Instituto de Biología y Medicina Experimental-Consejo Nacional de Investigaciones Cientificas y Técnicas, 1428 Buenos Aires, Argentina
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ABSTRACT |
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Activation of pituitary angiotensin (ANG
II) type 1 receptors (AT1) mobilizes intracellular
Ca2+, resulting in increased prolactin secretion. We first
assessed desensitization of AT1 receptors by testing ANG
II-induced intracellular Ca2+ concentration
([Ca2+]i) response in rat anterior pituitary
cells. A period as short as 1 min with 107 M ANG II was
effective in producing desensitization (remaining response was
66.8 ± 2.1% of nondesensitized cells). Desensitization was a
concentration-related event (EC50: 1.1 nM). Although
partial recovery was obtained 15 min after removal of ANG II, full
response could not be achieved even after 4 h (77.6 ± 2.4%). Experiments with 5 × 10
7 M ionomycin
indicated that intracellular Ca2+ stores of desensitized
cells had already recovered when desensitization was still significant.
The thyrotropin-releasing hormone (TRH)-induced intracellular
Ca2+ peak was attenuated in the ANG II-pretreated group.
ANG II pretreatment also desensitized ANG II- and TRH-induced inositol
phosphate generation (72.8 ± 3.5 and 69.6 ± 6.1%,
respectively, for inositol triphosphate) and prolactin secretion
(53.4 ± 2.3 and 65.1 ± 7.2%), effects independent of PKC
activation. We conclude that, in pituitary cells, inositol triphosphate
formation, [Ca2+]i mobilization, and
prolactin release in response to ANG II undergo rapid, long-lasting,
homologous and heterologous desensitization.
angiotensin type 1 receptor; thyrotropin-releasing hormone; calcium; homologous desensitization; protein kinase C
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INTRODUCTION |
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ANGIOTENSIN II (ANG
II) type 1 (AT1) receptors belong to the family of plasma
membrane receptors that couple to guanine nucleotide regulatory
proteins [guanine nucleotide protein-coupled receptors (GPCR)]. Two
subtypes have been described: AT1A and AT1B. In
the rat pituitary, activation of AT1B receptors coupled to
Gq/11 protein increases phospholipase C- (PLC-
)
activity, resulting in increased inositol 1,4,5-triphosphate
[Ins(1,4,5)P3] and diacylglycerol formation. A biphasic
increase in intracellular calcium concentration ([Ca2+]i) triggered by
Ins(1,4,5)P3 ensues. In a previous study (16) we showed that the initial transient [Ca2+]i
spike, which is primarily due to Ins(1,4,5)P3-mediated
release of intracellular Ca2+, is followed by a sustained
[Ca2+]i elevation that results from
increased Ca2+ influx through voltage-sensitive
calcium channels and capacitative Ca2+ entry. The initial
[Ca2+]i spike is terminated as
Ins(1,4,5)P3 concentration declines, intracellular
Ca2+ stores become exhausted, and cytoplasmic
Ca2+ is sequestered by intracellular organelles
or pumped out from the cells. DAG, together with the
increased [Ca2+]i, stimulates protein
kinase C (PKC) activity. Activation of this pathway by ANG II in
lactotrophs results in prolactin secretion (10).
It is well recognized that, in the presence of agonists, many transmembrane GPCRs not only display rapid activation of signal transduction but also rapid desensitization of response (31). Receptor desensitization is potentially a physiologically important process, as it provides a means of regulating continuous receptor stimulation. Rapid receptor desensitization does not necessarily involve receptor loss but rather receptor phosphorylation (36). This acute process (seconds to minutes) may be supplemented by internalization (minutes to hours) and by downregulation (hours to days) of membrane receptors.
Both AT1 receptor subtypes (AT1A and
AT1B) undergo rapid homologous desensitization in response
to ANG II. This has been reported in the adrenal gland
(28), vascular smooth muscle cells (2), rat
cardiomyocytes (1), renal afferent arterioles
(21), brain (14), and Chinese hamster ovary
(CHO) cells (34). In particular, desensitization of ANG II
receptors in the pituitary has not been documented. Different degrees
and temporal patterns of desensitization have been reported for
PLC--coupled receptors, and it is believed that desensitization may
be a cell-specific phenomenon, regulated by differential expression of
elements involved in the process. Moreover, it is well established that
signaling and desensitization of GPCRs can be influenced by the cell
type in which they are expressed, as in the case of the mouse
thyrotropin-releasing hormone (TRH) receptor (12). This
raises the possibility that ANG II desensitization may be a function of
the cell type rather than of AT1 receptor structure.
