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Department of Molecular Neurobiology (No.K., S.T., K.N.A.), Tokyo Metropolitan Institute for Neuroscience, Fuchu-shi, Tokyo 183; and the Department of Molecular Biology (Na.K.), Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173, Japan
Address all correspondence and requests for reprints to: Dr. Nobuko Kimura, Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, 2–6 Musashidai, Fuchu-shi, Tokyo-183, Japan. E-mail: kimura{at}tmin.ac.jp
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the rat anterior pituitary, messenger RNAs (mRNAs) of all five subtypes are detected using a ribonuclease protection assay (12), RT-PCR (13), and an in situ hybridization method (14, 15). However, the expression patterns of each subtype mRNA level are not consistent, partly because quantitative analyses of all subtype transcripts have not been performed (12, 13, 14, 15). For example, one group (12) showed that sst2 mRNA is expressed more than other subtype mRNAs in the rat pituitary, whereas other groups have shown that sst5 mRNA is expressed more abundantly than sst2 (14, 15). Also, there has been an argument on the physiological relevance of SRIF receptor subtypes related to GH release. It has been proposed that in somatotropes, inhibition of GH release is mediated by only sst2 receptors (16), but the physiological importance of sst5 in the pituitary has also been suggested from the predominant action of SRIF28 in both GH secretion (17) and binding affinity to the pituitary membrane (18).
On the other hand, based on the finding that 17ß-estradiol (E2) induced PRL response to SRIF (19), we revealed two kinds of SRIF receptors in the rat pituitary, E2-dependent and E2-independent receptors, which differed in some biochemical properties (20). However, it remains unknown which subtypes correspond to E2-dependent and E2-independent receptors. Further, there has been a paradoxical observation in which chronic E2 treatment increased the GH trough level [baseline level of GH secretion (21)], which is known to be suppressed by SRIF (22), despite the fact that E2 increased the number of SRIF receptors on pituitary membranes (20).
To delineate SRIF receptor subtype(s) under the regulation of E2, we determined the amount of mRNA of all SRIF receptor subtypes by a semiquantitative method using RT-PCR with an internal standard (23, 24) and compared the results with their functional receptors on the pituitary cellular membranes assessed by subtype-preferential SRIF analogs. The results show that both sst5 and sst2 mRNAs are expressed abundantly in E2-untreated rat pituitaries and that E2 treatment increased transcripts of sst2 isoforms and decreased those of sst5. The results were supported from the analysis of the functional binding characteristics of SRIF receptor subtypes on the pituitary plasma membranes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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32P]Deoxycytidine triphosphate (dCTP) (3000
Ci/mmol) and [125I]Tyr11-SRIF (2000 Ci/mmol)
were obtained from Amersham International Plc (Bucks, U.K.). All
oligonucleotides were obtained from Sawady Technology (Tokyo, Japan).
Primers of rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and PCR
MIMIC construction kit were obtained from Clontech Laboratories, Inc.
(Palo Alto, CA). The Optimizer Kit for PCR, including PCR buffer, was
obtained from Idaho Technology Inc. (Idaho Falls, ID); 10xPCR buffer
contains 500 mM Tris-HCl, pH 8.3, 2.5 mg/ml BSA, 5% Ficoll
400, and 10 mM tartrazine dye including 10 mM
MgCl2 (Low Mg) or 30 mM MgCl2 (High
Mg). Moloney murine leukemia virus reverse transcriptase (MMLV-RT,
RNaseH Minus) was obtained from Toyobo Co., Ltd.(Osaka, Japan).
Taq DNA polymerase (Thermus aquaticus) was from
Pharmacia Biotech (Uppsala, Sweden). RC160 was obtained from Peninsula
Laboratories, Inc. (Belmont, CA). BIM23052, BIM23056, and BIM23060 were
custom peptides synthesized by BEX Co., Ltd. (Tokyo, Japan). NC8–12
was a generous gift from Dr. D. H. Coy, Tulane University Medical
Center (New Orleans, LA).
