Cytosolic Progesterone Research Articles

High affinity progesterone binding sites of human uterine microsomal membranes

Microsomal membranes sedimented at 40000 g were prepared from human myometrium samples. The progesterone binding properties of microsomal suspensions were determined by incubating microsomes and [ 3H]progesterone at 4°C. Dextran-coated charcoal was used for the separation of bound and free steroids. Membrane-associated progesterone binding sites of high affinity were identified in microsomes prepared from pregnant and nonpregnant uteri. The binding was saturable ( K d ~4 × 10 −9 M, concentration of binding sites 400–900 fmol/mg microsomal protein) and specific for natural progesterone. Of 21 steroids tested only 21-hydroxy-4-pregnene-3,20-dione, 17α-hydroxyprogesterone and testosterone showed moderate competition against progesterone with relative affinities between 7.0–20.0% (R.A. of progesterone 100%). 5α-Dihydroprogesterone and 5α-dihydrotestosterone showed weak cross reaction (relative affinities 2.5 and 2.0%, respectively). Corticosteroids, estrogens and the 5 synthetic progestins tested showed only weak competition with relative affinities lower than 1.0%. These microsomal progesterone binding sites of high affinity and limited capacity resemble steroid hormone receptors but they are different from the soluble cytosolic progesterone receptor of human uterus in terms of steroid specificity. The physiological function of this microsomal progesterone receptor is unknown.

Uterine steroid receptor changes associated with progesterone withdrawal during pregnancy and pseudopregnancy in rabbits.

Cytosolic and nuclear progesterone (P) and estrogen (E) receptors (PR and ER, respectively) were measured in uterine tissues of rabbits at the end of pregnancy (days 25, 27, 29, 30, and 31 and 0-10 h postpartum) and pseudopregnancy (days 13 and 18). At both times, plasma P concentrations fell dramatically after a period of prolonged elevation, effecting changes in uterine function. Plasma concentrations of P remained elevated until day 29 of pregnancy and then fell continuously until postpartum, whereas plasma estradiol levels did not change. The numbers of nuclear ER and PR were constant from days 25-29. Concomitant with the fall in plasma P on day 30, levels of nuclear ER and PR doubled and remained elevated on day 31 and postpartum. In all cases, the numbers of nuclear receptors were negatively correlated with P concentrations. The number of cystosolic ER in the myometrium increased on day 30 of pregnancy and remained elevated on day 31 and postpartum. Although levels of cytosolic ER in the endometrium appeared to rise similarly, the change was not significant. The number of cytosolic PR in endometrium and myometrium did not change significantly. Between days 13 and 18 of pseudopregnancy, plasma P declined while plasma estradiol remained constant, as during pregnancy. At this time, cytosolic PR increased 4-fold, and nuclear PR doubled. Nuclear ER also increased between days 13 and 18, but the apparent increase in cytosolic ER was not significant. These data suggest that receptors for both steroids increase as the ratio of plasma estradiol to P increases at the end of pregnancy and pseudopregnancy. Surprisingly, nuclear PR levels are highest at term, when the influence of P in suppressing myometrial activity and preventing the onset of labor is removed. Thus, in the rabbit, the level of PR does not directly reflect the physiological response to P.

Steroid hormone receptors in breast tumours presenting during pregnancy or lactation

Cytosol oestrogen receptor (ER) and progesterone receptor (PR) levels were measured in tumours from three patients with breast cancer during pregnancy and from three patients developing breast cancer while lactating. All lactating patients were ER-positive and two were PR-positive, whereas pregnant patients were uniformly ER-negative and PR-negative. Pregnant patients had a significantly shorter disease-free survival compared with matched nonpregnant women with breast cancer. Of five patients developing metastatic disease, one from the lactating group had a complete remission with chemotherapy and one had static disease with endocrine treatment, whereas all others had progressive disease despite a variety of treatments. Although numbers are too small to permit generalisation, these provisional data suggest that patients presenting with breast cancer during pregnancy may have mainly receptor-negative tumours, a short disease-free interval, and may be relatively resistant to treatment of metastatic disease. By comparison, patients with breast tumours during lactation have receptor-positive disease and metastases may respond to systemic therapy.

