Growth hormone regulates the distribution of L-type calcium
channels in rat adipocyte membranes
Shikha
Gaur1,
Mary E.
Morton2,
G. Peter
Frick1, and
H. Maurice
Goodman1
1 Department of Physiology,
University of Massachusetts Medical School, Worcester 01655; and
2 Department of Biology, College
of the Holy Cross, Worcester, Massachusetts 01610
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ABSTRACT |
Earlier studies demonstrated that deprivation of growth hormone
(GH) for
3 h decreased basal and maximally stimulated cytosolic Ca2+ in rat adipocytes and
suggested that membrane Ca2+
channels might be decreased. Measurement of L-type
Ca2+ channels in purified plasma
membranes by immunoassay or dihydropyridine binding indicated a two- to
fourfold decrease after 3 h of incubation without GH. No such decrease
was seen in unfractionated adipocyte membrane preparations. The
decrease in plasma membrane channel content was largely accounted for
by redistribution of channels to a light microsomal membrane fraction.
Immunoassay of
1-,
2/
-, and
-channel
subunits in membrane fractions indicated that the channels
redistributed as intact complexes. Addition of GH during the 1st h of
incubation prevented channel redistribution, and addition of GH after 3 h restored channel distribution to the GH-replete state of freshly
isolated adipocytes. The studies suggest that GH may regulate the
abundance of Ca2+ channels in the
adipocyte plasma membrane and thereby modulate sensitivity to signals,
the expression of which is Ca2+
dependent.
immunoassay; tritiated PN-200-110; cell fractionation; intracellular calcium concentration
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INTRODUCTION |
GROWTH HORMONE (GH) regulates not only the metabolism
of carbohydrate and lipid in adipocytes (12) but also the concentration of intracellular free Ca2+ (9, 10,
32, 33). Earlier studies in this laboratory demonstrated at least two
apparently independent effects of GH on
Ca2+ in rat epididymal adipocytes
(33). At ~2 min after addition of GH to freshly isolated adipocytes,
intracellular Ca2+ concentrations
([Ca2+]i)
increased about twofold (10, 32). Similar responses to GH were
subsequently reported in human IM9 lymphocytes (16), in rat insulinoma
cells (4), and in Chinese hamster ovary cells that express transfected
GH receptors (34). In adipocytes this response to GH was abolished by
exclusion of Ca2+ from the
extracellular fluid, addition of 100 nM nimodipine, or treatment with
protein kinase C inhibitors (10). Conversely, the effect of GH was
mimicked by the dihydropyridine
Ca2+ channel activator BAY K 5552, partial depolarization of the adipocyte membrane with 30 mM KCl, or
treatment with
1,2-dioctanoyl-sn-glycerol (DOG),
suggesting strongly that GH activates voltage-sensitive L-type
Ca2+ channels secondary to
activation of protein kinase C (10).
In adipocytes that were maintained in vitro for
3 h in the absence of
hormones (GH-deprived cells), resting intracellular Ca2+ concentrations
([Ca2+]i)
declined from ~200 to ~100 nM (9, 33). The decline in [Ca2+]i
was prevented by brief exposure of the cells to GH (GH-treated cells)
during the 1st h of a 3- or 4-h incubation period (33). The ability of
GH to sustain resting levels of
Ca2+ appeared to require ongoing
transcription and was blocked by actinomycin D (33). The decline in
[Ca2+]i
in GH-deprived cells was accompanied by a decline in
thapsigargin-insensitive Ca2+-ATPase activity and
Mn2+ permeability through
dihydropyridine-sensitive channels (9) compared with freshly isolated
or GH-treated cells, suggesting that maintenance of resting
[Ca2+]i
depends on maintenance of Ca2+
influx rather than interference with
Ca2+ extrusion. Furthermore, the
decline in
[Ca2+]i
in GH-treated cells to levels seen in GH-deprived cells after treatment
with nimodipine suggested that activity of L-type
Ca2+ channels might account for
the effect of GH on basal levels of Ca2+ (9).
Although voltage-sensitive L-type
Ca2+ channels probably have little
influence on resting
[Ca2+]i
in most cells in which the resting membrane potential is more negative
than
60 mV, they are likely to play a crucial role in this
regard in adipocytes in which the resting membrane potential has been
measured with microelectrodes to be in the
20- to
40-mV range (1, 3, 17, 29, 37). This membrane potential is in the range of
the "window" in which there is sustained
Ca2+ channel activity (8). L-type
channels in heart, skeletal muscle, and nerve are activated only after
the plasma membrane has been depolarized to about
40 to
30 mV (27). The increase in
[Ca2+]i
that results from partial membrane depolarization, treatment with BAY K
5552, or DOG was also attenuated in GH-deprived cells (10). In
addition, no acute change in
[Ca2+]i
was seen for at least 30 min after GH was added to GH-deprived adipocytes (10, 32). Together with the decrease in resting [Ca2+]i,
these observations suggest that GH-deprived adipocytes might have fewer functional L-type
Ca2+ channels in their plasma
membranes. The experiments described here were undertaken to test the
hypothesis that GH maintains the abundance of functional L-type
Ca2+ channels in adipocyte plasma
membranes.
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MATERIALS AND METHODS |
Animals.
Male rats of the Charles River CD strain were obtained from the
breeding facilities at Kingston, NY, and maintained in the vivarium
under conditions of constant lighting (lights on from 0600 to 1800) and
temperature (23°C). Rats were fed Purina 5008 laboratory chow
(Ralston Purina, St. Louis, MO) and water ad libitum and studied when
they attained weights of 150-200 g. The animals were killed by
cervical dislocation, and the epididymal and perirenal fat bodies were
immediately harvested. All procedures were in accord with protocols
approved by the University of Massachusetts Medical School
Institutional Animal Care and Use Committee.
Isolation and handling of cells.
Tissues pooled from 2-30 rats were finely minced, suspended in
Krebs-Ringer bicarbonate incubation medium enriched with 5.5 mM glucose
and 1% (wt/vol) albumin (Intergen, Purchase, NY), and incubated for 20 min with 1 mg/ml collagenase (lot 143710, type A, Boehringer Mannheim
Biochemicals, Indianapolis, IN) according to the procedure of Rodbell
(31) as modified in this laboratory (13). After filtration through
silk, the fat cells were washed three times and resuspended at a
dilution of 1:3 in incubation medium. Some cells were studied without
further processing and hence are referred to as freshly isolated. Other
fat cells from the same pools were incubated at 37°C in a shaking
water bath under an atmosphere of 95%
O2-5%
CO2 in the absence or presence of
100 ng/ml recombinant human GH (generously provided by Genentech, S. San Francisco, CA). After 1 h the fat cells were again washed, resuspended in fresh medium, and incubated for an additional 2 h in the
absence of GH. Adipocytes that were incubated with GH in the 1st h are
referred to as GH treated; those that were incubated without GH are
referred to as GH deprived. In a few experiments, adipocytes were
incubated for 5 h at a dilution of 1:10.