We therefore assessed whether rapid desensitization of the AT1 receptor occurs in anterior pituitary cells by use of repetitive stimulation of intracellular Ca2+ mobilization, inositol triphosphate generation, and prolactin secretion as end points. We also determined whether heterologous desensitization occurred by use of TRH as a second alternative stimulus and the participation of PKC in the desensitization process.
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MATERIALS AND METHODS |
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Animals.
Female 60-day-old Sprague-Dawley rats were housed in an air-conditioned
room with lights on at 0700 and off at 1900. They had free
access to laboratory chow and tap water. Female rats were checked by
vaginal cytology for regular estrous cycles during 10 days previous
to the experiment. Rats in diestrus were used.
Cell dispersion. Unless specified, all drugs were purchased from Sigma (St. Louis, MO).
Rats were killed by decapitation at 9:00 AM, and pituitaries were removed on ice, separated from the neurointermediate lobe, and placed in chambers containing freshly prepared Krebs-Ringer bicarbonate buffer (KRBGA) without Ca2+ or Mg2+. Buffer contained 14 mM glucose, 1% bovine seroalbumin, MEM amino acids 2%, MEM vitamins 1% (GIBCO, Buenos Aires, Argentina), 2 mM glutamine, and Phenol Red 0.025%. It was previously gassed for 15 min with 95% O2-5% CO2 and adjusted to pH 7.35-7.40. Buffer was sterilized by filtration through a 0.45-µm pore diameter membrane (Nalgene). Pituitaries were washed three times with KRBGA and then cut into 1-mm pieces. Fragments obtained were washed and incubated in the same buffer containing 0.5% trypsin for 30 min at 37°C, with 95% O2-5% CO2. They were treated for two additional minutes with 50 µl DNase I (1 mg/ml, Worthington, Lakewood, NJ). Digestion was ended by addition of 1 mg/ml LBI. Fragments were dispersed in individual cells by gentle trituration through siliconized Pasteur pipettes. Resulting suspension was filtered through a nylon gauze (160 µm) and centrifuged for 10 min at 120 g. Before centrifugation, an aliquot of cellular suspension was taken to quantify hypophysial cell yield by use of a Neubauer chamber. Viability of cells, determined by trypan blue exclusion, was always >90%. In a series of experiments, cells were freshly used for intracellular Ca2+ measurements; alternatively, they were cultured for 4 days (as will be described).Intracellular Ca2+ measurements. Measurements were made as previously described (11). Briefly, the tetraacetoxymethyl ester (AM) of fura 2 was used as a fluorescent indicator. The pellet of anterior pituitary cells of each experimental group was redispersed and incubated in a buffered saline solution (BSS: 140 mM NaCl, 3.9 mM KCl, 0.7 mM KH2PO4, 0.5 mM Na2HPO4 · 12H2O, 1 mM CaCl2, 0.5 mM MgCl2, and 20 mM HEPES, pH 7.4) in the presence of 1.5 µM fura 2-AM, 10 mM glucose, and 0.1% BSA. Cells were incubated for 30 min at 37°C in an atmosphere of 95% O2-5% CO2, a time during which fura 2 is trapped intracellularly by esterase cleavage. Cells were then washed twice in BSS without fura 2-AM and brought to a density of 1.7-2 × 106 cells/ml BSS. Fluorescence was measured in a spectrofluorometer (Jasco, Tokyo, Japan) provided with the accessory CA-261 to measure Ca2+ with continuous stirring, a thermostat adjusted to 37°C, and an injection chamber. Intracellular Ca2+ levels were registered every second by exposure to alternating 340- and 380-nm light beams, and the intensity of light emission at 505 nm was measured. In this way light intensities and their ratio (F340/F380) were followed. Drugs were injected (5 µl) into the chamber as a 100-fold concentrated solution without interruption of recording. The preparation was calibrated, and maximal fluorescence (Fmax) was determined by addition of 0.1% Triton X-100 and minimal fluorescence (Fmin) in the presence of 6 mM EGTA (pH adjusted to >8.3). [Ca2+]i was calculated according to Grynkiewicz (17). Unless specified, basal values were considered those measured during 20 s before the addition of ANG II. Values were corrected for dye leakage, as described in Refs. 15 and 17, and for autofluorescence with unlabeled cells. Both dye leakage and autofluorescence were minimal.