Oestradiol and progesterone receptors in carcinoma of the human fallopian tube

SummaryIt is not known whether or not malignantly transformed epithelium of the fallopian tube retains its receptors or if tubal carcinoma is hormone dependent. Cytosol oestradiol and progesterone receptor contents of four carcinomata originating in the tubal epithelium are described. There was one patient with an oestradiol and progesterone receptor positive tumour, in which the progesterone receptor concentration was high.

Effects of calcium on the chick oviduct progesterone receptor

The chick oviduct cytosol progesterone receptor can be transformed to a small form (R s = 21 A ̊ , S 20, w: 2.9 ) denoted “mero-receptor” by incubation in the presence of Ca 2+ [8]. In the molybdate-free cytosol all the progestin binding components could be completely transformed to mero-form by 1 h treatment with 100 mM Ca 2+ at 0°C. If EDTA was secondarily added, the ligand was rapidly released. If molybdate (20 mM) containing cytosol was incubated with Ca 2+, no radioactivity was found in the meroposition on the Agarose A 0.5 m column, but the bound steroid sedimented at 2.9 S in sucrose gradients containing Ca 2+ (and no molybdate). When 20 nM molybdate was added to cytosol containing receptor activated by 0.3 M KCl, complete mero-transformation by Ca 2+ was obtained also by the gel filtration criterion, indicating that molybdate does not inhibit the mero-transforming factor. Ligand-free progesterone receptor could also be completely converted to mero-form by endogenous cytosolic transforming factor and calcium. The transforming factor was completely inactivated, when cytosol was run through Agarose A 0.5 m gel. Mero-transformation was found to be irreversible. The purified progesterone receptor subunit 110 K (B) was partially converted to smaller forms by calcium alone (100 mM, 0°C, 1 h) whereas addition of a small amount of cytosol allowed complete conversion to mero-form.

Nuclear and cytosol steroid hormone receptor levels in the myometrium during pregnancy in the sheep

Methods for the measurement of nuclear receptors for oestradiol and progesterone in sheep myometrium have been established. Scatchard analysis of nuclear receptors gave dissociation constants ( nM) on days 0 and 112 of pregnancy of 1.95 and 1.76 for oestradiol and 4.20 and 4.12 for progesterone, respectively. The concentration of nuclear and cytosol high-affinity receptors for oestradiol and progesterone has been determined during the first 112 days of gestation; and possible roles of oestradiol and progesterone in the regulation of myometrial hypertrophy and function are discussed. The rate of hypertrophy, as measured by changes in protein : DNA and RNA : DNA ratios, was maximal during days 56–84 and declined thereafter. The level of cytosol oestradiol receptor decreased rapidly between day 0 (oestrus) and day 28, and then more slowly between days 28 and 112, when expressed per unit of cytosol protein. However, when expressed per unit of DNA the level increased after day 28 to a peak at day 84, then decreased markedly to day 112. The level of nuclear oestradiol receptor declined from a peak at oestrus to very low levels on days 56–84, then increased markedly to day 112. The concentration of cytosol progesterone receptor declined from a peak at oestrus to low levels on days 28–112. The changes in the level of nuclear progesterone receptor were more complex; the level increased between oestrus and day 28, declined markedly to day 56, then increased again to high levels on days 84–112. The data suggest that oestradiol does not have any important role in stimulating myometrial growth, since the level of nuclear receptor for oestradiol was low when the rate of hypertrophy was maximal. The changes in nuclear progesterone receptor level were less clearly separated, temporally, from changes in rate of hypertrophy, and the possible influence of progesterone on myometrial growth remains unclear.

Activated oestrogen receptors in breast cancer and response to endocrine therapy

The status of oestrogen and progesterone receptors has been measured in 147 primary breast tumours. In addition to the measurement of cytoplasmic oestrogen receptors, the ability of these receptors to bind to oligo(dT)-cellulose has been assessed. This indicates the capability for activation of cytoplasmic receptors to a form able to bind in the nuclear compartment in vivo and thus be part of a functional receptor pathway. All the receptor concentrations measured were increased in the postmenopausal group of patients. All nuclear oestrogen receptors in this group were available for labelling at 4°C, in contrast to the premenopausal group. The apparent functionality of the oestrogen receptor pathway could be equally assessed either by the co-presence of cytosol progesterone receptor with nuclear oestrogen receptor (30 or 4°C) or with activated cytosol oestrogen receptor. The presence of activated cytosol oestrogen receptor was as reliable (80%) as the presence of either nuclear oestrogen receptor at 30 (83%) or 4°C (81%) in predicting the response of breast tumours to endocrine therapy.

