Thyroid hormones modulate zinc transport activity of rat
intestinal and renal brush-border membrane
Rajendra
Prasad1,
Vivek
Kumar1,
Rajinder
Kumar1, and
Kiran Pal
Singh2
Departments of 1 Biochemistry
and 2 Endocrinology, Postgraduate
Institute of Medical Education and Research, Chandigarh-160012,
India
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ABSTRACT |
Thyroid hormone status influences the
Zn2+ and metallothionein levels in
intestine, liver, and kidney. To evaluate the impact of thyroid
hormones on Zn2+ metabolism,
Zn2+ uptake studies were carried
out in intestinal and renal brush-border membrane vesicles (BBMV).
Steady-state Zn2+ transport in
intestinal and renal cortical BBMV was increased in hyperthyroid
(Hyper-T) rats and decreased in the hypothyroid (Hypo-T) rats relative
to euthyroid (Eu-T) rats. In both the intestinal and renal BBMV,
Hyper-T rats showed a significant increase in maximal velocity compared
with Eu-T and Hypo-T rats. Apparent Michaelis constant was unaltered in
intestinal and renal BBMV prepared from the three groups. Fluorescence
anisotropy of diphenyl hexatriene was decreased significantly in
intestinal and renal brush-border membrane (BBM) isolated from Hyper-T
rats compared with Hypo-T and Eu-T rats. A significant reduction in the
microviscosity and transition temperature for
Zn2+ uptake in intestinal and
renal BBM from Hyper-T rats is in accordance with the increased
fluidity of these BBMs. These findings suggest that the increased rate
of Zn2+ transport in response to
thyroid hormone status could be associated with either an increase in
the number of Zn2+ transporters or
an increase in the active transporters due to alteration in the
membrane fluidity. Thus the thyroid hormone-mediated change in membrane
fluidity might play an important role in modulating Zn2+ transport activity of
intestinal and renal BBM.
zinc metabolism; fluorescence polarization studies; metallothionein; Arrhenius plot
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INTRODUCTION |
ZN2+ is essential in many
biochemical processes and may have a relevant part in the control of
both cell proliferation and cell loss (5, 50).
Zn2+ is essential for enzymes
involved in DNA synthesis and mitosis (54), is a component of many
transcription factors and proteins that control the cell cycle (12,
45), and can inhibit apoptosis (29).
Zn2+ deficiency affects cell cycle
progression (38). Zn2+ is
indispensable for biological processes like development, growth, differentiation, and function of the endocrine and nervous system and
is also important for the maintenance of membrane structure and
function (50). Zn2+ and thyroid
function are related in several ways. Thyroid dysfunction influences
Zn2+ metabolism (18) and vice
versa. Thyroid hormones have a significant role in controlling growth
and functions of intestine and kidney (23, 49). Thyroid hormones
regulate cell proliferation and stimulate epithelial cell production
(49). Thyroid hormones also regulate expression of brush-border
membrane (BBM) enzymes of intestine (8, 19, 21). Dramatic structural
and functional alterations in intestine induced by
Zn2+ deficiency have been shown to
be repaired by thyroxine (T4)
treatment (36). Thyroid hormones also regulate renal plasma flow,
glomerular filtration rate (GFR), reabsorption of phosphate and
Ca2+ (25),
Na+-K+-ATPase
activity (30), and
Na+-Pi
and
Na+/H+
exchange activities in renal BBM (13, 26). In secondary
Zn2+ deficiency in humans,
decreased triiodothyronine (T3)
and T4 levels and unresponsiveness
of thyroid gland to thyroid-stimulating hormone have been reported
(35). On the other hand, hyperthyroid rats showed a variable
distribution of Zn2+ in different
organs with decreased levels of
Zn2+ in kidney (11).
Intestine, liver, and kidney are of particular importance in
maintaining Zn2+ homeostasis.
Intestine and kidney are the major target organs for various regulators
of mineral metabolism. The intestinal and renal absorptive cells are
polar in nature. The membrane exposed to lumen (BBM) is functionally
and structurally distinct from the basolateral membrane, which is in
contact with extracellular fluid. The BBM is the first barrier
encountered by various solutes during absorption in intestine and
kidney (52). Zn2+ transport
systems in renal and intestinal BBM have been well characterized (39,
41, 48). Zn2+ transport by
brush-border membrane vesicles (BBMV) is a carrier-mediated, temperature-dependent, and saturable process. However, no information is available about the influence of putative regulators of
Zn2+ metabolism at important steps such as
absorption, reabsorption, and storage in intestine and
kidney. The interaction between thyroid hormones and
Zn2+, which are both required for
growth and differentiation, prompted us to study the effect of thyroid
hormone status on Zn2+ metabolism,
especially with regard to the regulation of transport across the BBM in
the intestine and kidney of rats.
