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Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada
Address all correspondence and requests for reprints to: Dr. Gaétan Guillemette, Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, Quebec, Canada J1H 5N4.
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Introduction |
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It is well recognized that exposure of cells to agonists often leads to desensitization of cell surface receptors. This phenomenon has been shown to exist for a number of different G protein-coupled receptors and has been well characterized for ß2-adrenergic receptor (ß2-AR). Three different mechanisms have been proposed for the desensitization of ß2-AR (for review, see 13 . In the first few minutes of agonist stimulation, ß2-AR uncouples from its G protein, Gs. This uncoupling is caused by phosphorylation of ß2-AR by either protein kinase A or ß-adrenergic receptor kinase. The second mechanism, also activated in the first few minutes of receptor stimulation, is the internalization or sequestration of ß2-AR. Sequestration of ß2-AR is defined as the translocation of receptors to a membrane compartment that is inaccessible to hydrophilic ligands (14). The receptor is then dephosphorylated and can recycle back to the cell surface (15, 16). It is hypothesized that the sequestration and recycling of the receptor are needed to permit resensitization of ß2-AR. The third mechanism occurs after prolonged exposure to high concentrations of agonist. In this case, a decrease in cellular levels of ß2-AR is observed. This causes a profound desensitization of ß2-AR-mediated responses that is termed down-regulation. At the molecular level, down-regulation of ß2-AR is caused by phosphorylation of the receptor and regulation of its messenger RNA (mRNA) level (17). Each one of the three mechanisms of ß2-AR desensitization results in a reduced production of the second messenger cAMP. The extent of desensitization is, therefore, determined by the concentration of agonist used and the duration of stimulation.
The mechanisms involved in the desensitization of AT1 receptor are not as well characterized. We previously observed that a 20-min pretreatment with 10 nM Ang II resulted in a loss of Ang II-binding activity on bovine adrenal glomerulosa cells (18). This loss of binding activity corresponded to a decreased affinity of the AT1 receptor without any loss of binding sites. The lower affinity was due to the uncoupling of AT1 receptor from its cognate G protein. Recently, we also demonstrated that a short term stimulation with 10 nM Ang II activates a dynamic process in which AT1 receptor is rapidly internalized and recycled back to the cell surface (19). AT1 receptor down-regulation after long term stimulation with high concentrations of Ang II has been observed in bovine adrenal cells, rat hepatocytes, and vascular smooth muscle cells (20, 21, 22). However, the functional consequences of these different treatments have not been directly investigated for the AT1 receptor.
The present study was undertaken to evaluate the functional significance of AT1 receptor desensitization in bovine adrenal glomerulosa cells. We provide evidence that the cellular responses elicited by AT1 receptors are resistant to short term desensitization and that long term pretreatments with high concentrations of Ang II are needed to desensitize AT1-mediated cellular responses.
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Materials and Methods |
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Cell culture
Bovine adrenal glands were obtained at a nearby slaughterhouse.
Bovine adrenal glomerulosa cells were prepared as described by Boulay
et al. (24). Briefly, the outer 0.5-mm portion of the bovine
adrenal gland was excised with a Thomas tissue slicer (ESBE Scientific,
Markham, Ontario, Canada), then minced into 1-mm2 fragments
and digested in medium 199 containing 2 mg/ml collagenase, 0.2 mg/ml
deoxyribonuclease I, 50 U/ml penicillin, and 60 mg/ml streptomycin,
followed by mechanical dispersion with a 11-ml serological pipette.
This procedure was repeated five times. A 20% (vol/vol) Percoll
gradient was prepared by centrifugation at 35,000 x g
for 30 min at 4 C. Glomerulosa cells were then placed on the gradient
and purified by centrifugation at 500 x g for 15 min.