Monoclonal antibodies to rabbit progesterone receptor: crossreaction with other mammalian progesterone receptors.

A mouse was immunized with purified rabbit uterine cytosolic progesterone receptor (specific activity: 3 nmol of steroid bound per mg of protein). After fusion of its spleen cells with Sp2-OAg myeloma cells, supernatants of 11 hybrid cultures were found to react in both an immunoenzymatic test and a double-immunoprecipitation test with the progesterone receptor. Clones were obtained from the five hybrid cells that gave the strongest response in both tests. Antibodies from cell culture supernatants and ascitic fluids were characterized. Three are of the IgG1 and two of the IgG2a isotype. Their apparent affinity for the progesterone receptor was measured by immunoprecipitation in physiological salt conditions. The equilibrium dissociation constants were between 0.1 and 4 nM. All five monoclonal antibodies crossreacted with the rabbit nuclear receptor, the human cytosolic receptor, and other mammalian (rat, guinea pig) but not avian (chicken) cytosolic progesterone receptors. There was no interaction with the glucocorticoid receptor and corticosteroid binding globulin.

Evaluation of androgen antagonism of estrogen effect by dihydrotestosterone

The antagonism of estrogen effect on the immature rat uterus by dihydrotestosterone (DHT) was evaluated in vivo. One-hundred micrograms of DHT which has been shown by previous workers [4] to saturate the androgen receptor but not bind or translocate estrogen receptor was injected daily into immature rats with or without 5 μg of 17β estradiol (E 2) for three consecutive days. The animals were sacrificed on the fourth day, the uteri were excised and the uterine wet weight, cytosol protein content, [ 3H]-leucine and [ 3H]-UTP incorporation into TCA-precipitable material, and cytosol and nuclear levels of estrogen and progesterone receptors were measured. When DHT was given in conjunction with estradiol, estrogen induced uterine growth as measured by increases in uterine weight, cytosol protein content, and [ 3H]-leucine incorporation was significantly reduced. However, cytosol and nuclear concentrations of estrogen receptors and cytosol progesterone receptor concentration were not significantly affected, whereas progesterone treatment significantly reduced estrogen and progesterone receptor concentrations in both the cytosol and the nucleus. DHT administration did not change the binding affinity of the estrogen receptor populations and the ratio between the higher ( K d = 10 −10 M) and lower ( K d = 10 −9 M) affinity components of estrogen receptor remained unchanged. DHT treatment was also shown to significantly reduce estrogen-induced [ 3H]-UTP incorporation. These findings suggest that the mechanism of DHT antagonism of estrogen effect in this organ system does not involve inhibition of synthesis of estrogen receptor as has been shown for progesterone, but appears to occur by decreasing estrogen-induced RNA transcription at a point subsequent to estrogen receptor binding.

Effects of urea and molybdate on the chick oviduct progesterone receptor

1. 1. The chick oviduct cytosol progesterone receptor, when complexed with ligand, can be exposed to urea concentrations as high as 3 M (at 0°C) without loss of steroid binding capacity. The ligand dissociation rate is increased ≥ 10 fold under these conditions. 2. 2. The “native” 8 S form of the receptor is progressively converted to a 4 S species by urea (> 2M) as seen in ultracentrifugation analysis. This conversion is inhibited by Na 2MoO 4 (5–50 mM) suggesting that molybdate stabilizes the 8 S molecule by direct interaction. 3. 3. At urea concentrations above 2 M, the ligand-free receptor looses progressively its binding capacity. The “transformed”, 4 S receptor was less stable than the 8 S species, and could not be protected by molybdate.