Preparation of adipocyte membranes.
Adipocyte plasma membranes were prepared according to the protocol of
McKeel and Jarett (23) and microsomal membrane fractions according to Simpson et al. (35). Briefly, 3-5 ml (packed cell volume) of adipocytes were homogenized with a Dounce homogenizer in
three volumes of 0.25 M sucrose containing 50 mM Tris buffer, 1 mM
phenylmethylsulfonyl fluoride, and 5 mM
CaCl2, pH 7.4. All steps were
performed at 4°C. The homogenate was centrifuged at 16,000 g for 20 min. The fat cake was removed
and discarded, and the supernate and pellet were processed further. The
supernate was centrifuged at 48,000 g
for 20 min to obtain a "heavy" microsomal pellet and again at
212,000 g for 1 h to obtain a
"light" microsomal pellet. The
16,000-g pellet was resuspended in 10 ml of homogenization buffer, layered over a 9-45% sucrose
gradient, and centrifuged for 1 h at 100,000 g. The plasma membrane fraction was
visible as a white band near the top of the gradient. To obtain a
"combined" membrane fraction, cell homogenates were centrifuged
at 16,000 g to remove the fat cake and
then applied over a 38% (wt/vol) sucrose cushion. After
centrifugation at 100,000 g for 1 h,
the mitochondria and nuclei formed a pellet. The supernate was then centrifuged at 212,000 g for 1 h to
sediment a combined plasma membrane and microsomal pellet. Suspensions
of plasma membranes and heavy and light microsomes containing ~2.5 mg
protein/ml as measured by the procedure of Lowry et al. (22) were
stored at
95°C for later use.
Ligand binding studies.
Specific binding of
(+)-[3H]PN-200-110
(Amersham Life Science, Arlington Heights, IL), a dihydropyridine that
binds specifically to the
-subunit of L-type
Ca2+ channels (11), was used to
quantify Ca2+ channels. Binding
studies were carried out in triplicate by a modification of the
procedure of Glossman and Ferry (11). Briefly, cell membranes (400 µg
protein/tube) were incubated with 8.4 nM [3H]PN-200-110
(specific activity 86 Ci/mmol) in the dark for 2 h at 37°C in the
absence or presence of 1,000-fold molar excess of unlabeled PN-200-110
(generously donated by Sandoz, East Hanover, NJ) or nimodipine
(Research Biochemicals International, Natick, MA). Membrane-bound and
free ligand were separated by filtration through 24-mm Whatman (GF/C)
glass microfiber filters, as described by Morton and Froehner (25), and
the dried filters were counted by liquid scintillation spectroscopy.
Specific binding is defined as the difference between total
3H-ligand binding and the binding
obtained in the presence of unlabeled ligand.
Immunoassays.
Adipocyte membranes were extracted with 1% Triton X-100 plus 0.1%
(final concentration) SDS. After clarification by centrifugation at
16,000 g for 10 min, aliquots
containing 200-300 µg of protein were incubated for 18 h at room
temperature in 96-well microtiter plates that were coated with 1 µg/ml of the capture antibody, monoclonal antibody (MAb) 2B, raised
against the
1-subunit of the
L-type Ca2+ channel purified from
rabbit skeletal muscle (24). The plates were washed to remove unbound
antigen and further incubated for 2 h with 1 µg/ml of biotinylated
MAb 1A, which recognizes an epitope of the
1-subunit that is distinct from
that recognized by MAb 2B (24). Biotinylation was carried out by
incubating the antibody with sulfosuccinimidyl 6-(biotinamide)hexanoate
(Pierce, Rockford, IL) for 30 min at room temperature according to the
vendor's recommended protocol. After they were washed to remove excess
secondary antibody, the plates were incubated for 2 h with 0.1 µg/ml
streptavidin-alkaline phosphatase conjugate (Pierce), washed again, and
incubated for 2-16 h with 100 µl of
p-nitrophenyl phosphate substrate (0.5 mg/ml). Substrate hydrolysis was estimated from light absorbance at 405 nm. The optical density values were compared with values obtained for
standards prepared from crude t-tubule extracts of rat skeletal muscle
in which the specific activity of
Ca2+ channels had been determined
by [3H]PN-200-110
binding. In some experiments the
-subunit was also quantified using
a monoclonal antibody obtained from SWant (Bellizona, Switzerland), and
the
2/
-subunit was measured
using MAb 20A (25).
Measurement of
[Ca2+]i.
Immediately after cellular isolation or after a designated incubation
period, freshly isolated, GH-deprived, and GH-treated adipocytes were
incubated for 30 min with the
Ca2+-sensitive fluorescent dye
(13) fura 2-AM (20 µM; Molecular Probes, Eugene, OR).
[Ca2+]i
was measured according to procedures described previously (32, 33).
Briefly, 100 µl of a 1:30 (vol/vol) suspension of fura 2-loaded cells
were pipetted into a temperature-controlled Plexiglas perifusion
chamber mounted on the stage of an inverted microscope (Nikon Diaphot).
The buoyant cells were held in place by adhesion to a coverslip coated
with Cell-Tak (Collaborative Research, Bedford, MA) and perifused at a
flow rate of 1 ml/min with Krebs-Ringer bicarbonate medium enriched
with glucose and containing 0.1 mg/ml BSA. The adipocytes were imaged
with a ×10 ultraviolet lens (Nikon, glycerin immersion) and
sequentially illuminated with 3-ns pulses of light (337 or 380 nm)
delivered every 33 ms through a bifurcated quartz fiber extending from
a nitrogen laser and a tunable dye laser (Laser Science, Cambridge, MA)
to the epiport of the inverted microscope. Images were recorded with a
charge-coupled device camera (CCTV, New York, NY) and resolved into 512 × 240 pixels with an image processor (Recognition Technology,
Westborough, MA). Data processing was performed using a computer (NEC
Power Mate). Data were selected for processing, and background
fluorescence was subtracted from an area of interest (11 × 7 pixels) usually positioned over the perinuclear region of chosen
adipocytes. In a typical experiment, cells were observed for 10-15
s so that 30-40 cells could be examined during a 9-min scanning
period. [Ca2+]i
was estimated by comparison with an in situ calibration curve constructed from the fluorescence ratios measured in the cytosol of
GH-treated and GH-deprived adipocytes that were made permeable to
Ca2+ by treatment with 1 µM
ionomycin and equilibrated with
10
8-10
4
M extracellular Ca2+ (39).
