Indian Journal of Experimental Biology
Vol. 45, November 2007, pp. 1003-1006
Angiotensin converting enzyme from sheep
mammary, lingual and other tissues
N Mallikarjuna Rao* & E G Padmanabha Udupa1
*Department of Biochemistry, Katuri Medical College,
Guntur 522 019, India
1
Department of Biochemistry, Kasturba Medical College,
Manipal 576 104, India
Received 27 February 2007; revised 18 June 2007
Occurrence of angiotensin converting enzyme (ACE) in
mammary gland and tongue taste epithelium was demonstrated for
the first time. Six times higher ACE activity in lactating
mammary gland, than non-lactating mammary gland, suggested
pregnancy and lactation hormonal dependent expression of ACE
in female mammals. ACE activity was highest in choroid plexus,
less in spinal cord and moderate in cerebrum, medulla, cerebellum
and pons. Distribution of ACE in different regions of skin, kidney
and among other tissues was different. Presence of ACE in
adrenal glands, pancreas, bone marrow and thyroid gland
indicated functions other than blood pressure homeostasis for this
enzyme.
Keywords: Angiotensin Converting enzyme, Body tissues, Sheep
Angiotensin converting enzyme (ACE, EC. 3.4.15.1)
is a key enzyme of rennin-angiotensin-aldosterone
system (RAS) that is involved in blood pressure and
electrolyte homeostasis. It cleaves dipeptides from
C-terminus of several peptide substrates1,2. However,
recent research has shown that this enzyme is
involved in reproduction of male mammals and
appears at puberty in testis, epididymis, prostate,
seminal fluid under the influence of androgens and
pituitary hormones3-5. Even though this enzyme was
identified in female reproductive organs like uterus,
ovary, fallopian tubes, hormonal influence on
expression of ACE in female mammals is unknown.
Recently, gender differences in the expression of
circulating as well as tissue RAS has been reported6-8.
In females, mammary glands are rudimentary before
puberty and their development begins at puberty. Full
development of mammary gland occurs during
pregnancy and lactation under the influence of
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androgens and pituitary hormones9. However, in male
mammals mammary glands remain rudimentary.
ACE inhibitors are widely used in the treatment of
hypertension, congestive heart failure, coronary artery
disease and diabetic nephropathy2. However, taste
disturbance, skin rash, neutropenia are some of the side
effects associated with use of ACE inhibitors. Taste
disturbance includes absence of taste discrimination and
metallic or sour taste10, 11. Different regions of tongue are
responsible for various taste sensations due to presence
of many types of epithelial cells12. No information is
available on ACE activity of epithelial cells of tongue
from any animal species. Skin is the largest organ of the
body and consists of epidermis and dermis, each with
different functions and enzyme distributions13. An
unique feature of ACE is chloride dependent substrate
specific hydrolysis14. However, role of chloride on
mammary and lingual ACE activity is unknown.
In this study ACE activity was measured in nonlactating, lactating mammary gland of Ewe and
mammary gland of Ram. ACE activity in different
regions of tongue, skin, brain and other tissues was
also measured. Influence of chloride on mammary
and lingual ACE activity was determined. Permission
from the ethic committee of the institute was sought
to undertake the present study.
Tissues were obtained from local farm house and
kept deep frozen at −70°C. Only parts of the tissues
not involved in any disease process were used in the
study. Lactating and non-lactating mammary glands
were separated based on lactation and gender. Tongue
was separated into anterior tongue, lateral tongue,
central tongue and posterior tongue. Epithelial layer
of each part of the tongue was obtained by incubating
in (1 M) KCl for 5 h at 4°C and used for the
preparations of tissue extract. Skin was removed from
ear lobe . Dermis and epidermis were separated by
treating with NH4Cl (0.24 M) for 10 min at 4°C and
used for preparation of tissue extract as described15.
For the preparation of tissue extracts of different parts
of brain skull was opened exposing brain and spinal
cord which were carefully excised and placed on ice.
Different brain parts cerebrum, cerebellum, medulla,
pons and choroid plexus were separated within
30 min and stored at −70° C. A part of tissue was
excised from each of the thawed tissues and used for
INDIAN J EXP BIOL, NOVEMBER 2007
1004
preparation of tissue extract. Renal regions were
macroscopically separated into medulla and cortex as
described16 and part of tissue was excised from each
region and used for the preparation of tissue extract.