Evidence for two structurally related progesterone receptors in chick oviduct cytosol

The existence of two progesterone receptor forms present in crude cytosol of chick oviduct has been demonstrated by photoaffinity labelling using [ 3H]R5020. On SDS—polyacrylamide gels these two forms exhibit app. M r-values of 79 000 and 109 000 corresponding to the progesterone receptor forms A and B. Peptide maps of photoaffinity-labelled steroid receptors have been established by limited proteolysis with α-chymotrypsin. The peptide map obtained for chick oviduct cytosol progesterone receptor crosslinked with [ 3H]R5020 proved to be the sum of peptides obtained from partially purified preparations of forms A and B. The peptide maps of both progesterone receptor forms were identical for peptides below the M r-value of form A, indicating extensive homology of the two forms. A significantly different peptide pattern was observed for the rat liver glucocorticoid receptor crosslinked with [ 3H]triamcinolone acetonide. Prolonged proteolysis with chymotrypsin gave rise to peptides with M r-values of 6000 and 10 000 from the hormone-binding domain of progesterone and glucocorticoid receptors, respectively.

Temperature dependence of the dissociation rate constants for the nonactivated (molybdate-stabilized) and activated progesterone receptor-hormone complexes from the chick oviduct

Both the nonactivated and activated forms of the chick oviduct cytosol progesterone receptor-hormone complexes displayed first-order dissociation kinetics at temperatures between 0 and 25°C. The rate constant was always 2—3-times greater for the nonactivated than for the activated complex. The thermodynamic parameters calculated from the Eyring plot for the nonactivated and activated forms, respectively, were: ΔH † = 28.6 and 29.9 ± 1.5 kcal/mol; −T†S † = 7.4 ± 0.6 and 7.7 ± 1.6 kcal/mol; and ΔG † = 21.3 ± 0.5 and 22.1 ± 0.1 kcal/mol . These values suggest that activation results in an increase in enthalpy of the ligand-receptor interaction, thus stabilizing the complex. The dissociation rate constants for the native complex obtained by two different experimental approaches, namely, isotope dilution (‘chase’) and dissociation against charcoal, indicated the absence of cooperativity in the receptor-ligand binding.

Modulation of progestin binding activity in cultured human breast carcinoma cells: the effect of serum type and concentration.

Progesterone receptor levels in MCF-7 human breast cancer cells increase as a specific response to estrogen and to some nonsteroidal antiestrogens. In the present study we demonstrate that the type and quantity of serum present during culture of these cells modifies the level of progestin binding activity, but not the level of estradiol binding activity. MCF-7 cells maintained in media supplemented with 5% charcoal-dextran treated calf serum (CDCS) contain 0.3 - 0.4 pmol of cytosol progesterone receptor (PRc) per mg DNA. When cells previously maintained in 5% CDCS-media are shifted to media containing 5% charcoal-dextran treated fetal calf serum (CDFCS), the level of progestin binding increases after day 16, and stabilizes at 2 - 3 pmol/mg DNA at days 30 to 40. Shifting these cells back to 5% CDCS-media, reduces PRc to 0.2 - 0.4 pmol/mg DNA within 3 days. This reduction is dose dependent with a half-optimal decrease at 1% CDCS, and a full decrease at 2% CDCS (4d incubation). Nuclear progestin binding was uniformly low (0.2 - 0.4 pmol/mg DNA) and unaffected by type or concentration of serum, and no consistent change in cytosol or nuclear estrogen receptor levels was observed. These cytoplasmic progestin binding sites are translocated to the nucleus by progesterone, and are similar to estradiol (E2) induced sites by Scatchard binding and sucrose gradient analysis. Similar serum-dependent changes are also observed in the T47D human breast cancer cell line where growth in CDFCS-media results in 4-fold higher progestin binding levels than observed in CDCS-media. Our findings suggest the presence of non-dialyzable stimulatory factor(s) in CDFCS that influence the progestin receptor level and highlight the fact that serum components can alter dramatically the cellular progestin binding activity.

The interaction of canrenone with oestrogen and progesterone receptors in human uterine cytosol.

1 Canrenone, the major active metabolite of spironolactone, decreased [3H]-progesterone binding to isolated uterine cytosolic progesterone receptors. The inhibition was concentration-dependent. 2 Canrenone did not alter [3H]-oestradiol binding to isolated uterine cytosolic oestrogen receptors. 3 Canrenone inhibition of progesterone binding to isolated cytosolic receptors was strictly competitive: Kd (apparent dissociation constant for progesterone binding) was increased in a concentration-dependent manner by canrenone, whereas Bmax (maximal number of progesterone binding sites/mg cytosolic protein) was unaltered. There was marked cooperativity in progesterone binding at high canrenone and low progesterone concentrations. The implication is that canrenone alters the subunit interaction of the receptor protein. 4 Kd for progesterone was 3.2 X 10(-9)M. Ki (the inhibition constant for canrenone with respect to progesterone binding) was 300 X 10(-9)M. Reports in the literature suggest that, following spironolactone administration, canrenone may rise to concentrations sufficiently high to inhibit progesterone binding. This action may contribute to the effect of spironolactone in inducing menstrual disturbances in female patients.