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MATERIALS AND METHODS |
Chemicals. 65ZnCl2 (483 mCi/g
Zn2+),
D-[U-14C]glucose (292 mCi/mmol),
and
110mAg+
(10 GBq/g Ag+) were purchased
from Bhabha Atomic Research Center (Trombay, Mumbai, India). Thyroid
powder (3 times recrystallized) was obtained from ICN Pharmaceuticals
(Costa Mesa, CA). T3 and
T4 ELISA kits were purchased from
IFCI Clone Systems [Casselchio Di Reno(Bo)]. Ionophore A-23187, HEPES, EGTA, and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma Chemical (St. Louis, MO). Diphenyl hexatriene (DPH) was procured from Molecular Probes (Eugene, OR). Millipore filters (pore size 0.45 µm) were obtained from Millipore (Bedford, MA). Glucose oxidase peroxidase kit was procured from Boehringer Knoll. All other chemicals were analytical grade compounds obtained from commercial sources.
Animals. Young male Wistar strain rats
were obtained from the animal breeding colony of the Postgraduate
Institute of Medical Education and Research. The animals were
acclimatized to laboratory conditions for a few days before
commencement of the experiments. All animals were housed individually
in plastic cages with stainless steel lids and were fed rat chow
(Hindustan Lever, Bombay, India) containing 20% protein, 0.7%
Ca2+, 0.5% phosphorus, 0.004%
Zn2+, and tap water ad libitum.
Zn2+ content in the diet was
measured by atomic absorption spectrophotometry as described earlier
(42).
The rats were randomly segregated into three groups as follows:
hypothyroid (Hypo-T), hyperthyroid (Hyper-T), and euthyroid (Eu-T).
Hypo-T and Hyper-T status was induced essentially as described by
Kinsella and Sacktor (26). Hypo-T rats were fed thiouracil added to the
chow (3 g/kg) and water (0.25 g/l). Hyper-T rats were fed thyroid
powder (1-2 g/kg) added to the thiouracil-containing chow. All the
rats were kept on the test diets for 3 wk. At the end of study, the
animals were killed under light ether anesthesia, and aortic blood was
collected. Serum concentrations of
T3 and T4 were determined using
T3 and
T4 ELISA kits. Small portions of
the liver, kidney, and small intestine were stored at
70°C for the measurement of Zn2+ and
metallothionein (MT).
Measurement of
Zn2+ and MT in
different organs.
Zn2+ levels in the tissues were
estimated by the wet digestion method using an atomic absorption
spectrophotometer (Perkin Elmer-4000) fitted with a hollow cathode lamp
of Zn2+ at 310 nm (42). MT levels
in the tissues were determined in 10,000 g supernatant, which was assayed by
the silver hemoglobin method as described earlier (43).
Preparation of intestinal and renal
BBMV. Renal and intestinal BBMV were prepared by
differential centrifugation procedures essentially as described
elsewhere (3, 40, 48). The BBM pellet was resuspended in 300 mM
mannitol and 15 mM HEPES buffer, pH 6.8, for
Zn2+ transport studies. For
measurement of membrane fluidity, the BBM were suspended in 300 mM
mannitol, 5 mM EGTA, 0.1 mM PMSF, and 18 mM Tris, pH 7.4. EGTA, a
chelator of Ca2+, and PMSF, a
protease inhibitor, were used to minimize the potential effect of
Ca2+, phospholipases, and
proteases. The purity of the BBM was checked by measuring the specific
activities of marker enzymes in intestinal and renal BBM and in
original homogenates (40, 48). Maltase (EC 3.2.1.20) and alkaline
phosphatase (EC 3.1.3.1) activities were enriched 15- to 17-fold in the
intestinal and 9- to 12-fold in renal BBM compared with the respective
homogenates in all three groups. The contamination of basolateral
membranes was checked by assaying
Na+-K+-ATPase
(EC 3.6.1.3), which was found to be negligible and similar in the three
groups. Protein content in the BBM was determined by the method of
Lowry et al. (31) after solubilization of the sample in 2% sodium
lauryl sulfate as described earlier (39). The protein yields in
intestinal and renal BBM isolated from Eu-T, Hypo-T, and Hyper-T rats
were similar (4-5 mg/g intestinal mucosa; 7-8 mg/g kidney cortex).