ACE was extracted from tissues as described17
using 0.1 M phosphate buffer (pH 8.3) with (50 mM)
NaCl. The protein content of tissue extracts was
measured by the method18 using bovine serum
albumin (Sigma Chemical Company, USA) as
standard.
Determination of ACE activity in tissues⎯Tissue
ACE activity was measured with hippuryl-L-histidylL-leucine (HHL; Sigma Chemical Company, USA) as
substrate by a modified method19. The reaction
mixture (0.175 ml) contained 0.1 ml of 5 mM HHL
dissolved in 0.2 M phosphate buffer (pH 8.3)
containing 0.6 M NaCl, tissue extract and distilled
water. The tissue extract in a volume of 25 μl or less
was used to initiate the reaction. After 30 min
incubation at 37°C , the reaction was stopped by
addition of 0.175 ml of 1 M HCl. Hippuric acid was
extracted with ethyl acetate, dried and resuspended in
ethanol and measured spectrophotometrically at
228 nm17. The specificity of the reaction for ACE was
tested by adding 50 μl of 10 μM captopril (Sigma
Chemical Company, USA) in the incubation mixture.
One unit of ACE was defined as the amount of
enzyme catalyzing release of 1 n mole of hippuric
acid from HHL per min at 37°C.
Effect of chloride on the ACE activity of mammary
gland and tongue was studied by preparing tissue
extracts in chloride free buffer and measuring ACE
activity in the absence and in the presence of 300 mM
NaCl. The Ethical Committee of the institute
approved this study.
ACE activity has been shown in mammary gland,
tongue and skin (Table 1). ACE activity was more in
lactating mammary gland and less in non-lactating
mammary gland and skin. ACE activity was not found
in non-lactating mammary gland of Ram. In Ewe
lactating mammary gland ACE activity was six times
higher than non-lactating mammary gland. These
results indicated that mammary ACE was subjected to
hormonal regulation and appeared at puberty in
female mammals. Further, mammary ACE seemed to
be more sensitive to hormones related to pregnancy
and lactation. Almost equal ACE activity was found
in anterior, lateral and central tongue epithelial cells.
However in posterior tongue epithelium, ACE activity
was slightly less. This suggested specific function for
ACE in tongue. To our knowledge mammary ACE
and lingual ACE had not been determined in any
animal species. ACE activity was same in both
epidermis and dermis of the skin. Captopril caused an
inhibition of above 97% of ACE activity in all the
tissues.
ACE activity in different brain tissues are
presented in Table 2. Captopril inhibited ACE
activity in all brain tissues. ACE activity was high in
choroid plexus and low in spinal cord. In cerebrum,
medulla, cerebellum and pons ACE activity was
moderate. High ACE level in choroid plexus
strongly suggested role for ACE in cerebrospinal
fluid of brain.
Table 1⎯ Angiotensin converting enzyme activity in sheep mammary gland, tongue epithelium and skin
[Values are mean ± S.D of 6 individuals]
Tissue
Specific activity (Units/mg protein)
Inhibition (%)
0.10 ± 0.04
0.66 ± 0.37
100 ± 20.0
97.0 ± 25.0
N.D
0.22 ± 0.07
0.22 ± 0.06
0.21 ± 0.07
0.17 ± 0.06
0.080 ± 0.024
0.034 ± 0.021
0.036 ± 0.020
---100 ± 25.0
100 ± 24.0
100 ± 27.0
100 ± 23.0
97.0 ± 28.0
100 ± 20.0
100 ± 21.0
Ewe mammary gland
Non-lactating
Lactating
Ram mammary gland
Non-lactating
Anterior tongue taste epithelium
Lateral tongue taste epithelium
Central tongue taste epithelium
Posterior tongue taste epithelium
Whole skin
Epidermis
Dermis
ND⎯not detected
SHORT COMMUNICATION
ACE activities of brush border epithelial cells,
vascular endothelial cells and other tissue cells are
presented in Table 3. ACE activity was more
concentrated in renal cortical epithelial cells than
intestinal epithelial cells, vascular endothelial cells of
pulmonary vein and aorta. However, in kidney ACE
activity was ten-folds higher in cortex than medulla.
Likewise, pulmonary vein ACE activity was twice
than aortic ACE activity. Bone marrow, pancreas,
adrenals and lymph nodes exhibited less ACE
activity. Lowest ACE activity was detected in the
thyroid gland. These results indicated that distribution
of ACE varies among tissues and within tissues of
sheep.