Electrophoresis.
Membrane fractions were solubilized in Laemmli sample buffer (19), and
10 µg of protein from each were applied to each lane in 7.5%
polyacrylamide slab gels. The proteins were resolved by electrophoresis
and visualized with Coomassie blue.
Enzyme assays.
,
-Methylene adenosine diphosphate-inhibitable
5'-nucleotidase was used as an enzyme marker for the plasma
membranes (5). Assays were performed as described by Burger and
Lowenstein (6) in the absence or presence of
,
-methylene
adenosine diphosphate. Briefly, aliquots of membrane fractions
(1-15 µg protein) were incubated in 1 ml of 60 mM
Tris · HCl (pH 7.4) containing 100 µM ATP and 0.015 µCi of [3H]AMP
(Dupont-NEN, Boston, MA; specific activity 0.2 µCi/pmol) for 15 min
at 37°C. The reaction was terminated by addition of 200 µl of 5%
(wt/vol) zinc sulfate and 200 µl of 0.3 N barium hydroxide. After
centrifugation the supernate was removed and counted by liquid
scintillation.
Ca2+-ATPase activity was measured
by monitoring Ca2+-dependent
release of
32PO4
from [
-32P]ATP as
described by Pershadingh and McDonald (28). Briefly, aliquots of
membrane fractions (10-20 µg protein) were incubated for 30 min
at 37°C in 0.5 ml of 12 mM Tris-PIPES (pH 7.4) that contained 200 µM EGTA, 1 mM
[
-32P]ATP
(Dupont-NEN; specific activity 0.2 µCi/mmol), and 20 mM sodium azide
in the absence or presence of 150 µM
CaCl2. The reaction was terminated
by addition of 100 µl of SDS.
32Pi
was extracted in 300 µl of phosphate reagent [2 vol of 10 N sulfuric acid, 2 vol of 10% (wt/vol) ammonium molybdate, and 1 vol of
0.1 M silicotungstic acid] and 5 ml of 65:35 (vol/vol) xylene-isobutanol. After vigorous shaking, 2 ml of the organic phase
were counted by liquid scintillation.
Ca2+-ATPase activity was
determined as the difference in
32Pi
produced in the absence or presence of
CaCl2. Sensitivity to inhibition
by 100 nM thapsigargin (Sigma Chemical, St. Louis, MO) was used to
distinguish between Ca2+-ATPase
isoforms present in the plasma membrane and the endoplasmic reticulum
(7).
Statistics.
Data were analyzed by Student's
t-test for paired or unpaired values
as appropriate with the Bonferroni adjustment (36) to compensate for
type I errors when multiple comparisons were made.
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RESULTS |
Measurement of specific binding of
3H-labeled dihydropyridines to
Ca2+ channels in intact adipocytes
was not feasible because of low channel abundance and a partition
coefficient that favors sequestration of the ligand in the fat droplet.
However, specific binding of [3H]PN-200-110 was
obtained with a sucrose gradient-purified plasma membrane fraction
prepared from disrupted adipocytes (23). Scatchard analysis of
[3H]PN-200-110 binding
to plasma membranes derived from freshly isolated adipocytes (Fig.
1) indicated ~1.2 × 1011 binding sites/µg membrane
protein and half-saturation at a concentration of ~3.5 nM PN-200-110.
This affinity is similar to reported dissociation constants of 3.87 nM
for skeletal muscle L-type Ca2+
channels (11) and 2.15 nM for clonal pituitary cells (20). If it is
assumed that 250-300 µg of membrane protein can be recovered from 1 ml of packed cells (~2 × 107 cells) and that 50-100%
of the plasma membranes is recovered in our purification procedure,
each adipocyte contains ~2,000-3,000 specific binding sites.

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Fig. 1.
Concentration-dependent binding of
(+)-[3H]PN-200-110 to
plasma membranes prepared from freshly isolated adipocytes from 30 rats. Triplicate aliquots of membranes (400 µg protein/tube) were
incubated with 1 nM
[3H]PN-200-110 and
0-10 nM unlabeled (+)-PN-200-110 in darkness for 2 h at 37°C.
Linear regression analysis gives a slope
(r2 = 0.966)
corresponding to dissociation constant of 3.5 nM and an
x-intercept corresponding to a
Bmax of 76 fmol. Similar data were
obtained in 2 experiments.
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Because of the relatively low specific activity of the available
tritiated ligand and the low abundance of membrane binding sites, it
was not practical to perform multiple Scatchard analyses for each
experimental condition. Ca2+
channel abundance was therefore estimated in plasma membranes prepared
from freshly isolated, GH-deprived, and GH-treated adipocytes using
ligand at the near-saturating concentration of 8.4 nM (Fig. 2). Deprivation of GH for 3 h resulted in
an ~80% decrease in specific binding compared with freshly isolated
or GH-treated cells. The time course for the decrease in PN-200-110
binding and for the decline in
[Ca2+]i
is shown in Fig. 3. After 2 h of GH
deprivation, binding was reduced by about one-half, and by 4 h it
declined even further to ~20% of that seen in freshly isolated cells
(Fig. 3A). By comparison, [Ca2+]i
as measured with fura 2 was unchanged at 2 h of GH deprivation but fell
precipitously to ~25% of that seen in freshly isolated cells in the
succeeding 2 h (Fig. 3B), suggesting
that changes in channel abundance may precede changes in
[Ca2+]i.

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Fig. 2.
L-type Ca2+ channels in adipocyte
plasma membranes as determined by
[3H]PN-200-110
binding. Plasma membranes (400 µg protein) prepared from freshly
isolated, growth hormone (GH)-deprived, or GH-treated adipocytes were
incubated in triplicate with 8.4 nM
[3H]PN-200-110 in
presence or absence of 8.4 µM unlabeled PN-200-110. Bars, means ± SE for binding of
[3H]PN-200-110;
n, number of independent measurements.