To assess the concentration of ANG II at which homologous desensitization occurs, experiments were conducted by applying an initial stimulus of ANG II in concentrations ranging from 1 × 10Effect of ionomycin and TRH on
[Ca2+]i in ANG
II-desensitized cells.
Desensitization of the ANG II response was elicited by exposing
dispersed cells to 1 × 107 M ANG II for 10 min.
Cells were then washed repeatedly to remove ANG II and maintained in
ANG II-free medium for 2 h before application of 5 × 10
7 M ionomycin or 1 × 10
7 M TRH and
testing of the Ca2+i response. Control cells
were pretreated with buffer and similarly treated thereafter.
Cell culture. Cell pellets were resuspended in DMEM, supplemented with 10% horse serum, 2.5% fetal calf serum, 1% MEM nonessential amino acids, 25,000 U/l of nystatin, and 25 ng/l gentamicin. Cells were plated in sterile tissue culture plates (Cluster 24, Corning; 500,000 cells per well) and incubated with 1 ml DMEM (GIBCO) in a metabolic incubator at 37°C with 5% CO2-95% O2.
Desensitization of ANG II-induced cellular inositol phosphate
accumulation and prolactin secretion.
After 4 days in culture, cells were washed twice with DMEM and F-12
Nutrient Mixture (GIBCO), supplemented with 1% BSA, 2 mM glutamine,
and the same concentration of antibiotics, to remove all traces of
serum. Fresh medium containing 4 µCi/ml
myo-[2-3H(N)]inositol (specific activity: 20 Ci/mmol; New England Nuclear, Boston, MA) was added, and cells were
incubated at 37°C for 24 h. At the end of the labeling period,
cells were washed twice with DMEM-F-12 supplemented with 1% BSA, 2 mM
glutamine, 25,000 U/l nystatin, and 25 ng/l gentamicin. Cells were
pretreated with buffer or first stimulated with 1 × 107 ANG II for 10 min, and stimulus was removed by
washing. Thereafter, cells were further incubated for 105 min in
DMEM-F12. Medium was replaced, and cells were incubated with 20 mM LiCl
for 15 min to allow accumulation of inositol phosphate species. A
second stimulus of 1 × 10
9 or 1 × 10
7 M ANG II, 1 × 10
7 M TRH, or buffer
(control) was then applied to the cells in duplicate. For analysis of
prolactin secretion, samples were taken 30 min after drug
administration. Samples were stored at
20°C until analyzed by RIA
after appropriate dilution in 0.01 M PBS with 1% egg albumin. Cells
were placed on ice, treated with 0.5 M cold perchloric acid, and
scraped for inositol phosphate determination. Experiments were repeated
four times. Times and concentrations were chosen according to our
previous experience (4).
Inositol phosphate accumulation. Inositol phosphates were measured as previously described (3), with minor modifications. Well contents were transferred to tubes and centrifuged for 10 min at 3,000 rpm (4°C). Pellets were kept for DNA measurement. The supernatants were neutralized (0.72 M KOH-0.6 M HKCO3) and chromatographed on Dowex columns (AG-1-X8, 200-400 mesh, formate form, Bio-Rad, Buenos Aires, Argentina) to elute inositol monophosphate (InsP), diphosphate (InsP2), and triphosphate (InsP3). Phosphate esters were eluted by the stepwise addition of solutions containing increasing levels of formate. Specifically, they were sequentially eluted with 10 mM inositol (for free [3H]inositol), 0.1 M formic acid and 0.2 M ammonium formate (for InsP), 0.1 M formic acid and 0.4 M ammonium formate (for InsP2), and 0.1 M formic acid and 1.0 M ammonium formate (for InsP3). Two-milliliter aliquots of each wash fraction were mixed with 6 ml Optiphase "Hisafe" 3 (Wallac Oy, Turku, Finland) and counted in a liquid scintillation counter.
