100 µM 0.658 ± 0.02* 1.342 ± 0.01*
Orciprenaline 10 nM 0.476 ± 0.03* 1.203 ± 0.01*
1 µM 0.611 ± 0.01* 1.376 ± 0.02*
100 µM 0.688 ± 0.02* 1.488 ± 0.01*
Table 2 - Kinetic data for interaction between alpha- and beta-adrenergic agonists and
purified carbonic anhydrase (CA) I.
CA I concentration = 3.68 x 10 nM, pH 7.5, T = 25
o
C. Data are reported as means ± SD
(N = 5 assessments). *P<0.05 compared to purified CA I (Newman-Keuls multiple
comparison test).
System V
max
(mM s
-1
) K
m
(mM)
CA I 1.332 ± 0.01 8.99 ± 0.2
CA I + adrenaline (100 µM) 1.796 ± 0.02* 8.87 ± 0.1
CA I + noradrenaline (100 µM) 1.733 ± 0.01* 8.91 ± 0.1
CA I + isoprenaline (100 µM) 1.710 ± 0.02* 8.95 ± 0.2
CA I + orciprenaline (100 µM) 1.742 ± 0.02* 8.89 ± 0.1
343
Braz J Med Biol Res 34(3) 2001
Carbonic anhydrase and catecholamine activation
In group 3, isoprenaline increased red
cell CA I activity from 0.247 ± 0.015 to
0.450 ± 0.020 EU/ml (82%) (P<0.001)
and CA II activity from 0.983 ± 0.105 to
1.474 ± 0.208 EU/ml (50%) (P<0.05) (Fig-
ure 1). Arterial blood pressure rose from 120
± 10 to 160 ± 5 mmHg (P<0.05) (Figure 2).
Vessel studies
In animals, after the acute experiment the
activity of vascular smooth muscle CA I was
0.812 ± 0.062 EU/ml and arterial blood pres-
sure was 120 ± 10 mmHg in the control
group. In all adrenergic-treated groups, vas-
cular smooth muscle CA I activity and arte-
rial blood pressure increased significantly
(P<0.05) compared to controls, as follows:
group 1 - CA I was 1.696 ± 0.124 EU/ml and
arterial blood pressure was 180 ± 15 mmHg;
group 2 - CA I was 1.408 ± 0.136 EU/ml and
arterial blood pressure was 165 ± 10 mmHg;
group 3 - CA I was 1.519 ± 0.143 EU/ml and
arterial blood pressure was 170 ± 10 mmHg
(Figure 3).
Discussion
Our group has studied the relationship
between adrenergic agonists and CA (15,16),
showing that CA was activated by a direct
mechanism. The erythrocyte studies proved
that alpha- and beta-adrenergic agonists are
direct and strong CA I activators which have
less effect on CA II.
Clinical and vessel studies have shown
that adrenergic agonists are powerful CA I
activators both in erythrocytes and in vascu-
lar smooth muscles. In humans, noradrena-
line, orciprenaline and isoprenaline increased
arterial blood pressure in volunteer subjects,
in parallel with an increase of erythrocyte
CA I activity. Parallelism between the in-
crease in arterial blood pressure and erythro-
cyte CA I activation was observed in all
groups. The most potent activating effect on
CA was induced by noradrenaline which
Figure 1 - Effect of noradrena-
line (4 µg/min, over 30 min, iv),
orciprenaline (0.5 mg, iv) and
isoprenaline (0.2 mg, sc) on red
blood cell CA I and CA II activity.
Values are reported as means ±
SEM; N = 12-16 patients.
*P<0.05 compared with values
before treatment (paired t-test).
Figure 2 - Effect of noradrena-
line (4 µg/min, over 30 min, iv),
orciprenaline (0.5 mg, iv) and
isoprenaline (0.2 mg, sc) on ar-
terial blood pressure. Values are
reported as means ± SEM; N =
12-16 patients. *P<0.05 com-
pared with values before treat-
ment (paired t-test).
Figure 3 - Increase of vascular
smooth muscle CA I activity and
of arterial blood pressure values
in piglets after treatment with
noradrenaline (group 1), orci-
prenaline (group 2) and iso-
prenaline (group 3) compared to
control. Values are reported as
means ± SEM; N = 5 piglets.
*P<0.05 compared with control
(paired t-test).