Adipocytes obtained from 10 rats were used for each experiment. Values
correspond to ~2,250, 560, and 2,100 channels/cell in freshly
isolated, GH-deprived, and GH-treated adipocytes, respectively, with
assumptions that 1 ml of packed cells contains 250 µg of plasma
membrane protein and 2 × 107
cells and that 70% of binding sites are occupied at this concentration
of ligand.
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Fig. 3.
Time course for development of effects of GH deprivation.
A: specific binding of
[3H]PN-200-110 to
adipocyte membranes prepared 2 and 4 h after incubation without GH
shown as percentage of values obtained in freshly isolated cells. Bars,
means ± SE for 3 populations of cells.
P values are for comparison with
freshly isolated cells. B:
intracellular Ca2+ concentration
([Ca2+]i)
in adipocytes after 2 and 4 h of incubation without GH expressed as
percentage of value obtained for freshly isolated cells. Bars, means ± SE for 8 populations of adipocytes, each including 20 cells.
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Decreased binding to membranes prepared from GH-deprived adipocytes
might reflect decreased affinity for the ligand as a result of channel
inactivation or a decrease in channel abundance due to altered channel
turnover or cellular distribution. To resolve this issue, using two
monoclonal antibodies (MAb 1A and MAb 2B) that recognize distinct
epitopes of the purified
1-subunit of skeletal muscle
L-type Ca2+ channels, we developed
a sandwich immunoassay to estimate the abundance of channel proteins.
Ca2+ channels in a crude t-tubule
preparation of rat skeletal muscle and quantified by binding and
sandwich assays were used as standards (Fig.
4). In agreement with the results of the
binding studies shown in Fig. 2, immunoassays of plasma membrane
extracts revealed a significant reduction in
Ca2+ channel protein in membranes
prepared from GH-deprived adipocytes compared with freshly isolated or
GH-treated adipocytes (Fig. 5). Estimates
of immunoassayable channel abundance in freshly isolated and GH-treated
cell membranes were in close agreement with the results of binding
experiments, but the decline observed in GH-deprived cells appeared
less severe by immunoassay than by ligand binding.

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Fig. 4.
Quantification of Ca2+ channels by
specific ligand binding and optical density readings obtained in
immunoassays. Pooled rat quadriceps muscles (10 g) were homogenized,
and a membrane preparation was obtained by differential centrifugation
(6, 23). Specific binding of 8.4 nM
[3H]PN-200-110 was
assayed as described for adipocyte membranes. Solubilized extracts of
muscle membranes (0-25 µg) were incubated in microtiter plates
with monoclonal antibody (MAb) 2B as capture antibody and biotinylated
MAb 1A as detecting antibody.
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Fig. 5.
L-type Ca2+ channels in adipocyte
plasma membranes as determined by immunoassay for
1-subunit.
Ca2+ channel proteins were
extracted from plasma membranes prepared from freshly isolated,
GH-deprived, or GH-treated adipocytes and assayed in triplicate in
96-well microtiter plates with MAb 2B as capture antibody and
biotinylated MAb 1A for detection. Bars, means ± SE;
n, number of independent measurements.
Values correspond to ~2,900, 1,600, and 2,500 channels/cell in
freshly isolated, GH-deprived, and GH-treated adipocytes,
respectively. P value is for
comparison with freshly isolated or GH-treated cells.
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In contrast with findings of fewer
Ca2+ channels in membranes
purified from GH-deprived adipocytes, binding of
[3H]PN-200-110 in a
combined plasma membrane-microsomal membrane fraction failed to detect
any differences in abundance in GH-deprived or freshly isolated
adipocytes (Fig. 6). This finding suggested that GH might regulate channel distribution rather than channel synthesis or degradation. To assess this possibility, the presence of
Ca2+ channels in microsomal and
plasma membrane fractions obtained from freshly isolated and
GH-deprived adipocytes was estimated by ligand binding assays. With the
assumption of 70% occupancy of binding sites at the ligand
concentration of 8.4 nM, these data correspond to 3,800 ± 640 binding sites in the combined membranes of freshly isolated cells and
3,300 ± 570 binding sites in the combined membranes of GH-deprived
adipocytes. Nearly 80% of those binding sites (3,000 ± 300) were
recovered in the plasma membrane fraction of freshly isolated cells
compared with only ~24% (780 ± 120) in the plasma membrane
fraction of GH-deprived cells. Conversely, <20% of the binding sites
were in the microsomal fraction of freshly isolated cells (720 ± 170) compared with 42% (1,400 ± 210) in the microsomes of
GH-deprived cells.

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Fig. 6.
Binding of
[3H]PN-200-110 by
membrane fractions prepared from freshly isolated and GH-deprived
adipocytes. Combined membranes, pellet obtained when
"postmitochondrial" supernate was centrifuged for 1 h at 212,000 g; microsomes, combined heavy and
light microsomal fractions. Data were corrected for different amounts
of protein recovered in each fraction and normalized to packed cell
volume to provide insight into actual cellular distribution of
channels. In terms of specific activity, plasma membrane fraction of
freshly isolated adipocytes bound 280 ± 27 fmol of
[3H]PN-200-110 per mg
protein compared with 73 ± 12 for GH-deprived cells; microsomal
fraction prepared from freshly isolated cells bound 88 ± 21 fmol of
PN-210-110 compared with 170 ± 26 for GH-deprived cells. Bars,
means ± SE of results of 5 independent experiments, each including
adipocytes pooled from 30 rats.
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Immunoassay of channel distribution in freshly isolated, GH-treated,
and GH-deprived adipocytes yielded similar conclusions (Fig.
7). GH deprivation significantly decreased
the abundance of Ca2+ channel
proteins in the plasma membrane fraction and increased their recovery
in the light microsomes. These changes were prevented by GH treatment.
Once again the decrease in Ca2+
channels in the plasma membrane measured immunologically (Fig. 7) was
not as profound as that estimated from binding studies (Fig. 6) and
corresponds to ~600 channels per cell, ~75% of which could be
accounted for by the increase in channel proteins in the light
microsomes. GH had little if any effect on the abundance of channel
proteins found in the heavy microsomes, which probably include some
plasma membranes and some light microsomes.

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Fig. 7.
Immunoassay of Ca2+ channels in
membrane fractions of freshly isolated, GH-deprived, and GH-treated
adipocytes. Bars, means ± SE of 7 experiments with freshly isolated
cells and 14 experiments each with GH-deprived and GH-treated cells.
Each experiment included adipocytes pooled from 10 rats.