The cells were resuspended in DMEM and washed by centrifugation. For
culture, glomerulosa cells were suspended in DMEM supplemented with
10% (vol/vol) FBS, 1% (vol/vol) ITS-X (Life Technologies medium
supplement containing 1 g/liter insulin, 0.55 g/liter transferrin, 0.67
mg/liter sodium selenite, and 0.2 g/liter ethanolamine), 50 U/ml
penicillin, 60 mg/ml streptomycin, and 2 mM
L-glutamine and were seeded at a density of 200,000
cells/ml and cultured at 37 C in a CO2 incubator (5%
CO2-95% air). The medium was changed 24 h after
seeding and every other day until cells reached apparent confluence
(6–7 days).
Pretreatment with Ang II
At confluence, glomerulosa cells were washed twice with PBS (140
mM NaCl, 3.5 mM KCl, 0.9 mM
CaCl2, 0.9 mM MgCl2, 5.5
mM dextrose, and 20 mM sodium phosphate buffer,
pH 7.4) and incubated in medium 199 containing 25 mM HEPES,
0.1% BSA, and 0.01% bacitracin (pH 7.4). Incubations were performed
for the desired time at 37 C with various concentrations of Ang II.
During long term stimulations, fresh Ang II was added every 4 h.
The cells were then washed twice and incubated for 20 min in an
ice-cold acid solution (90 mM NaCl and 50 mM
sodium citrate, pH 4.5) to dissociate and remove residual Ang II (18).
Cells were then washed twice with ice-cold PBS and used for subsequent
experiments.
Binding assays
Glomerulosa cells in multiwell plates (48 wells;
300,000–350,000 cells/well) were incubated in medium 199 with
[125I]Ang II (0.1 nM) at 12 C for 2 h.
Incubations were terminated by immersion of culture plates in two
successive baths containing ice-cold PBS. The cells were then
solubilized with 0.1 N NaOH, and the radioactive content
was quantified by 
-counting. Nonspecific binding was determined in
the presence of 1 µM Ang II.
RNA isolation and Northern blot
Total RNA was isolated from glomerulosa cells with the use of
the single step Tri Reagent method according to the manufacturer’s
instructions. RNA samples were quantified by spectrophotometry, and 10
µg total RNA samples were separated by electrophoresis on 1%
denaturing formaldehyde agarose minigels (25). RNA was then transferred
to a Hybond-N hybridization membrane and immobilized by UV
cross-linking. Blots were prehybridized for at least 4 h at 42 C
in the following solution: 5% SDS (wt/vol), 400 mM
NaH2PO4, 1 mM EDTA, 50% formamide
(vol/vol), and 0.1% BSA (wt/vol). The medium was replaced with fresh
hybridization medium, and denatured 32P-labeled probe was
added. Hybridization was performed overnight at 42 C. After
hybridization, the blots were washed twice in 1 x SSC (150
mM NaCl and 15 mM sodium citrate pH 7), 0.1%
SDS (wt/vol), and 1 mM EDTA for 15 min at 52 C, followed by
two additional washes with 0.2 x SSC, 0.1% SDS (wt/vol), and 1
mM EDTA. The membranes were then autoradiographed for
5 h with Kodak BioMax film (Eastman Kodak, Rochester, NY) at -80
C. Results were quantified with the use of a GS-250 Molecular Imager
(Bio-Rad).
The complementary DNA (cDNA) used to prepare the AT1 receptor probe corresponded to entire codant region of the human AT1 receptor. The probes were prepared with a random priming oligolabeling kit using [32P]deoxy-CTP. Unincorporated label was removed by gel filtration on a Sephadex G-50, DNA grade column.
Inositol phosphate production
Labeling of adrenal glomerulosa cells was performed in multiwell
plates (24 wells; 450,000–500,000 cells/well) according to the method
of Balla et al. (26) with some modifications. Briefly, the
culture medium was replaced with inositol-free DMEM containing 5
µCi/ml myo-[3H]inositol. In desensitization
experiments, Ang II was added simultaneously with
myo-[3H]inositol for different periods of time. After a
24-h labeling period, cells were washed twice with ice-cold PBS and
subjected to a 20-min acid wash, followed by two subsequent washes with
ice-cold PBS. Cells were then incubated in medium 199 containing 20
mM LiCl, 25 mM HEPES, 0.1% BSA, and 0.01%
bacitracin with 1 µM Ang II for 10 min at 37 C.