SDS-PAGE. SDS-PAGE of intestinal and
renal BBM proteins was performed by the method of Laemmli (28) using a
MINI-PROTEAN II electrophoresis apparatus (Bio-Rad). The separating gel
contained 10% (wt/vol) acrylamide and 0.23% bis-acrylamide before
polymerization, and resolving gel was of 5% polyacrylamide. Intestinal
and renal BBM samples (6 µg protein) were dissolved in 25 µl of
0.625 M Tris · HCl, pH 6.8, containing 20% (wt/vol)
glycerol, 1% SDS (wt/vol), 25 mM
-mercaptoethanol, and 0.05%
bromphenol blue and were analyzed by electrophoresis. Broad-range
molecular weight markers (Sigma) were also run along with the test
samples. Electrophoresis was performed at 10 mA/0.75 mm gel for ~1.5
h until the tracking dye reached the lower end of the gel. After the
electrophoresis, the protein bands were visualized by the silver
staining procedure (32).
Transport measurements. Uptakes of
Zn2+ and D-glucose
were measured at 22°C by the Millipore filtration technique using
0.45-µm filters (22, 39). For
Zn2+ uptake, 10 µl of BBMV
(80-120 µg protein) prepared in 300 mM mannitol and 15 mM
HEPES-KOH (pH 6.8) were incubated in 40 µl of uptake buffer
containing 300 mM mannitol, 15 mM HEPES-KOH (pH 6.8), 1 mM
ZnCl2, and 1.0 µCi 65Zn2+. The
uptake was terminated by the addition of 3 ml of ice-cold stop solution
consisting of 150 mM KCl, 15 mM HEPES, and 5 mM EGTA (pH 6.8). The
filters were rinsed two times with this solution. Radioactivity
retained on the filters was measured by an autogamma scintillation
counter (1282 Compugamma, Universal Gamma Counter). For glucose uptake,
10 µl of the membrane suspension (80-120 µg of protein) were
preincubated at 22°C for 1 min, and uptake was initiated by
addition of 40 µl of uptake medium containing a final concentration
of 150 mM NaCl, 10 mM HEPES-KOH, and 25 µM glucose (0.1 µCi
[U-14C]glucose), pH
7.5. The uptake was terminated by addition of 30 vol of ice-cold uptake
medium followed by two washings. The filters were then counted for
radioactivity using liquid scintillation spectrometry
(22).
Fluorescence polarization studies. The
steady-state fluorescence anisotropy (r) of DPH in BBM samples was
measured using a polarization spectrofluorometer (24). In brief, BBM
samples were diluted with a phosphate and HEPES-buffered saline (pH
7.4) to a concentration of 0.4, 0.2, and 0.1 mg protein/ml. One
microliter of DPH was then added from a 1 mM stock solution in
tetrahydrofuran, and the sample was vortexed vigorously. The suspension
was incubated for 30 min at 37°C. Fluorescence was measured at an
excitation wavelength of 360 nm and emission wavelength of 430 nm. All
measurements were done in a 3-mm square quartz cuvette that minimizes
the depolarization of the emitted light due to scattering. The
circulating water bath was used to regulate the temperature of membrane
suspension within ±0.1°C. The intensities of the parallel
(III) and perpendicular (I
) components
of the emission were measured for both parallel and perpendicular
components of excitation wavelength. The polarization (P) of
fluorescence was obtained from the values of intensities by the
following relation: P = III
I
/III + I
(34). The anisotropy parameter
([r0/r]
1)
1 was calculated using
the limiting anisotropy of DPH (r0 = 0.362; see Ref. 46). The apparent microviscosity (ñ, poise) of
the lipid region of the membrane preparation was estimated using the modification of the Perrin equation (46), whereby approximate expression for ñ can be obtained by ñ = 2P/0.46
P = 2.4 r/0.362
r = III/I
1/0.73
0.27 III/I
(8).