* P < 0.05 compared with
freshly isolated or GH-treated adipocytes.
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Modifications of the immunoassay procedure to detect the
2/
-subunit or the
-subunit provided similar data on channel redistribution (Fig.
8). To assay for the
2/
-subunit, MAb 2B was again
used as the capture antibody, and biotinylated antibody to the
2/
-subunit (MAb 20A) was
used for detection. To assay for the
-subunit, an MAb directed
against an epitope on the
-subunit was used as the capture antibody,
and biotinylated MAb 1A was used for detection. Like the
1-subunit, the
- and
2/
-subunits of the
Ca2+ channels were decreased in
the plasma membrane fractions prepared from GH-deprived adipocytes and
increased in the light microsomal fraction. Although recoveries of
partially purified membrane fractions varied, the data shown in Figs. 7
and 8 indicate that most (>50%) of the
Ca2+ channels that disappeared
from the plasma membranes of GH-deprived cells could be accounted for
in the light microsomal fraction. Table 1
shows the nearly identical distribution ratios of the three channel
proteins in the plasma membrane and light microsomal fractions of
freshly isolated and GH-deprived adipocytes. These data are the means
of three independent experiments, each including adipocytes from 15 rats. Thus it appears that the channels remain intact and may be
translocated as functional complexes.

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Fig. 8.
Effects of GH treatment and deprivation on abundance of L-type
Ca2+ channel subunits in plasma
membranes (A) and light microsomes
(B). Bars, means ± SE of 3 experiments, each including adipocytes from 15 rats. All 3 subunits
were significantly higher (P < 0.05)
in plasma membranes of GH-treated than GH-deprived adipocytes
(A) and significantly higher
(P < 0.05) in light microsomes of
GH-deprived adipocytes (B).
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Table 1.
Ratios of channel subunit proteins recovered in plasma membrane and
light microsomal fractions calculated from data in Fig. 8
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Clearly, the fractionation scheme used did not yield pure plasma
membranes and a pure light vesicular membrane fraction. The specific
activity of the plasma membrane marker enzymes 5'-nucleotidase and thapsigargin-insensitive
Ca2+-ATPase was at least three
times higher in the plasma membrane fraction than in the microsomal
fraction, whereas the heavy microsomal fraction contained the bulk of
the thapsigargin-sensitive
Ca2+-ATPase characteristic of the
endoplasmic reticulum (Fig.
9). The relatively low
abundance of plasma membrane and endoplasmic reticulum marker enzymes
is consistent with the findings of Simpson et al. (35), who reported
that this fraction consisted largely of Golgi membranes. GH deprivation
did not affect the distribution of any of the three enzymes measured,
although the amount of the Ca2+-ATPase recovered in the
plasma membrane fraction was significantly lower than that recovered in
GH-treated cells in accord with earlier findings (9). Qualitative
differences among the three fractions were also evident from the array
of the most abundant proteins, as seen in each fraction after
electrophoretic separation on polyacrylamide gels and staining with
Coomassie G200. Each membrane fraction showed a distinct pattern of
darkly staining bands, but there were no obvious differences between
GH-deprived and GH-treated cells, indicating that GH deprivation did
not produce any global rearrangement of cellular proteins (data not
shown).

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Fig. 9.
Distribution of enzyme activities in plasma membranes, heavy
microsomes, and light microsomes prepared from GH-treated and
GH-deprived adipocytes. Bars, means ± SE of 4 experiments each
involving 8 rats. Plasma membrane fractions contained significantly
greater (P < 0.01) activities of
5'-nucleotidase (A) and
thapsigargin-insensitive
Ca2+-ATPase
(B) than light or heavy microsomes.
Activity of thapsigargin-sensitive
Ca2+-ATPase
(C) was significantly greater
(P < 0.05) than zero only in heavy
microsomal fraction. Thapsigargin-insensitive
Ca2+-ATPase activity was
significantly (P < 0.05)
higher in plasma membranes of GH-treated adipocytes, but GH treatment
did not significantly affect activity of either enzyme in any
fraction.
|
|
To determine whether the translocation of
Ca2+ channels can be reversed by
treatment with GH, adipocytes were incubated for a total of 5 h in the
absence of GH or with GH present for various times (Fig.
10), and
[Ca2+]i
or the
1-subunit was measured.
[Ca2+]i
declined by a factor of ~2 during the first 3 h of incubation without
GH and remained at that low level for the subsequent 2 h. Addition of
GH at 3 h restored
[Ca2+]i
by the 5th h (Fig. 10A). When GH was
present during the first 3 h of the 5-h incubation,
[Ca2+]i
remained constant or rose slightly (data not shown). Similarly, Ca2+ channels measured by
immunoassay declined by ~40% in the plasma membrane fraction after 5 h without GH and increased by nearly twofold in the light microsomes.
Exposure of adipocytes to GH for 1 h at the end of the 3rd h resulted
in a nearly complete reversal of these changes by the end of the 5th h.
The distribution of Ca2+ channel
protein measured at the end of the 5-h incubation period was unchanged
compared with freshly isolated cells when GH was present for the first
3 h of incubation (data not shown). The data suggest that GH not only
prevents the migration of Ca2+
channels out of the plasma membrane but also promotes their
reinsertion.

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|
Fig. 10.
Reversibility of effects of GH deprivation on
[Ca2+]i
and channel protein distribution. A:
[Ca2+]i
was measured at start of 5-h incubation and 3 and 5 h later. All cells
were deprived of GH for first 3 h. Some cells were then incubated with
100 ng/ml GH for 1 h (GH @ 3 h). Each point represents mean ± SE
for number of cells indicated in parentheses.
[Ca2+]i
was decreased significantly at 3 h (P < 0.01) and declined further (P < 0.05) at 5 h. Treatment with GH increased
[Ca2+]i
significantly (P < 0.01) above 3- and 5-h values in untreated cells. Cells from same populations that
were treated with 100 ng/ml GH for first 3 h showed no decline in
[Ca2+]i
(data not shown). B:
adipocytes were incubated for 5 h without GH, with 100 ng/ml GH
present only for first 3 h or only during 4th h. Bars, means ± SE
for 5 independent experiments each involving adipocytes from 30 rats.