Modulation of rat uterine progesterone receptor levels and peroxidase activity by tamoxifen citrate, LY117018, and estradiol.

The biochemical and morphological effects on the immature rat uterus of tamoxifen citrate (TAM) and LY117018 were compared with those of estradiol. A single dose of TAM (100 μg) increased uterine wet weight and peroxidase activity 12,24,36,48,60, and 72 h postinjection, while the uterine DNA content was increased after 36 h compared to controls at 72 h. A single sc dose of LY117018 (100 fig) also increased uterine wet weight and peroxidase activity, although to a lesser extent than TAM, 36–72 h postinjection. DNA content was increased to the same extent as that observed after TAM treatment. Coadministration of progesterone (1.0 mg) with 100 μg TAM or LY117018 completely blocked the antiestrogen-induced increase in uterine peroxidase activity and elongation of the luminal epithelium observed at 36 h, while the increase in uterine wet weight was only partially blocked. Cytosol progesterone receptor (PR) levels were increased significantly at 36 h by estradiol (10 μg) or TAM (100 μg), but no increase was obs...

Cytosol and nuclear estrogen and progesterone receptors in the rabbit endocervix

This report describes the measurement of estrogen and progesterone receptors in cytosols and nuclear fractions from endocervical tissue components. Unoccupied cytosol estrogen receptor levels as determined by Scatchard analysis of [ 3H]-estradiol binding data indicated a single class of high affinity binding sites for the epithelial-stromal complex ( K D = 0.74 × 10 −9 M). Binding was specific for estrogen (estradiol > estriol > estrone) and unaffected by desoxycorticosterone, dihydrotesterone and progesterone. Assays for total estrogen receptor verified that 71.6 ± 5.3% of this 8S estrogen receptor is in the epithelial-stromal complex while the remaining approximately 28% is localized in the stroma and fibromuscular wall, with the cells of the complex containing the highest receptor concentration. In 5-day pseudopregnant and ovariectomized rabbits compared to estrous rabbits there was a 50% decrease in the cytosol estrogen receptor in the epithelial-stromal complex and a 30% decrease in the concentration of nuclear receptor. Cytosol and nuclear progesterone receptors were measured as an indicator of estrogen action in the rabbit endocervix. Cytosol progesterone receptor concentrations (fmol/mg DNA) in 5-day pseudopregnant and ovariectomized animals were reduced to approximately 35% of the concentration in estrous animals. Nuclear progesterone receptor concentrations decreased 65% in 5-day pseudopregnant and 90% in ovariectomized animals suggesting decreased receptor synthesis. Collectively these data support the concept that the rabbit endocervix may be directly regulated by estrogens.

Androgen Control of Cytosol Progesterone Receptor Levels in the MT-W9B Transplantable Mammary Tumor in the Rat<xref ref-type="fn" rid="fn2">2</xref><xref ref-type="fn" rid="fn3">3</xref>

The effect of androgen on the levels of the cytosol progesterone receptor was examined in the transplantable rat mammary tumor MT-W9B and in the normal uteri of inbred WF rats. Progesterone receptor levels were barely detectable in tumors grown in male WF rats but were increased after castration or administration of 17 beta-estradiol. Both effects were blocked by testosterone. In tumors grown in intact female rats, both testosterone and dihydrotestosterone decreased progesterone receptor levels in a dose-dependent manner, and testosterone completely blocked the estradiol-induced increase in progesterone receptor levels in tumors from ovariectomized rats. The inhibitory effect of testosterone in female rats was blocked by the antiandrogen flutamide, suggesting an androgen receptor-dependent mechanism. Neither dihydrotestosterone nor testosterone had any effect on basal levels of progesterone receptor in tumors from ovariectomized rats. In uterus, up to 5 mg dihydrotestosterone/kg did not affect progesterone receptor levels, and a dose of 5 mg/kg was also uterotropic. This fact plus the finding that testosterone only partially blocked the estradiol-induced increase in uterine progesterone receptor levels suggested stimulation of different cell types by testosterone and estradiol. This did not appear to be the case in the tumor, however. Androgen is suggested to act as a negative modulator of progesterone receptor levels, which might have clinical relevance in terms of hormone therapy of breast cancer.