PKC downregulation. PKC was downregulated in vitro by a 24-h pretreatment of cultured cells with 1 µM phorbol 12-myristate 13-acetate (PMA). Homologous and heterologous desensitization of prolactin response by ANG II pretreatment was tested as we have described. Downregulation of PKC was confirmed by the inability of a 30-min stimulation with 1 µM PMA (PMA30) to release prolactin in PMA-pretreated cells (PMA24), (PMA30: 274.5 ± 52.0; PMA30+PMA24: 9.4 ± 12.6 prolactin increase in ng/ml, P = 0.023).
RIA. Prolactin was measured by RIA using kits provided by the National Institute of Diabetes and Digestive and Kidney Diseases. Results are expressed in terms of prolactin PRL RP3. Intra- and interassay coefficients of variation were 7.2 and 12.8%, respectively.
Statistical analyses. Results are expressed as means ± SE. For [Ca2+]i measurements, peak values were analyzed by one-way ANOVA for repeated measures. Individual means were then compared by Duncan's test (desensitized vs. control). Prolactin secretion and inositol phosphate generation in vitro were analyzed by two-way ANOVA for repeated measures for the effects of drug treatment and group (control or desensitized). If an F of interaction was found significant, individual means were compared by Scheffé's test; if it was not significant, groups of means were analyzed by the same test. Basal prolactin and inositol phosphates were analyzed by Student's t-test. P < 0.05 was considered significant.
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RESULTS |
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Desensitization of ANG II-mediated increase in
[Ca2+]i .
The amplitude of the [Ca2+]i spike depended
on ANG II concentration, reaching an apparent maximum at 10 nM. At
higher concentrations, the [Ca2+]i response
occurred earlier and more synchronically, with a steeper upstroke (Fig.
1). The EC50 for peak
amplitude was 2.3 nM.
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Incubation time required for desensitization.
To determine the briefest ANG II exposure that would cause
desensitization, 1 × 107 M ANG II was applied for
varying periods of time (1, 5, 10, or 20 min) to pituitary cells in
culture. Cells were washed repeatedly and subjected to a second 1 × 10
7 M ANG II pulse after 120 min. ANG II-induced
[Ca2+]i increase was already desensitized
after 1 min of pretreatment (Fig. 3). The
response obtained was 66.8 ± 2.1% of maximal response (P = 0.044), and this percentage did not change
significantly with increasing incubation periods. For subsequent
experiments, a 10-min pretreatment was chosen.
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Recovery of Ca2+ response.
In light of the susceptibility of cells to desensitization, we further
assessed the ability of desensitized cells to recover. Cells were
subjected to a 10-min pretreatment with 1 × 107 M
ANG II and then washed with medium devoid of ANG II and
incubated for varying periods before the second pulse of 1 × 10
7 M ANG II. The Ca2+ response
recovered partially after 15 min but remained at only 41.4 ± 2.6% of the control response (P = 0.00016 vs.
control). The response to ANG II was still different from the
control value 4 h after the initial ANG II stimulus
(77.6 ± 2.4%, P = 0.00022; Fig.
4).
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Recovery of intracellular Ca2+ stores
after a desensitizing stimulus of ANG II.
To test the possible involvement of intracellular Ca2+ pool
depletion, we compared the effects of the Ca2+ ionophore
ionomycin (5 × 107 M) on
[Ca2+]i mobilization in control cells and in
cells desensitized with a 10-min pretreatment of 1 × 10
7 M ANG II 2 h earlier. We found that the increase
in [Ca2+]i in response to ionomycin was not
different in control and desensitized cells (Fig.
5), suggesting that intracellular
Ca2+ stores had already recovered by the time
desensitization was still significant.
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Heterologous desensitization of intracellular
Ca2+ mobilization.
To investigate whether a pretreatment with 1 × 107
M ANG II produced heterologous desensitization, 1 × 10
7 M TRH was applied 2 h after a 10-min
pretreatment with buffer or 1 × 10
7 M ANG II, and
[Ca2+]i was monitored. As observed in Fig.
6, the TRH-induced Ca2+ peak
was attenuated in the ANG II-pretreated group and reached only 60.3%
of levels attained in the control group (P = 0.001).
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ANG II-mediated homologous and heterologous desensitization of
inositol phosphate generation.