Phase transition in lipids is characterized by an abrupt change with
temperature; therefore, Zn2+
uptake was measured at 10, 15, 20, 25, 30, 35, and 40°C in the presence of 1 mM 65Zn2+ as described in
Transport measurements. The activation energy was calculated
using the Arrhenius equation as described earlier (39).
Statistical analysis. The statistical
analysis was done by using one-way ANOVA. Significance was calculated
using preplanned orthogonal contrasts comparing two groups.
F values having a
P < 0.05 were considered significant.
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RESULTS |
Metabolic effects in relation to thyroid hormone
status. As shown in Table 1, serum
T3 and
T4 levels were reduced
significantly in Hypo-T rats compared with Hyper-T and Eu-T rats. The
body weights of Hyper-T rats were significantly lower than those
of Hypo-T and Eu-T rats. There was no significant difference in body
weights between Hypo-T and Eu-T rats. In spite of decreased body
weight, the food intake was significantly higher in Hyper-T rats
compared with Hypo-T and Eu-T rats. This alteration in food intake in
different groups was associated with basal metabolic rate (16). We
measured endogenous creatinine clearance as an index of GFR. GFR
and GFR per 100 gram body weight were increased significantly in
Hyper-T rats compared with either Hypo-T or Eu-T rats, indicating a
substantial increase in filtered load. However, there was no
significant change in serum creatinine levels in either group.
Effect of thyroid hormone status on
Zn2+ and MT
content in intestine, liver, and kidney.
Zn2+ and MT contents in intestine,
liver, and kidney cortex of different groups are presented in Table
2. Zn2+ content in
the intestine, liver, and kidney cortex of Hyper-T rats was
significantly higher than the corresponding organs of Hypo-T and Eu-T
rats. Zn2+ content in these organs
of Hypo-T rats was significantly lower than that in Eu-T rats. These
observations suggest that accumulation of
Zn2+ in intestine, liver, and
kidney cortex varies in parallel to the thyroid hormone status of the
animals. MT levels were found in proportion to the
Zn2+ content in these organs of
different groups, reflecting the induction of MT either in response to
Zn2+ status of the cell or to
thyroid hormone status.
SDS-PAGE of BBM protein. A comparison
of BBM protein pattern from the three groups based on SDS-PAGE is shown
in Fig. 1. It showed striking similarity with respect to
number of bands recovered and their electrophoretic mobilities.
However, intensity of some bands varied between the groups, indicating
that thyroid hormone status altered the expression of various proteins
in intestinal and renal BBM.

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Fig. 1.
SDS-PAGE of renal (A) and intestinal
(B) brush-border membrane (BBM)
proteins. Protein (6 µg) was applied in each lane and resolved on
10% polyacrylamide gel. The gel was silver stained.
A: lane
1, standard molecular mass markers;
lane 2, hypothyroid rats (Hypo-T);
lane 3, euthyroid rats (Eu-T),
lane 4, hyperthyroid rats (Hyper-T).
B: lane
1, Eu-T; lane 2,
Hyper-T; lane 3, Hypo-T. Symbols + and denote increase and decrease, respectively, in
intensity of bands in the indicated lane compared with other two lanes.
* Band visualized in the indicated lane but not in other lanes.
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Effect of thyroid hormones on
Zn2+ uptake.
Zn2+ transport activity in
intestinal and renal BBMV was affected by the thyroid hormone status of
the rats from which the membranes were derived (Table
3). Hyper-T rats showed a significant increase in the
initial (1-min) uptake in both the intestinal and renal BBMV compared
with Hypo-T and Eu-T rats. In contrast, Hypo-T rats showed a
significant decrease in initial uptake of
Zn2+. Interestingly, equilibrium
(2-h) uptake was also significantly higher in the Hyper-T rats in
intestinal and renal BBMV compared with Hypo-T and Eu-T rats. This
finding indicated a probable alteration in the intravesicular volume of
the BBMV in different groups, which could have been responsible for the
altered initial uptake of Zn2+
observed in these groups. To find out whether intravesicular volume is
the factor for a significant accumulation of
Zn2+ at 2 h in intestinal and
renal BBMV of Hyper-T rats, initial and equilibrium uptake measurements
were carried out in the presence or absence of 10 µM A-23187 in the
uptake buffer. In the presence of the ionophore, initial uptake of
Zn2+ by intestinal and renal BBMV
was increased to the same extent in all three groups. However, the
initial uptake remained significantly different in these groups. In
contrast, equilibrium uptake values in the presence of the ionophore
were found not to be significantly different in either of the groups.