1-Subunit was significantly
diminished in plasma membrane fraction of cells incubated without GH
for 5 h compared with GH treatment for first 3 h
(P < 0.01) or GH treatment for only
4th h (P < 0.05) and significantly
increased in light microsomes compared with cells treated with GH for
first 3 h (P < 0.01) or only 4th h
(P < 0.05).
|
|
 |
DISCUSSION |
Results of the foregoing experiments are consistent with the hypothesis
that GH maintains functional L-type
Ca2+ channels in the adipocyte
plasma membrane. Channel abundance in partially purified plasma
membrane preparations was markedly reduced after GH deprivation as
determined by ligand binding assays and by immunoassays for three
different channel proteins. Although the particular isoforms of
Ca2+ channel subunits that are
present in adipocytes are unknown, the very fact that they could be
measured by immunoassay indicates that adipocytes express L-type
Ca2+ channels that must share at
least some immunologic determinants with L-type channels that are
expressed in skeletal muscle. The decrease in plasma membrane channels
was accompanied by an increase in channel abundance in the light
microsomal fraction in GH-deficient adipocytes. This shift in channel
distribution was prevented by including GH in the initial incubation
buffer and was reversed by treating GH-deprived cells with GH.
In these experiments the abundance of
Ca2+ channels in different
membrane fractions was estimated by ligand binding and by immunoassay. Estimates of channel abundance in ligand binding assays as carried out
in these studies assume that at least two important conditions are met:
1) all channels are accessible to
the ligand and 2) affinity for the
ligand is constant. Immunoassay provides an estimate of the amount of
channel proteins that are present in the sample and, because the
proteins are extracted from their native membrane environment, neither
accessibility nor configuration as related to ligand binding affinity
is a relevant concern. Agreement between the two assay procedures was
quite good for channel abundance in membranes of freshly isolated or
GH-treated cells, as evident from comparison of Figs. 2 and 5. However,
the abundance of Ca2+ channels in
the plasma membranes of GH-deprived adipocytes was two- to threefold
greater when estimated by immunoassay than by ligand binding.
Additionally, ~75% of the immunoassayable channel proteins lost from
the plasma membrane in GH-deprived cells could be accounted for by the
gain in the light microsomes (Fig. 7), but less than one-half can be
accounted for when measured by ligand binding assay (Fig. 6). Several
factors may contribute to these discrepancies. It is possible that the
discrepancies are artifactual because of the imprecision inherent in
measuring low levels of specific binding or that the immunoassay
overestimates channel protein abundance at the low end of the
detectable range. Alternatively, it is possible that GH deprivation
decreases affinity for the ligand as well as channel abundance. The
affinity of the
1-subunit for
dihydropyridines is not constant and varies with the activity state
(20, 21). Binding assays were performed at a single ligand
concentration of 8.4 nM, at which theoretically 70% of the ligand
binding sites in freshly isolated or GH-treated cells are occupied. A
fivefold decrease in affinity, for example, would produce an apparent
twofold decrease in Ca2+ channel
abundance. The possibility that GH maintains the activity as well as
the abundance of Ca2+ channels
will probably be resolved only by application of electrophysiological studies.
Another explanation for the discordant assay results in the GH-deprived
adipocytes derives from the finding that dihydropyridine binding sites
are accessible to the ligand only from the extracellular side of the
membrane (2). The binding sites of any L-type channels present in
intracellular (endosomic) vesicles that escaped rupture by the
homogenization procedure would face inward and, hence, be inaccessible
to [3H]PN-200-110.
These channels would escape detection in a ligand binding assay, but
not in an immunoassay. The observed higher specific activity of ligand
binding sites in microsomal vesicles than in plasma membranes of
GH-deprived cells (Fig. 6) indicates that contamination with plasma
membranes cannot account for all the binding detected in the microsomes
and that at least some of the channels in these vesicles were
accessible to
[3H]PN-200-110.
Nevertheless, the combined heavy and light microsomal fractions of
GH-deprived cells contained ~30% more channels when estimated by
immunoassay than by ligand binding, raising the possibility that as
many as one-third of the channels in the light microsomes may be
inaccessible to the ligand. If so, any intact light microsomal vesicles
that might contaminate the plasma membrane fraction would contribute
more to the immunoassay estimates of channel abundance than to the
ligand binding estimates. Such contamination would be quantitatively
less important for the plasma membranes of freshly isolated or
GH-treated cells, because the light microsomes of these cells contain
fewer channels than those of GH-deprived cells. Our data provide no
estimate of light microsomal contamination of the plasma membrane
fraction, but assay of specific marker enzymes by Simpson et al. (35)
in comparable experiments suggests that as much as 20-25% of the
protein recovered in the plasma membrane fraction was derived from the
light microsomal fraction. This degree of contamination of the plasma
membrane fraction with intact microsomal vesicles that were impermeable
to the ligand could account for as much as 40% of the discrepancy
between the two assays.
The findings that the time and magnitude of the reversible decline in
[Ca2+]i
roughly paralleled the decline in
Ca2+ channel abundance in the
partially purified membranes lends further credence to the idea that GH
regulates the distribution of these channels and that resting
[Ca2+]i
in adipocytes are at least partly determined by rates of
Ca2+ influx through these
channels. The earlier decline in plasma membrane channel abundance than
in
[Ca2+]i
seen in Fig. 3 appears to indicate that the loss of plasma membrane
channels preceded the fall in
[Ca2+]i,
but it is important to note that
[Ca2+]i
is also partly determined by the rate of
Ca2+ extrusion by the
Ca2+-ATPase, which also declined
in GH-deficient adipocytes (8) (Fig. 9). The time course of that
decline is unknown. In this regard, it is noteworthy that the
Ca2+-ATPase did not translocate to
the light microsomes with the L-type Ca2+ channels in GH-deficient
cells (Fig. 9).