To ascertain whether homologous and heterologous desensitization of
PLC- function occurred, we determined the effect of a 10-min
pretreatment with 1 × 10
7 M ANG II on the ability
of ANG II or TRH to generate inositol phosphates 2 h later. Basal
levels of the three inositol phosphate species were increased in cells
previously treated with ANG II. Basal levels in buffer-pretreated cells
were 452.3 ± 37.3, 95.4 ± 8.7, and 32.63 ± 2.79 counts · min
1 (cpm) · µg
DNA
1 ± SE for InsP, InsP2, and
InsP3, respectively, and in ANG II-pretreated cells, levels
were 802.2 ± 57.3, 171.6 ± 12.8, and 52.65 ± 1.67 cpm/µg DNA (P = 0.038 control vs. desensitized for
the three inositol phosphate species). Such increment was probably
related to the effect of ANG II pretreatment, which was prolonged by
addition of LiCl, which prevents inositol phosphate breakdown. When we evaluated absolute increments after the second stimulus, similar patterns of response were found for the three inositol phosphate species. InsP3 production was lower in desensitized cells
(response to 1 × 10
9 and 1 × 10
7 M ANG II was 69.8 ± 3.5 and 72.8 ± 3.5%
of control cells, P = 0.045; Fig.
7, left), and both
InsP2 and InsP production was also lower in these cells
(P = 0.05 and 0.046, respectively; Fig. 7, middle and right). Interestingly, the response of
inositol phosphates to 1 × 10
7 M TRH was also
desensitized by previous treatment with ANG II (response was 69.6 ± 6.1% that of control response for InsP3, P = 0.045).
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ANG II-mediated homologous and heterologous desensitization of
prolactin secretion.
Response to the secretory activity of ANG II was also desensitized,
because a pretreatment with 1 × 107 M ANG II for 10 min significantly reduced ANG II-induced prolactin secretion 2 h
later (Fig. 8). The response was only
64.3 ± 9.9 and 53.4 ± 2.3% of that achieved in the control
group for the concentrations of 1 × 10
9 and 1 × 10
7 M ANG II, respectively (P = 0.022). Response of prolactin secretion to 1 × 10
7 M TRH was also decreased (65.1 ± 7.2% of
control response, P = 0.022) by a desensitizing
pretreatment with ANG II.
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Involvement of PKC on ANG II-mediated heterologous desensitization
of prolactin secretion and Ca2+
mobilization.
To assess the role of PKC in the agonist-induced desensitization of the
ANG II receptor, we investigated the effect of PMA pretreatment on the
ANG II-mediated desensitization of the prolactin and intracellular
Ca2+ response. PMA pretreatment (1 µM, for 24 h) did
not modify basal prolactin levels [control: 901.8 ± 73.4, PMA:
807.6 ± 63.0 ng/ml, not significant (NS)]. Prolactin increment
in response to ANG II and TRH was not significantly different in buffer
or PMA-pretreated cells. As expected, prolactin response to ANG II and
TRH was lower in ANG II-desensitized cells (Fig.
9; P = 0.043). In
PMA-pretreated cells, ANG II-induced homologous and heterologous
desensitization was still observed.
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DISCUSSION |
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The octapeptide ANG II binds and activates receptors in the plasma membrane of target cells, thereby mediating a variety of important cardiovascular, homeostatic, and neuroendocrine functions. A brain-renin-ANG II, as well as a pituitary-renin-ANG II, system has been well characterized, and there are particularly high levels of ANG II in the hypothalamus. ANG II receptors are present in both the hypothalamus and the anterior pituitary of several species. Studies from numerous laboratories have shown that ANG II affects pituitary prolactin, growth hormone, adrenocorticotropin, and luteinizing hormone acting at the hypothalamus and/or the pituitary, and receptor number varies in physiologically diverse conditions (29). We have demonstrated that ANG II activates AT1 pituitary receptors, and that inositol phosphate generation, [Ca2+]i increase, and prolactin stimulation ensue (10, 16).
In many cells or tissues, exposure of ANG II receptors to ANG II often leads to a rapid loss of receptor responsiveness, or receptor desensitization. This has been described in cells from the kidney, heart, adrenal gland, and brain (1, 2, 14, 21, 28, 34). In the present study, we describe for the first time ANG II-induced homologous and heterologous desensitization of intracellular Ca2+ mobilization, InsP3 generation, and prolactin release in dispersed anterior pituitary cells.
It has been documented that desensitization is an event associated with receptor structure as well as with the cell type in which the receptor is expressed (12). It is therefore understandable that different temporal and functional aspects of ANG II receptor desensitization have been described.