Incubations were stopped with perchloric acid (5%, vol/vol). Cells
were scraped and centrifuged at 15,000 x g for 5 min.
Pellets were kept to evaluate total cellular membrane labeling.
Inositol phosphates were extracted from the supernatant with an equal
volume of a mixture of 1,1,2-trichlorotrifluoroethane and
tri-n-octylamine (1:1). The samples were vigorously mixed
and centrifuged at 15,000 x g for 1 min. The upper
phase containing the inositol phosphates was applied to a AG 1-X8 resin
column. Inositol phosphates were sequentially eluted by the addition of
ammonium formate-formic acid mixtures of increasing ionic strength, as
described by Berridge et al. (27).
cAMP determination
Intracellular cAMP levels were determined by measuring the
conversion of [3H]ATP to [3H]cAMP,
according to the method of Weiss et al. (28). Briefly, cells
cultured in 35-mm2 culture dishes (1.5 x
106 cells/dish) were incubated at 37 C in medium 199
containing 2 µCi/ml [3H]adenine. In desensitization
experiments, Ang II was added before or simultaneously with
[3H]adenine for different periods of time. After a 3-h
labeling period, cells were washed twice with ice-cold HBS (130
mM NaCl, 3.5 mM KCl, 1.8 mM
CaCl2, 1 mM MgCl2, 5.0
mM HEPES, 0.5 mM EGTA, and 5.5 mM
dextrose, pH 7.4) and subjected to a 20-min acid wash, followed by two
subsequent washes with ice-cold HBS. Cells were equilibrated in HBS
containing 0.1% BSA (wt/vol), 0.01% bacitracin (wt/vol), and 1
mM isobutylmethylxanthine for 15 min at 37 C. Cells were
then preincubated with Ang II (1 µM) for 15 min at 37 C,
followed by a final stimulation with ACTH (1 µM) for 15
min at 37 C. The reaction was stopped with ice-cold perchloric acid
(5%, vol/vol). Cells were then removed with a rubber scraper, and 100
µl of a solution containing 5 mM ATP and 5 mM
cAMP were added to the mixture. Cellular membranes were pelleted at
5000 x g for 15 min, and the supernatants were
sequentially chromatographed on AG 50W-X8 resin and alumina columns,
which allowed separation of [3H]ATP from
[3H]cAMP. Formation of cAMP was expressed as the percent
conversion of ATP to cAMP =
([3H]cAMP/[3H]cAMP + [3H]ATP)
x 100.
Aldosterone production
After desensitization, cells in multiwell plates (24 wells) were
incubated in PBS medium containing 0.1% (wt/vol) BSA and 0.01%
bacitracin for 30 min at 37 C. The medium was then replaced with fresh
medium containing 1 µM Ang II. After 2 h at 37 C,
the aldosterone released into the medium was measured by RIA (29).
Data analysis
Experimental data resulting from representative experiments with
different cell preparations are expressed as the mean ±
SD of triplicate values. When the error bar is not seen,
the symbol is larger than the error. Binding data were analyzed by both
the Scatchard plot method and the curve-fitting program Ligand, using
weighted nonlinear least squares to find the values for each parameter
that minimized the weighted sum of the squares (30). The extra sum of
squares test (F statistic test) was used to compare the aptness of the
fit to models of one and two classes of receptors. When needed, the
experimental data were analyzed by use of Student’s t test.