Characteristics of the nuclear translocation of progesterone receptor in fetal guinea pig uterus “in vivo”, “in vitro” and in organ culture

The translocation of progesterone receptor from the cytosol into the nucleus was studied under “in vivo” and “in vitro” conditions in the uteri of guinea pig fetuses exposed to progesterone or a synthetic progestin, R5020. Progesterone treatment of estrogen-primed fetuses leads to a rapid (before lh) transfer of cytosol progesterone receptor into the nucleus which is, however, short-lived (<3h). A rapid decrease in the retention of the estrogen receptor in the nucleus also occurs. In the “in vitro” incubations of whole fetal uteri, translocation of progesterone receptor is temperature-dependent and specific for progesterone and R5020; estradiol and cortisol have no effect. Putative progesterone receptors can also be induced in explants of fetal guinea pig uteri in organ culture which translocate from the cytosol into the nucleus under the same “in vitro” conditions as in whole uteri. Fetal uterine progesterone receptor, either stimulated “in vivo” by estrogen-priming or induced in organ culture, translocates from the cytosol into the nucleus and this process seems to be accompanied by a decrease in retention of the estrogen receptor in the nucleus which appears to be the mechanism by which progesterone antagonises estrogen action in fetal guinea pig uterus.

Steroid-binding specificity of the progesterone receptor from rat placenta

We have attempted to delineate some salient features of the progesterone binding site of the cytosol progesterone receptor ( Rp) in rat placenta by studying the binding profile of various chemical modifications of the progesterone molecule (P). The relative competition ratio ( RCR) was used to calculate the relative affinity of P and the modified ligand for receptor ( K a prog/ K a inh). Cortisol exhibited no appreciable binding. Other corticoids (corticosterone, deoxycorticosterone, 11β-hydroxyprogesterone) had relative affinities 10–30-fold lower than P. Alterations in the structure of P which caused extensive declines in relative binding affinity (i.e. ⩾ 100-fold) include: reduction of A-ring to the Sa-stereoisomere (A/B trans), introduction of a 17α-hydroxyl group > removal of C17 side chain > reduction of C20 carbonyl. The binding profile of the rat placental Rp was similar to that described for uterine Rp from other species indicating a high degree of conservation of molecular structure for the progesterone receptor binding site from species to species.

UTERINE OESTROGEN AND PROGESTERONE RECEPTORS IN THE OVARIECTOMIZED RAT

The effect was studied of repeated injections of oestradiol-17 beta (5, 10, 25, 50 micrograms) given for various lengths of time (3, 5, 9 days) on total cell content of oestrogen receptors and cytosol progesterone receptors in the uteri of ovariectomized rats. An additional group of rats was injected daily with 50 micrograms oestradiol benzoate (OB) for 9 days in order to achieve a more sustained concentration of oestradiol in the blood. Injections were begun 24h after ovariectomy and the rats were killed 24h after the last injection. Daily administration of 5 micrograms oestradiol prevented the initial transient rise in oestrogen receptors which was observed in the uteri of untreated rats after ovariectomy. Repeated injections of 10 micrograms oestrogen produced an initial lowering in oestrogen receptors after 3 days of treatment which was followed by a prompt rise at 5 and 9 days when treatment was continued. A significant reduction in oestrogen receptors occurred at all times studied when rats were injected daily with 25 and 50 micrograms oestradiol. A more profound reduction in oestrogen receptors was observed in the group of rats treated for 9 days with 50 micrograms OB. Synthesis of progesterone receptors was stimulated by all doses of oestrogen studied. Concentrations of progesterone receptors were significantly higher after 3 and 5 days of treatment with 25 and 50 micrograms oestrogen. After 9 days of treatment, however, concentrations of progesterone receptors were virtually identical in all treated groups, including the group treated with OB. We have concluded that large doses of oestrogen significantly decrease oestrogen receptor content in the rat uterus, especially when OB is used. The degree of reduction, however, is only moderate under these experimental conditions and is insufficient to inhibit synthesis of progesterone receptors.