The desensitization of the ANG II-induced Ca2+
response was a concentration-related phenomenon reaching an
apparent maximum at 1 × 107 M, and desensitization
could not be overcome by using higher concentrations of ANG II (data
not shown). The EC50 that induced homologous
desensitization (1.1 nM) was in the range of the dissociation constant
Kd for the AT1 receptor in the
pituitary (8). It was also similar to that obtained
for ANG II-induced desensitization of Ins(1,4,5)P3
production in CHO cells (33).
In contrast to the present results, in CHO-K1 cells expressing the
AT1 receptor (34, 35) after an initial
stimulus with a subsaturating concentration (1 × 109 M) of ANG II, no response in
[Ca2+]i was observed to a second ANG II
challenge added 2 min later. We observed that 1 × 10
9 M ANG II reduced the subsequent response to 34.2%,
which was still a significant response. In human embryonal kidney, 293 cells stably expressing AT1A receptors, cells initially
stimulated with 1 × 10
7 M ANG II were refractory to
a second ANG II stimulus 4 min later, as in the present work
(18). Transfected cell systems represent a useful tool to
study cellular mechanisms, but they may not reliably reflect the actual
mechanism present in cells endogenously expressing AT1
receptors. Particularly when desensitization is considered, discrepant
results may be related to different amounts of expressed receptors or
differential cell expression of elements involved in the mechanism of desensitization.
We next evaluated the extent of desensitization in relation to the duration of the initial stimulus. When cells were stimulated by ANG II and then washed and tested for desensitization 2 h later, an initial stimulus as short as 1 min was effective in producing maximal desensitization. This time course resembles desensitization of the AT1A receptor in rat cardiomyocytes and in CHO cells, where rapid desensitization can occur even if receptor internalization is inhibited (1, 32). This suggests that receptor desensitization and internalization are distinct events, even though they can occur concurrently. In CHO cells expressing the AT1B receptor, there was also a rapid (few minutes) and dose-dependent homologous desensitization of receptor-mediated production of second messengers (25, 33). Nevertheless, it has been shown that in bovine adrenal glomerulosa cells, 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 (28). This may reflect the influence of cell type on the AT1 receptor sensitivity to desensitization.
When ANG II was washed, the cells recovered the ability to increase [Ca2+]i in response to a second ANG II stimulus already at 15 min, even though maximal response could not be obtained at that time. Full recovery from the desensitized state required more than 4 h. In CHO-K1 cells, a small response could be obtained 30 min after the initial stimulus, and an incomplete resensitization was observed 60 min after washing the initial stimulus. In contrast, in bovine adrenal glomerulosa cells, desensitization of the AT1B receptor was a reversible phenomenon, because most of the binding capacity was recovered after 60 min (28).
The lack of full recovery of Ca2+ response in desensitized pituitary cells could be related to a depleted intracellular Ca2+ pool. This was verified by using ionomycin 120 min after a desensitizing treatment with ANG II. We found that Ca2+ stores had fully recovered by that time, suggesting that Ca2+ pool depletion might not account for ANG II-induced desensitization. In contrast, it has been described that gonadotropin-releasing hormone-induced desensitization of calcium response in the pituitary rapidly recovers (37) in parallel with a rapid replenishment of Ca2+ stores (23).
Desensitization was accompanied by a decrease of PLC- function, as
assessed by measurement of InsP3 and by a decrease in prolactin release in response to a second stimulus of ANG II. Desensitization in all cases was not complete, because a response of 54 to 72% could be obtained depending on the parameter measured. A
similar degree of desensitization of Ins(1,4,5)P3
response to ANG II has been described in cultured smooth muscle cells
(20) and in CHO cells stably transfected with the
AT1A receptor (9). This
suggests that the desensitized Ca2+ response
could be due to impaired InsP3 production. The fact that
the prolactin response was also desensitized would indicate that
lactotropes are target cells of ANG II-induced desensitization. Nevertheless, response of corticotropes could also be involved, inasmuch as it has been described that AT1B receptors are
present in both lactotropes and corticotropes in the pituitary,
lactotropes being the predominant cell type in the female pituitary
(22).