Arterial blood pressure (mmHg)
300
Before treatment After treatment
Noradrenaline Orciprenaline Isoprenaline
250
200
150
100
50
0
CA I and CA II activity (EU/ml)
2.5
Noradrenaline
*
2.0
1.5
1.0
0.5
0
Orciprenaline Isoprenaline
CA I CA II
*
*
*
*
*
Blood pressure (mmHg)
320
CA I activity (EU/ml)
1.5
1.0
0.5
0
Blood pressure CA I activity
Control Group 1 Group 2 Group 3
240
160
80
0
also produced the main increase in blood
pressure. None of these patients presented
any major side effects during the experi-
ments.
In animals, administration of adrenergic
agonists significantly increased CA activity,
mainly of CA I in arteriolar smooth muscle
as compared to controls, in parallel with an
increase in arterial blood pressure.
These results agree with previous studies
by our group which showed that CA I is
involved in the modulation of vascular pro-
*
*
*
*
*
*
*
*
*
344
Braz J Med Biol Res 34(3) 2001
I. Puscas et al.
cesses in the organism. In our conception,
the pH increase induced by CA I inhibition
might influence the binding of hypotensive
stimuli to their specific receptors, followed
by signal transduction to the cytoplasm of
smooth muscle cells with subsequent vaso-
dilating effects (9,14). Similarly, the reduc-
tion in pH induced by vascular smooth muscle
CA I activation with hypertensive agents
may influence the membrane specific recep-
tor and signal transduction in the vascular
smooth muscle cytosol, with subsequent va-
soconstrictive effects (14).
Regarding the role of pH changes in hy-
pertension, our results support recent studies
which have shown that primary hyperten-
sion may be associated with perturbations of
acid-base status or intracellular pH, respec-
tively (19,20). Furthermore, the same stud-
ies demonstrated a decrease of intracellular
pH in hypertensive animal models as com-
pared to normotensive animals (19,21). An
evaluation of steady-state intracellular pH in
erythrocytes using a nuclear magnetic reso-
nance technique indicated that intracellular
pH is reduced in erythrocytes from untreated
patients with essential hypertension com-
pared to treated patients and normotensive
controls (22).
Other studies have reported that the blood
pressure-lowering effects of calcium chan-
nel blockade were inversely related to intra-
cellular pH, i.e., the lower the initial pH, the
greater the antihypertensive effect. Further-
more, nifedipine consistently elevated intra-
cellular pH values (23).
An enhanced activity of the H
+
-Na
+
anti-
porter (as a major mechanism of cell defense
against cellular acidification) has been re-
ported in lymphocytes (24) as well as in the
renal brush border membrane of spontane-
ously hypertensive rats (25) and in hyperten-
sive rats (26). Other authors have shown
enhanced responsiveness of the renal proxi-
mal Na
+
-H
+
antiporter of hypertensive rats to
stimulation with some hormones (27). It has
been assumed that an overactivity of this
antiporter is not a primary process but rather
reflects intracellular acidosis in hyperten-
sion, as studied in spontaneously hyperten-
sive rat models (20,21).
Catecholamine-induced CA I activation
has suggested a concept concerning the in-
volvement of pH changes (induced by acti-
vation) in vasoconstrictive processes due to
adrenergic agonists. In keeping with this
concept, catecholamines have a dual mech-
anism of action: the first consists of adrener-
gic agonists acting on their specific recep-
tors with subsequent formation of a stimu-
lus-receptor complex, followed by the cou-
pling of G proteins and information trans-
mission within the cell. The second mechan-
ism suggests that catecholamines directly
act on CA I (an isozyme that might be func-
tionally coupled with adrenergic receptors),
which by its activation accompanied by a
fall in pH may be favorable to catechol-
amine-binding to its specific receptor and by
G proteins to facilitate information transmis-
sion into the cell.
Our results suggest that CA I modulates
vascular tone by means of pH changes (14).
This role for pH in the vascular bed is sup-
ported by our data as follows: a) the already
known role of CA I in acid-base balance; b)
the effect of vasoconstrictive substances that
activate erythrocyte CA I and vascular smooth
muscle CA I by a direct mechanism of ac-
tion, and c) the effect of vasodilatory sub-
stances along with drugs used in the treat-
ment of hypertension which inhibit CA I by
a direct mechanism of action both in erythro-
cytes and in vascular smooth muscle.
Our results suggest that CA I activation
decreases intracellular pH while its inhibi-
tion increases it. These changes in intracel-
lular pH might influence ion channel activ-
ity, as well as symport and antiport pump
and ATPase activity, all being involved in
the modulation of vascular processes.
345
Braz J Med Biol Res 34(3) 2001
Carbonic anhydrase and catecholamine activation
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