These findings indicated that the intravesicular volume of BBMV
prepared either from the intestinal mucosa or renal cortex of the Eu-T,
Hypo-T, and Hyper-T rats was similar.
Effect of thyroid hormone status on kinetic constants of
Zn2+ transport
systems.
Next, the kinetic properties of the
Zn2+ transport in intestinal and
renal BBMV were examined. The initial uptake of
Zn2+ in the intestinal and renal
BBMV was measured at different concentrations of
Zn2+ in the uptake buffer (Figs.
2A and
3A). Hyper-T rats
showed a significant increase in the maximal
Zn2+ transport activity
(Vmax) in the
intestinal and renal BBMV compared with Hypo-T and Eu-T rats. Hypo-T
rats, on the other hand, showed a
Vmax
significantly lower than even the Eu-T rats (Table
4). However, thyroid hormone status did not
alter the apparent Michaelis constant
(Km) in the
intestinal and renal BBMV in either of the groups.

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Fig. 2.
A: Zn2+ (V) uptake as a function of
[Zn2+] in intestinal
BBMV from Hyper-T ( ), Hypo-T ( ), and Eu-T ( ) rats.
Zn2+ uptake into BBMV was measured
at varying concentrations of
65Zn2+
(0.3-3 mM) in the uptake buffer.
B: Hanes-Woolf transformation of data
illustrated in A.
Km and
Vmax for
Zn2+ uptake were determined by
linear regression analysis. Results are expressed as mean ± SE of 6 experiments.
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Fig. 3.
A: Zn2+ uptake (V) as a function of
[Zn2+] in renal BBMV from Hyper-T ( ), Hypo-T
( ), and Eu-T ( ) rats. Zn2+
uptake into BBMV was measured at varying concentrations of
65Zn2+
(0.3-3 mM) in the uptake buffer.
B: Hanes-Woolf transformation of data
illustrated in A. Michaelis constant
(Km) and
maximal velocity
(Vmax) for
Zn2+ uptake were determined by
linear regression analysis. Results are expressed as mean ± SE of 6 experiments.
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Table 4.
Kinetic constants of Zn2+ transport systems in
intestinal and renal BBM of hypo-, hyper- and euthyroid rats
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The effect of temperature on the
Zn2+ transport systems in
intestinal and renal BBMV isolated from different groups was carried out to correlate temperature-dependent changes in the activities of the
transmembrane proteins and the physical state of the membrane lipids.
The temperature dependence of Zn2+
transport activity was expressed as an Arrhenius plot (log
V vs.
1/k). The linear plots with two
slopes were observed in intestinal and renal BBMV from the three groups
(Fig. 4). There was a significant increase in the
transition temperatures of intestinal and renal BBM of Hypo-T rats
compared with Hyper-T rats (Table 5). However, no
significant change was found in transition temperature of intestinal and renal BBM between Hyper-T and Eu-T rats. The flow of activation energy below and above the transition temperature was not significantly different in Hypo-T, Hyper-T, and Eu-T rats.

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Fig. 4.
Arrhenius plots of Zn2+ transport
in intestinal (A) and renal
(B) BBMV from Hyper-T ( ), Hypo-T
( ), and Eu-T ( ) rats. Zn2+
uptake was determined at 1 mM
65Zn2+
in uptake buffer containing 300 mM mannitol and 15 mM HEPES-KOH (pH
6.8) at different temperatures (10-40°C). Data represent means ± SE of 3 experiments, each carried out in triplicate with separate
preparations.
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Table 5.
Activation energies and thermotropic transition temperature of
intestinal and renal BBM of hypo-, hyper- and euthyroid rats
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Fluorescence polarization studies. The
fluorescence polarization and anisotropy of DPH are inversely related
to membrane fluidity. These parameters were significantly lower in
intestinal and renal BBM from Hyper-T rats than from Eu-T and Hypo-T
rats (Fig. 5). The values of apparent
microviscosity and anisotropy parameter {[(ro/r)
1]
1} obtained
at 37°C from intestinal and renal BBM isolated from Hyper-T rats
were significantly lower than those from Eu-T and Hypo-T rats (Fig.
6). The lower microviscosity of intestinal and renal BBM
from Hyper-T rats could then be interpreted as an increased fluidity of
their lipids.

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Fig. 5.