Chloride influenced hydrolytic activity of
mammary ACE and lingual ACE. In presence of
chloride lingual ACE activity and mammary ACE
activity was enhanced by 4.2 and 6.6 times,
respectively. This suggested a non-essential
activator role for chloride in catalytic activity of
ACE.
The present study demonstrated existence of ACE
activity in mammary gland and tongue epithelium for
the first time. Further, lack of ACE activity in Ram
mammary gland, high ACE activity in the Ewe
lactating mammary gland suggest that ACE activity is
regulated in female mammals by androgens and by
pituitary hormones, like testicular ACE and
epididymal ACE, in male mammals5. Moreover, six
Table 2⎯ Angiotensin converting enzyme activity in sheep
brain
[Values are mean ± S.D of 6 individuals]
Tissue
Specific activity
(Units/mg protein)
Choroid plexus
Cerebrum
Medulla
Cerebellum
Pons
Spinal cord
0.73 ± 0.25
0.61 ± 0.19
0.58 ± 0.17
0.56 ± 0.18
0.48 ± 0.18
0.29 ± 0.19
Inhibition
(%)
96.3 ± 18.0
98.3 ± 16.0
98.5 ± 17.0
98.0 ± 18.0
98.5 ± 16.0
99.0 ± 18.0
1005
times higher ACE activity in lactating mammary
gland than non-lactating mammary gland suggested
that ACE expression increased several-folds in
presence of hormones of pregnancy and lactation. To
our knowledge, hormonal influence on female
mammal’s ACE activity has not been reported in any
one of the species. The mammary ACE may be
important for uptake of amino acids by mammary
epithelial cells for milk protein synthesis, blood flow
and nutrient supply in lactating mammals. Since taste
disturbance is associated with the use of ACE
inhibitors10,11 ACE activity found in tongue epithelium
suggested involvement of ACE in taste signal
transduction. Alternatively, ACE may be part of local
RAS of lingual epithelial cells. Equal ACE activity in
dermis and epidermis of sheep differs from that found
in murine skin15.
Distribution of ACE in different regions of sheep
brain is different from rat20, man21 and pig22.
However, high ACE activity found in choroid plexus
like by others in rat and man20,21 suggests that it may
be source for ACE in cerebrospinal fluid where it
hydrolyzes peptides other than angiotensin23.
Occurrence of ACE activity in cerebrum, medulla,
pons and cerebellum indicates a role for ACE in
angiotensin II mediated synaptic transmission in
brain24.
Regional distribution of ACE activity in kidney
was similar to that found in man25 and rat16. Further,
distribution of ACE activity in sheep kidney and
intestine was similar to that found in mouse, but
different from rat and guinea pig kidney and
intestine26. High ACE activity in pulmonary vein than
aorta was different from higher ACE activity found in
arterial than cultured venous endothelial cells27. This
suggests that distribution of ACE differs among blood
vessels endothelial cells. Presence of ACE in
adrenals, pancreas and thyroid gland indicates that
ACE may have role in angiotensin II dependent
actions like hormone secretion, pro-inflammatory,
anti-proliferation, cell growth etc. in these organs28,29.
Table 3⎯ Angiotensin converting enzyme activity in sheep tissues
[Values are mean ± S.D of 6 individuals]
Tissue
Kidney cortex
Kidney medulla
Small intestine
Pulmonary vein
Aorta
Bone marrow
Specific activity (Units/mg protein)
Tissue
Specific activity (Units/mg protein)
6.89 ± 1.50
0.79 ± 0.39
1.16 ± 0.63
0.62 ± 0.22
0.36 ± 0.12
0.38 ± 0.15
Pancreas
Adrenals
Lymph nodes
Spleen
Thyroid
0.22 ± 0.09
0.13 ± 0.04
0.12 ± 0.04
0.07 ± 0.03
0.04 ± 0.01
1006
INDIAN J EXP BIOL, NOVEMBER 2007
In contrast bone marrow ACE may regulate
haematopoietic stem cell differentiation and
proliferation by hydrolyzing peptides other than
angiotensin30. Since chloride partially activated
mammary and lingual ACE, it can be concluded that
ACE is involved in hydrolysis of peptides other than
angiotensin I in these tissues14.
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