ANG II pretreatment also desensitized TRH-induced Ca2+ mobilization, InsP3 generation, and prolactin release. This would indicate that a common element in the signal transduction pathway of ANG II and TRH was desensitized by ANG II. In this regard, both ANG II and TRH receptors interact with the pertussis toxin-insensitive Gq/G11 class of G proteins (19, 29). It has been proposed that, within any given cell, there is a limited pool of G proteins shared by a variety of endogenous receptors. It is possible that ANG II receptor stimulation and subsequent receptor uncoupling, or receptor G protein internalization, limits the G protein pool available to couple to other endogenously expressed receptors, in this case the TRH receptor.
Current evidence suggests that the initial, most rapid phase of desensitization occurs with a time course of seconds to minutes after exposure to an agonist and involves agonist-induced phosphorylation of GPCRs, uncoupling of the receptors from G proteins, and a loss of subsequent downstream events (7, 13). GPCRs can be phosphorylated by two different types of kinases: 1) second messenger-activated kinases, such as protein kinase A or PKC, which produce a negative feedback and a nonspecific mechanism of desensitization, and 2) specific kinases that form the growing family of G protein-coupled receptor kinases, which phosphorylate only the activated, or agonist-occupied, forms of GPCRs (13). Phosphorylation and regulation of GPCRs by second messenger-activated protein kinases are commonly suggested to play a role in heterologous desensitization of GPCRs.
Because both ANG II and TRH activate PKC, it is possible that this kinase might play an important role in heterologous desensitization. Because PKC does not discriminate between agonist-occupied and unoccupied receptors (13), activation of PKC is believed to be associated with both homologous and heterologous desensitization.
When PKC activation was prevented by 24 h of prior exposure to PMA, homologous and heterologous desensitization induced by ANG II still occurred, suggesting that this process is independent of PKC activation. Similar results for ANG II have been described in cardiomyocytes (38), bovine adrenal glomerulosa cells (6), and CHO cells transfected with the AT1A receptor (9). It has also been reported that PKC inhibition reduced agonist-induced phosphorylation of the AT1A receptor but did not affect its desensitization (25). Nevertheless, the PKC independence has not been a universal finding, because inhibitors of PKC prevented ANG II-mediated desensitization in vascular smooth muscle cells (26), glomerular mesangial cells (24, 27), and Xenopus oocytes (30), or they partially reverted ANG II-induced heterologous desensitization in CHO cells (33). These results confirm that different mechanisms of desensitization occur for the same agonist in different cell types.
Another possible mechanism involved in heterologous desensitization would be downregulation of the Ins(1,4,5)P3 receptor, which has been shown to participate in heterologous desensitization of responses to ANG II in a rat liver epithelial cell line (5). Nevertheless, that event was observed only after longer treatments. Results from the present study suggest that the observed reduction in InsP3 formation per se could explain the decrease in intracellular Ca2+ mobilization.
Desensitization is a complex process that plays an important role in turning off receptor-mediated signal transduction pathways. Although rapid desensitization appears to be a feature common to PLC-linked receptors, the extent and persistence of desensitization have been reported to vary from receptor to receptor. We provide evidence that ANG II induces homologous and heterologous desensitization of [Ca2+]i mobilization, InsP3 formation, and prolactin release in pituitary cells by a mechanism that does not involve PKC activation. Desensitization was rapid in onset and long lasting, although it was not complete because partial responses could be obtained. Time and concentration patterns of desensitization presented similarities and differences with respect to endogenously expressed or transfected AT1 receptors in different cell types. In light of the development of antihypertensive treatments that use selective AT1 receptor or angiotensin converting enzyme antagonists, it is important to gain an insight into the physiology of ANG II in the different organs in which it might have an impact.
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ACKNOWLEDGEMENTS |
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We thank the National Hormone and Pituitary Program of the National Institute of Diabetes and Digestive and Kidney Diseases and Dr. A. F. Parlow for the RIA kits.
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FOOTNOTES |
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This work was supported by Consejo Nacional de Investigaciones Cientificas y Técnicas, Agencia Nacional de Promoción Científica y Técnica, Centro de Investigaciones Quimicas y Farmacológias, and Fundación Antorchas, Buenos Aires, Argentina.
Address for reprint requests and other correspondence: D. Becú-Villalobos, Instituto de Biología y Medicina Experimental, CONICET, V. Obligado 2490, (1428) Buenos Aires, Argentina (E-mail: dbecu{at}dna.uba.ar).
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. Section 1734 solely to indicate this fact.
Received 26 May 2000; accepted in final form 31 October 2000.
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