P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Figure 1B
shows Scatchard analysis of the dose displacement data. When the
binding data were analyzed by the Ligand program, the only model that
was significantly relevant was the one-site fit model
(P < 0.05). For control cells, the analysis yielded a
linear plot showing a single population of receptors with an apparent
affinity of 2.7 ± 0.8 nM and a binding capacity
(Bmax) of 83 fmol/well. When a two-site model was
tested for the same data, a high affinity site with a Kd1
of 0.4 nM and a Bmax1 of 18
fmol/well, and a low affinity site with a Kd2 of 2.9
nM and a Bmax2 of 78 fmol/well were suggested.
However, the F statistic test revealed that experimental data cannot
accommodate the two-site fit model (P = 0.1140). These
data indicate that although the AT1 receptor may exist
under two affinity states, as previously reported in bovine glomerulosa
cells (18), it is sometimes difficult, depending on specific cell
preparations or culture conditions, to resolve these two affinity
states. This problem was thoroughly discussed in a recent publication
by DeBlasi (31).
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shows that pretreatment with 1
µM Ang II caused a rapid and time-dependent decrease in
cell surface receptors. The t1/2 of this loss was 20 min,
and a maximal loss of 76.4 ± 7.7% was attained after 14 h.
These results suggest a down-regulation of AT1 receptor. As
in many cases receptor down-regulation has been attributed to a loss of
cell surface receptors and a decreased expression of receptor mRNA, we
evaluated the effect of pretreatment of glomerulosa cells with 1
µM Ang II on AT1 receptor mRNA levels. The
representative autoradiogram shown in Fig. 3 indicates that exposure of glomerulosa
cells to 1 µM Ang II for 12–24 h markedly reduced
(58.0 ± 8.6%) their content of AT1 receptor mRNA.
Interestingly, the level of glyceraldehyde phosphate dehydrogenase
(GAPDH) message increased by 200 ± 29% during the same
treatment. Increased GAPDH message was also observed after prolonged
treatment of vascular smooth muscle cells with high concentrations of
Ang II (22, 32). Gel loading in our experiments was uniform, as the
amount of total ribosomal RNA evaluated by methylene blue coloration
was similar in each lane (data not shown). These results suggest that
long term stimulations with high concentrations of Ang II (1
µM) cause down-regulation of AT1
receptor.
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shows that
short pretreatment periods (up to 30 min) did not reduce Ang II-induced
InsP3 production by glomerulosa cells. A slight elevation
of InsP3 production was observed after the first few
minutes of pretreatment. This elevation was possibly due to increased
incorporation of myo-[3H]inositol in cells, as shown in
the inset of Fig. 4. However, longer pretreatment periods
(between 30 min and 24 h) caused a gradual reduction of Ang
II-induced InsP3 production, with a t1/2 of
approximately 9 h and a maximal desensitizing effect of 83.1
± 4.9% after 24 h. Dose-response experiments revealed that when
cells were pretreated for 24 h with low concentrations of Ang II,
a slight increase in Ang II-induced InsP3 production was
observed (Fig. 5). This increase was also
possibly due to the increased incorporation of
myo-[3H]inositol (Fig. 5, inset). Pretreatment
with Ang II concentrations ranging from 0.1 nM to 1
µM, caused a gradual decrease in Ang II-induced
InsP3 production. The half-maximal effect was obtained with
a concentration of 90 ± 5.4 nM Ang II. An important
reduction of Ang II-induced InsP3 production capacity
(83.7 ± 5.6%) was observed after a 24-h pretreatment with 1
µM Ang II. These results show that desensitization of Ang
II-induced phospholipase C activation necessitates long term
pretreatment with high concentrations of Ang II.
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shows that pretreatment of
adrenal glomerulosa cells for 24 h with 1 µM Ang II
did not reduce but, rather, increased NaF- and BK-induced
InsP3 production, suggesting a homologous desensitization
of AT1-mediated inositol phosphate production. As
previously discussed, the increased InsP3 production by Ang
II-desensitized cells is probably related to increased incorporation of
myo-[3H]inositol resulting from an increased turnover of
phosphoinositides during the pretreatment with Ang II.