Fluorescence polarization (P) and anisotropy of diphenyl hexatriene
(DPH) in intestinal and renal BBM isolated from Hyper-T, Hypo-T, and
Eu-T rats. All values are means ± SD of 6 independent experiments.
Statistical analysis was conducted using preplanned orthogonal contrast
after ANOVA. * and § F
values having P < 0.05 compared with
the Eu-T and Hypo-T rats, respectively, were considered significant.
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Fig. 6.
Anisotropy parameter
{[r0/r 1] 1} and
microviscosity (ñ, poise) in intestinal and renal BBM isolated
from Eu-T, Hypo-T, and Hyper-T rats. All values are means ± SD of 6 independent experiments. Statistical analysis was conducted using
preplanned orthogonal contrast after ANOVA. * and
§ F values having
P < 0.05 compared with
Eu-T and Hypo-T rats, respectively, were considered significant.
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Thyroid hormone and
Na+-dependent
uptake of D-glucose.
To examine the specificity of the effect of thyroid hormone on
Zn2+ transport activity,
Na+-dependent uptake of
D-glucose was also determined in membrane vesicles from
Hypo-T, Hyper-T, and Eu-T rats (Fig. 7). The initial (30-s) uptake of Na+-dependent
D-glucose was similar in either of the groups.
Additionally, there was no change in passive
(Na+-independent) transport of
D-glucose in these groups (data not shown). Equilibrium
(60-min) uptakes of D-glucose in the three groups were
similar in intestinal and renal BBMV, substantiating our earlier
finding that there was no change in the intravesicular volume of the
BBMV prepared from either group.

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Fig. 7.
Effect of thyroid hormones on
Na+-dependent
D-glucose transport in intestinal
(A) and renal
(B) BBMV from Hyper-T,
Hypo-T, and Eu-T rats. D-Glucose (25 µM) uptake
in BBMV was measured as the difference between glucose uptake in the
presence of an initial Na+
gradient (extracellular Na+
concentration = 150 mM, intracellular
Na+ concentration = 0 mM) and an
initial K+ gradient (extracellular
K+ concentration = 150 mM,
intracellular K+ concentration = 0 mM). Values are expressed as mean ± SE of 6 experiments.
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DISCUSSION |
The present study demonstrated that thyroid hormones alter the
Zn2+ transport activity in both
intestinal and renal BBMV. The membranes from Hypo-T rats had reduced
Zn2+ uptake activity in contrast
to Hyper-T rats, which had greater activity compared with preparation
from Eu-T rats. The mechanism by which
T3 and
T4 regulate
Zn2+ uptake is not known. However,
incubating the intestinal and renal BBMV from the Eu-T rats with
T3 and
T4 (up to 50 nM) in vitro for 1 h
at 25°C did not alter the Zn2+
uptake significantly (data not shown). Presumably, the intact cell was
necessary to demonstrate the effect of thyroid hormones on
Zn2+ transporters. Thyroid hormone
is known to exert cellular effects through binding to a receptor
protein located within the nucleus of target tissues such as intestine,
kidney, and other tissues. These receptors are expressed in a highly
regulated tissue-specific manner (20, 21, 53).
Na+-Pi
cotransport and
Na+/H+
exchanger (NHE) activity in renal BBM have been correlated with thyroid
hormone status of animals (13, 26). Recently,
T3 has been shown to regulate the
renal NHE3 and NHE2 isoform mRNAs, both likely to be apical exchangers
(2).
Our results demonstrated that the Hyper-T state increased
Vmax for
Zn2+ without affecting
Km in either
intestinal or renal BBM. The thyroid hormone-mediated increase in
Zn2+ uptake may be attributable to
the following two factors: 1)
increase in number/turnover of
Zn2+ transporter and
2) alteration in the lipid
environment leading to conversion of inactive transporters into active
ones. Indeed the increased
Vmax could be
associated with increased expression of protein involved in
Zn2+ transport, as thyroid
hormones are known to exert the cellular effects through receptors,
that specifically recognize and bind DNA sequence, thereby acting at
the transcription level (37). In our study, we have also observed a
different pattern of protein expression in renal and intestinal BBM on
SDS-PAGE in all three groups. However, it was not possible to pinpoint
whether the expression of protein involved in
Zn2+ transport is increased or
not, since none of the Zn2+
transporters in the intestinal and renal BBM have been characterized to
date. Recently, in the human intestinal cell line,
Zn2+ transport like
Ca2+ has been shown to occur via
both saturable and nonsaturable processes; in addition,
lysosome-mediated transcellular movement of
Zn2+ has also been suggested (14).