The findings of reciprocal and reversible changes in the abundance of
channels in the plasma membrane and the light microsomal fraction
strongly support an argument for hormone-dependent shuttling of
channels between the surface membrane and some less dense intracellular membranous structures. Similar reversible internalization of L-type Ca2+ channels in response to
depolarization was reported by Liu et al. (20) in clonal pituitary
cells. The reversible downregulation of these channels in rat
GH4C1
pituitary cells after prolonged treatment with epidermal growth factor
(15) may represent a similar phenomenon. GH may now be added to the
growing list of hormones, including insulin (30), antidiuretic hormone
(ADH) (18), and parathyroid hormone (26), the actions of which include regulation of the insertion and removal of intrinsic membrane proteins
that determine membrane permeability characteristics. Likewise, L-type
Ca2+ channels may join GLUT-4,
aquaporin, and the sodium-phosphate cotransporter as proteins that are
shuttled into and out of the plasma membrane in a hormone-dependent
manner. Many questions remain to be answered, however. The successful
immunologic assays that measure the expression of
2/
- and
-subunits along
with the
1-subunit indicate
that the channel complex remained intact in the translocation process
and was not disrupted by our solubilization procedure. The
internalization of Ca2+ channels
in GH-deprived cells differs in this regard from the internalization of
Ca2+ channels brought about by
prolonged depolarization of
GH4C1
cells (21). In the latter case, the
1- and
-subunits appeared to dissociate from the
2/
-subunit, which remained
associated with the plasma membrane. Failure to account for a
relatively small amount of channel proteins lost from the plasma
membrane and not recovered in microsomes may be ascribed to
methodological limitations, but some dissociation and loss to the
soluble cell extract cannot be ruled out. In the adipocyte, unlike the
GH4C1
cell, there was no evidence of selective retention of the
2/
-subunit in the plasma
membrane. Translocation of intact or partially dissociated channel
complexes might best be envisioned as an endocytic process akin to that
described for GLUT-4 (30). However, the nature, composition, and
cellular location of the putative vesicles that harbor the
Ca2+ channels in the GH-deficient
state remain to be determined.
There are some notable differences in the GH-dependent translocation of
Ca2+ channels and in the
translocation of GLUT-4 in response to insulin or translocation of
aquaporin in response to ADH. The actions of insulin on GLUT-4
translocation and of ADH on aquaporin are apparent within minutes (18,
30) and are independent of protein synthesis, whereas the actions of GH
on Ca2+ channels require hours and
may depend on transcription (33). The nature of any induced protein(s)
that may be required for translocation and their role in the putative
translocation process are unknown. It is possible, however, that the
delay in the GH effect reflects time needed for synthesis of some
short-lived protein that signals a translocation process similar to
that involved in GLUT-4 or aquaporin translocation.
Although the precise role of L-type
Ca2+ channels in adipocyte
physiology has yet to be established, it is clear that cellular Ca2+ levels are associated with,
and probably govern, responsiveness to the insulin-like actions of GH
(33). Because changes in intracellular Ca2+ are involved in signaling and
executing many different cellular functions, adjustment of the
abundance of Ca2+ channels may
provide a means for adjusting the sensitivity of many cellular
processes to hormones and other agonists. For example, if the abundance
of L-type Ca2+ channels in the
plasma membranes of secretory cells were increased by GH, a given
concentration of agonist might be expected to produce a greater
secretory response. Further studies are required to ascertain whether
GH or other hormones enhance the sensitivity of target cells to
agonists in this manner.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Hiroshi Yamaguchi for technical support,
helpful discussions, and encouragement.
 |
FOOTNOTES |
This publication was made possible by National Institute of Diabetes
and Digestive and Kidney Diseases Grant DK-19392. S. Gaur was supported
by National Institute of Diabetes and Digestive and Kidney Diseases
Training Grant DK-07302. The contents are solely the responsibility of
the authors and do not necessarily represent the official views of the
National Institutes of Health.
A preliminary report of these findings was presented at the 79th Annual
Meeting of the Endocrine Society, Minneapolis, MN, June 1998.
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 reprint requests: H. M. Goodman, Dept. of Physiology,
University of Massachusetts Medical School, 55 Lake Ave. N., Worcester,
MA 01655.
Received 2 February 1998; accepted in final form 29 April 1998.
 |
REFERENCES |
1.
Akiyama, Y.,
C. Kamei,
and
K. Tasaka.
Histamine lipolysis. II. Effects of histamine and related compounds on membrane potentials of rat adipocytes.
Methods Find. Exp. Clin. Pharmacol.
12:
457-465,
1990[Medline].
2.
Bangalore, R.,
N. Baindur,
A. Rutledge,
D. J. Triggle,
and
R. S. Kass.
L-type calcium channels: asymmetrical intramembrane binding domain revealed by variable length, permanently charged 1,4-dihydropyridines.
Mol. Pharmacol.
46:
660-666,
1994[Abstract].
3.
Biegelman, P. M.,
and
M. J. Shu.
Adipose resting membrane potential: in vitro responses to Cl
and K+.
Proc. Soc. Exp. Biol. Med.
141:
618-621,
1972.
4.
Billestrup, N.,
P. Bouchelouche,
G. Allevato,
M. Ilondo,
and
J. H. Nielsen.
Growth hormone receptor C-terminal domains required for growth hormone-induced intracellular free Ca2+ oscillations and gene transcription.
Proc. Natl. Acad. Sci. USA
92:
2725-2729,
1995[Abstract].
5.
Brake, E. T.,
P. C. Will,
and
J. S. Cook.
Characterization of HeLa 5'-nucleotidase: a stable plasma membrane marker.
Membr. Biochem.
2:
17-46,
1977.
6.
Burger, R. M.,
and
J. M. Lowenstein.
Preparation and properties of 5'-nucleotidase from smooth muscle of small intestine.
J. Biol. Chem.
245:
6274-6280,
1970[Abstract/Free Full Text].
7.
Fernandez, J. L.,
M. Rosemblatt,
and
C. Hidalgo.
Highly purified sarcoplasmic reticulum vesicles are devoid of Ca2+-independent ("basal") ATPase activity.
Biochim. Biophys. Acta
599:
552-568,
1980[Medline].
8.
Fleishmann, B. K.,
R. K. Murray,
and
M. I. Kotlikoff.
Voltage window for sustained elevation of cytosolic calcium in smooth muscle cells.
Proc. Natl. Acad. Sci. USA
91:
11914-11918,
1994[Abstract/Free Full Text].
9.
Gaur, S.,
H. Yamaguchi,
and
H. M. Goodman.
Growth hormone regulates cytosolic free calcium in rat fat cells by maintaining L-type calcium channels.
Am. J. Physiol.
270 (Cell Physiol. 39):
C1478-C1484,
1996[Abstract/Free Full Text].
10.
Gaur, S.,
H. Yamaguchi,
and
H. M. Goodman.
Growth hormone increases calcium uptake in rat fat cells by a mechanism dependent on protein kinase C.
Am. J. Physiol.
270 (Cell Physiol. 39):
C1485-C1492,
1996[Abstract/Free Full Text].
11.
Glossman, H.,
and
D. R. Ferry.
Assay for calcium channels.
Methods Enzymol.
109:
513-559,
1985[Medline].
12.
Goodman, H. M.