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demonstrate that although Ang II alone
did not significantly increase the production of cAMP, it very
significantly potentiated the effect of ACTH (443 ± 35%). After
cells were subjected to a 24-h desensitizing pretreatment with 1
µM Ang II, the potentiating effect of Ang II on
ACTH-induced cAMP production was completely abolished. This
desensitizing pretreatment with Ang II did not affect the response to
ACTH alone. Interestingly, a 24-h desensitizing pretreatment with 10
nM Ang II did not noticeably reduce the potentiating effect
of Ang II on ACTH-induced cAMP production. We also verified that a
short term desensitizing pretreatment (up to 4 h) with 1
µM Ang II did not significantly reduce the potentiating
effect of Ang II on ACTH-induced cAMP production (data not shown).
These results demonstrate again that AT1 receptor-mediated
cellular responses of bovine glomerulosa cells are very resistant to
desensitization by Ang II.
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shows that a 2-h stimulation of adrenal
glomerulosa cells with 1 µM Ang II increased aldosterone
production by approximately 6.5-fold. After cells were subjected to a
24-h desensitizing pretreatment with 1 µM Ang II, their
steroidogenic response to Ang II was significantly reduced by 47.3
± 5.2%. The basal level of aldosterone production was not
significantly altered by the desensitizing pretreatment (data not
shown). When cells were desensitized for 24 h with 10
nM Ang II, no significant reduction in Ang II-induced
aldosterone production was observed. These results indicate that long
term pretreatment with high concentrations of Ang II is needed to
desensitize bovine adrenal glomerulosa cells to the steroidogenic
effect of Ang II.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we demonstrated that a pretreatment of bovine adrenal glomerulosa cells with a high concentration of Ang II (1 µM) caused a rapid loss of cell surface receptors. This phenomenon is different from the uncoupling and internalization-recycling pathways previously observed in our laboratory when adrenal glomerulosa cells were stimulated with a lower concentration of Ang II (10 nM) (18, 19). Although our experiments were performed under conditions where recycling is possible, in this study we demonstrated that recycling is not likely to occur or at least it does not play a significant role at these higher concentrations of Ang II, as we invariably observed an important time-dependent loss of cell surface receptors. We also demonstrated that this pretreatment with high concentrations of Ang II caused a reduction of total AT1 receptor mRNA. These are characteristic features of receptor down-regulation, as demonstrated for the ß2-adrenergic receptor (17). Other studies have previously reported that Ang II down-regulates its own binding sites in cultured hepatocytes, vascular smooth muscle cells, and adrenal fasciculata cells (20, 21, 22). In vascular smooth muscle cells, it was also previously shown that Ang II down-regulates AT1 receptor mRNA (22, 32). In adrenal fasciculata-reticularis cells, Ang II-induced AT1 receptor mRNA down-regulation was also observed, at a rate similar to that seen in adrenal glomerulosa cells (34, 35).
The functional significance of a desensitizing pretreatment with Ang II was investigated on the production of second messengers. We demonstrated that long term pretreatments with high concentrations of Ang II were necessary to desensitize inositol phosphate production in bovine adrenal glomerulosa cells. Interestingly, even if an important decrease of about 50% of the total AT1 receptors was observed after a 30-min stimulation, Ang II-induced InsP3 production was not significantly disrupted. It was previously shown that in cellular models in which AT1 receptor had been transfected, second messenger production could be rapidly desensitized in the first few minutes of stimulation (36, 37). These discrepant results could be due to the large amount of spare AT1 receptors expressed by adrenal glomerulosa cells. They could also be due to differential cell expression of elements involved in the mechanism of desensitization. These results show that although the transfected cell system represents a useful tool to study cellular mechanisms, it may not reliably reflect the actual mechanisms present in cells endogenously expressing AT1 receptor.