Vitamin D has been shown to increase Zn2+ transport across the cell
with a simultaneous increase in MT content.
T3 in vitro has been shown to
potentiate the genomic effects of vitamin D on
Ca2+ and
Pi transport in chick intestine
(10). Thus it is possible that the thyroid hormone-mediated increase in
Zn2+ transport activity in
intestinal and renal BBM occurs as a result of interaction between
thyroid hormones and vitamin D. Schräder et al. (44) demonstrated
that T3 modulates the vitamin
D-mediated expression of human osteocalcin and mouse osteopontin genes.
Therefore, the observed changes in
Vmax of
Zn2+ transport activity could be a
result of interaction of thyroid hormone and vitamin D at genomic
levels manifesting itself as increased expression of protein involved
in Zn2+ transport in intestinal
and renal BBM.
However, a strong correlation has also been observed between the
Vmax and membrane
fluidity. Indeed thyroid hormones appear to stimulate virtually all
aspects of lipid metabolism, including synthesis, mobilization, and
degradation (1, 27, 47). In general, thyroid hormone excess is
associated with a decrease in stores of most lipids, including
triglycerides, phospholipids, and cholesterol. Thyroid hormones have
also been shown to change the lipid composition of rat colonic plasma
membrane (7). It is well accepted that membrane undergoes many
functional changes when subjected to various intrinsic and extrinsic
stimuli by modification of its physical state (4). The sensitivity of
Zn2+ transport in intestinal and
renal BBM to the physical state of its lipid environment is evident
from the observations of Arrhenius plots (Fig. 4). Existence of breaks
at different temperatures in Hyper-T and Hypo-T rats suggests a lipid
phase separation within the membrane. Increased fluidity of intestinal
and renal BBM from Hyper-T rats is also associated with a decrease in
transition temperature (Table 5). Fluorescence anisotrophy of DPH,
inversely related to membrane fluidity, denotes the structural and
dynamic properties that determine the relative motions and order of
lipid molecules in the membrane (9, 51). The membrane fluidity can
modulate transport processes at different levels, i.e., accessibility and translocation steps. In both of the steps, the
Vmax of the transport system can be modified even without changing the total number
of carriers. A direct effect of membrane fluidity on the accessibility
of protein and freedom of protein conformational changes has been
documented (4, 6, 33). In isolated BBM and cultured renal cells, a
moderate fluidization stimulates the Na+-phosphate transport (15, 55).
This stimulation resulted from an increase in the
Vmax of the
transport system, leaving its affinity unchanged. Taken together, these
findings are consistent with the present observation that increased
Zn2+ uptake is associated with the
increase in Vmax
due to increased fluidity in Hyper-T rats.
Zn2+ and MT are inextricably
linked, and their levels in intestine, kidney, and liver of Hyper-T
rats were significantly higher compared with either Hypo-T or Eu-T
rats. The MT occurs in vivo with its component of bound metal. The
5'-untranslated region of MT genes in humans and mice contains
elements responsive to metals
(Cd2+,
Zn2+,
Hg2+, etc.) and hormones (17).
Zn2+, next to
Cd2+, is the most successful
MT-inducing agent. Generally, a close relationship exists between
Zn2+ status and levels of MT, as
observed in the present study; however, direct action of thyroid
hormones in induction of MT is not yet known.
Further studies are required to elucidate the effect of thyroid
hormones on the expression of proteins involved in transport and
storage of Zn2+ in intestine and
kidney. However, the above evidence that thyroid hormones modulate
Zn2+ transport activity in
intestinal and renal BBM is of importance, as
Zn2+ is a very potent trace
element, and its transporter response to the hormones provides
attractive models to examine the cellular and molecular mechanisms for
endocrine control of transport processes.
 |
ACKNOWLEDGEMENTS |
This project was financed by the Council of Scientific and
Industrial Research, New Delhi, and the Postgraduate Institute of
Medical Education and Research, Chandigarh, India.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for correspondence and reprint requests: R. Prasad, Dept. of
Biochemistry, PGIMER, Chandigarh-160012, India (E-mail:
medinst{at}pgi.chd.nic.in).
Received 14 July 1998; accepted in final form 15 December 1998.
 |
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