Growth hormone and metabolism.
In: The Endocrinology of Growth, Development, and Metabolism in Vertebrates, edited by M. P. Schreibman,
C. G. Scanes,
and P. K. T. Pang. San Diego, CA: Academic, 1993, p. 93-115.
13.
Grichting, G.,
L. K. Levy,
and
H. M. Goodman.
Relationship between binding and biological effects of growth hormone in rat adipocytes.
Endocrinology
113:
1111-1120,
1983[Abstract].
14.
Grynkiewicz, G.,
M. Poenie,
and
R. Y. Tsien.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J. Biol. Chem.
260:
3440-3450,
1985[Abstract].
15.
Hinkle, P. M.,
E. J. Nelson,
and
A. A. Haymes.
Regulation of L-type voltage-gated calcium channels by epidermal growth factor.
Endocrinology
133:
271-276,
1993[Abstract].
16.
Ilondo, M. M.,
P. De Meyts,
and
P. Bouchelouche.
Human growth hormone increases cytosolic free calcium in cultured human IM-9 lymphocytes: a novel mechanism of growth hormone transmembrane signalling.
Biochem. Biophys. Res. Commun.
202:
391-397,
1994[Medline].
17.
Kamei, C.,
T. Mukai,
and
K. Tasaka.
Histamine-induced depolarization and the cyclic AMP-protein kinase A system in isolated guinea pig adipocytes.
Jpn. J. Pharmacol.
60:
179-186,
1992[Medline].
18.
Knepper, N. A.,
J. G. Verbalis,
and
S. Nielsen.
Role of aquaporins in water balance disorders.
Curr. Opin. Nephrol. Hypertens.
6:
367-371,
1997[Medline].
19.
Laemmli, U. K.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[Medline].
20.
Liu, J.,
R. Bangalore,
A. Rutledge,
and
D. J. Triggle.
Modulation of L-type Ca2+ channels in clonal rat pituitary cells by membrane depolarization.
Mol. Pharmacol.
45:
1198-1206,
1994[Abstract].
21.
Liu, J.,
A. Rutledge,
and
D. J. Triggle.
Short-term regulation of neuronal calcium channels by depolarization.
Ann. NY Acad. Sci.
765:
119-133,
1995[Abstract].
22.
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr,
and
R. J. Randall.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:
265-275,
1951[Free Full Text].
23.
McKeel, D. W.,
and
L. Jarett.
Preparation and characterization of a plasma membrane fraction from isolated fat cells.
J. Cell Biol.
44:
417-432,
1970[Abstract/Free Full Text].
24.
Morton, M. E.,
and
S. C. Froehner.
Monoclonal antibody identifies a 200-kDa subunit of the dihydropyridine-sensitive calcium channel.
J. Biol. Chem.
262:
11904-11907,
1987[Abstract/Free Full Text].
25.
Morton, M. E.,
and
S. C. Froehner.
The
1 and
2 polypeptides of the dihydropyridine-sensitive calcium channel differ in developmental expression and tissue distribution.
Neuron
2:
1400-1506,
1989.
26.
Murer, H.,
and
J. Biber.
Molecular mechanisms of renal apical Na/phosphate cotransport.
Annu. Rev. Physiol.
58:
607-618,
1996[Medline].
27.
Pelzer, D.,
S. Pelzer,
and
T. E. McDonald.
Properties and regulation of calcium channels in muscle cells.
Rev. Physiol. Biochem. Pharmacol.
114:
107-207,
1990[Medline].
28.
Pershadingh, H. A.,
and
J. M. McDonald.
A high-affinity calcium-stimulated magnesium-dependent adenosine triphosphatase in rat adipocyte plasma membranes.
J. Biol. Chem.
255:
4087-4093,
1980[Free Full Text].
29.
Ramirez-Ponce, M. P.,
J. Acosta,
and
J. A. Bellido.
Electrical activity of white adipose tissue of rat.
Rev. Esp. Fisiol.
46:
133-138,
1990[Medline].
30.
Rea, S.,
and
D. E. James.
Moving GLUT4: the biogenesis and trafficking of GLUT4 storage vesicles.
Diabetes
46:
1667-1677,
1997[Abstract].
31.
Rodbell, M.
Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis.
J. Biol. Chem.
239:
375-380,
1964[Free Full Text].
32.
Schwartz, Y.,
H. M. Goodman,
and
H. Yamaguchi.
Refractoriness to growth hormone is associated with intracellular calcium in rat adipocytes.
Proc. Natl. Acad. Sci. USA
88:
6790-6794,
1991[Abstract].
33.
Schwartz, Y.,
H. Yamaguchi,
and
H. M. Goodman.
Growth hormone increases intracellular free calcium ([Ca2+]i) in rat adipocytes: correlation with actions on carbohydrate metabolism.
Endocrinology
131:
772-778,
1992[Abstract].
34.
Sekine, N.,
S. Ullrich,
R. Regazzi,
W. F. Pralong,
and
C. B. Wollheim.
Postreceptor signalling of growth hormone and prolactin and their effects in the differentiated insulin-secreting cell line, INS-1.
Endocrinology
137:
1841-1850,
1996[Abstract].
35.
Simpson, I. A.,
D. R. Yver,
P. J. Hissin,
L. J. Wardzala,
E. Karnielli,
L. B. Salans,
and
S. W. Cushman.
Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: characterization of subcellular fractions.
Biochim. Biophys. Acta
763:
393-407,
1983[Medline].
36.
Snedecor, G. W.,
and
W. G. Cochran.
Statistical Methods (7th ed.). Ames: Iowa State University Press, 1980.
37.
Stark, R. J.,
P. D. Read,
and
J. O'Doherty.
Insulin does not act by causing a change in membrane potential of intracellular free sodium and potassium concentration of adipocytes.
Diabetes
29:
1040-1043,
1980[Abstract].
38.
Thastrup, O.,
A. P. Dawson,
O. Scharaff,
B. Foder,
P. J. Cullen,
B. K. Drobak,
P. J. Bjerrum,
S. B. Christensen,
and
M. R. Hanley.
Thapsigargin, a novel molecular probe for studying intracellular calcium release and storage.
Agents Actions
27:
17-23,
1989[Medline].
39.
Williams, D. A.,
K. Fogarty,
R. Y. Tsien,
and
F. S. Fay.
Calcium gradients in single smooth muscle cells revealed by the digital imaging microscope using fura 2.
Nature
318:
558-561,
1985[Medline].
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