It has been shown that a short term pretreatment with Ang II potentiates ACTH-induced cAMP production in bovine fasciculata and glomerulosa cells (33, 38). Protein kinase C and the Ca2+/calmodulin-activated protein phosphatase calcineurin were suggested to be involved in this phenomenon. In the present study, we evaluated the susceptibility of this pathway to desensitization after pretreatment with Ang II. We showed that long term pretreatments with 1 µM Ang II were necessary to desensitize the potentiating effect of Ang II on ACTH-induced cAMP production. As was the case for the production of inositol phosphates, these results indicated that the AT1 receptor-activated cellular signaling is relatively resistant to agonist-induced desensitization.
The ultimate cellular response evoked by Ang II in adrenal glomerulosa cells is the production and secretion of aldosterone. We showed that aldosterone production and secretion are relatively resistant to desensitization, as pretreatments with high concentrations of Ang II (0.1–1.0 µM) were necessary to reduce Ang II-induced aldosterone production. Penhoat et al. (20) also demonstrated that a long term pretreatment with high concentrations of Ang II was necessary to importantly reduce steroidogenesis in bovine adrenocortical cells (20). Considering how essential aldosterone is in the maintenance of electrolyte balance, it is not surprising and somehow reassuring to realize that the mechanism used by the most important stimulator of its production (Ang II) is relatively resistant to agonist-induced desensitization.
Desensitization of G protein-coupled receptors has been shown to be homologous (affecting only the responses mediated by the stimulated receptor) or heterologous (affecting the responses mediated by other receptors) (39). In this study we demonstrated that long term pretreatment with Ang II caused a homologous desensitization, as BK-induced cellular signaling was not affected. It is important to mention that the amount of B2 receptors expressed by adrenal glomerulosa cells is very low compared with that of AT1 receptors, and furthermore, B2 receptors are very rapidly desensitized after stimulation (see Footnote 1). It is interesting to note that a low abundance receptor that uses the same transduction mechanism as the AT1 receptor is not affected by the severe long term pretreatment with high concentrations of Ang II. We also showed that long term pretreatments with high concentrations of Ang II did not affect ACTH-induced cAMP production, ruling out another possible mechanism of heterologous desensitization. These results demonstrate that under conditions where an activating mechanism for aldosterone production is severely desensitized, adrenal glomerulosa cells are still perfectly responsive to other steroidogenic stimuli.
Taken together, the results of the study and of previous studies from our laboratory (18, 19) indicate that he regulation of the AT1 receptor proceeds through at least three different mechanisms of desensitization. The three mechanisms are similar to those reported for ß2-AR (13), which include uncoupling of the receptor from its G protein, internalization-recycling of the receptor (under mild short term stimulation), and down-regulation of the receptor (under intense long term stimulation). In bovine adrenal glomerulosa cells, however, the only mechanism that substantially reduces the production of intracellular messengers and the ultimate production of aldosterone is receptor down-regulation obtained with high concentrations of Ang II. Experiments are currently underway in our laboratory to better characterize the molecular mechanisms involved in AT1 receptor desensitization and their influence on cellular responses.
In conclusion, we have identified conditions under which the AT1 receptor can undergo down-regulation in bovine adrenal glomerulosa cells. This pathway causes an important decrease in the production of intracellular messengers. This study provides evidence that glomerulosa cells are functionally resistant to short term desensitization of the AT1 receptor and that long term down-regulation with high concentrations of Ang II is needed to desensitize AT1-mediated cellular responses.
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Acknowledgments |
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Footnotes |
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2 Recipient of a studentship from the Heart and Stroke Foundation of
Canada.
3 Scholar from the Fonds de la Recherche en Santé du
Québec.
4 Recipient of a Medical Research Council of Canada Scientist
Award.
5 Chrétien, L., D. Richard, S. N.
Poirier, and G. Guillemette, manuscript submitted for
publication.
Received March 13, 1997.
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