Animals
Male and female Wistar rats were obtained from Clea Japan, Inc.
(Tokyo, Japan) and used at 3–6 months of age. Female rats were used at
various stages of the estrous cycle, and ovariectomized rats were used
4 weeks postoperatively. Estrogenized rats were obtained by sc
injection of 1 mg of E2 dipropionate oil solution once a
week for 4 weeks, as previously described (20).
Methylmercury hydroxide treatment of RNA and complementary DNA
(cDNA) synthesis
Total RNA from the pituitaries was isolated by guanidium
thiocyanate/cesium chloride centrifugation method as described
(25).
In brief, pituitaries were homogenized in 5.5 M guanidium thiocyanate containing 25 mM sodium citrate (pH 7.0), 0.2 M 2-mercaptoethanol, and 0.5% sodium N-lauryl sarcosinate (1.0 ml per pituitary in the rats without E2 treatment). The homogenate was layered onto 2.0 ml of 5.7 M CsCl in 0.1 M EDTA (pH 7.0) in a Hitachi RPS50–2 tube, followed by ultracentrifugation at 36,000 rpm for 22 h at 15 C. Then the resulting pellets were used as total RNA samples. In these experiments, three pooled pituitaries from ovariectomized rats were used to prepared one RNA sample because the pituitaries were small in size. Three to seven pituitaries from intact rats were pooled for one RNA sample. In contrast, one pituitary was used from an estrogenized rat because the pituitary weight increases about 3- to 5-fold after treatment with E2 for 4 weeks.
Total RNA (2–4 µg) was denatured by treatment with 5 mM
methylmercury hydroxide for 10 min at 25 C (26). The optimum
concentration of methylmercury hydroxide was about 5 mM in
the present study. Methylmercury hydroxide was inactivated by the
addition of 8 mM dithiothreitol. The denatured RNA sample
and 0.5 µg of oligo(dT)24 was heated for 2 min at 70 C
and immediately chilled, after which cDNA synthesis was carried out at
37 C for 75 min with 50 mM Tris-HCl, pH 8.3, 10
mM dithiothreitol, 1.25 mM deoxynucleotide
triphosphate (dNTP), 75 mM KCl, 3 mM
MgCl2, 30 U of RNAase inhibitor, and 200 U of MMLV-RT.
After incubation, samples were heated at 95 C for 5 min and then
quickly chilled on ice, diluted in 1x PCR buffer, and stored at -20 C
until use. As a control experiment, cDNA synthesis reactions were
carried out without reverse transcriptase for every RNA sample to
evaluate contaminating DNA in RNA samples. Under the condition of PCR
amplification (32 cycles) of total RNA (80 ng) with
0.02x10-3 attomoles (amol) of sst4 competitor
(the lowest internal standard), no signal derived from the
contaminating DNA was observed. The amount of cDNA synthesized, which
was measured using [-32P]dCTP (24), was 15–22 ng from
1 µg total RNA.
PCR
The PCR mixture (20 µl) contained cDNA products, 1x PCR
buffer (1 mM MgCl2 for all subtypes, or 3
mM MgCl2 for GAPDH), 0.2 mM
deoxynucleotide triphosphate, 1 µM sense and antisense
primers, and 0.25 U of Taq DNA polymerase. PCR
amplifications were performed in a Perkin-Elmer DNA thermal cycler
PJ2000 (Perkin-Elmer Corp., Norwalk, CT) using Gene Amp tubes
(Perkin-Elmer Corp.). PCR cycle parameters for all subtypes and GAPDH
except for sst5 were: denaturation (94 C for 40
sec)/annealing (60 C for 1 min)/extension (72 C for 2 min). Parameters
for sst5 were: denaturation (94 C for 40 sec)/annealing (65
C for 1 min)/extension (72 C for 2 min). The following primers were
used: sst1 (6): 5'-CTCATCATTGCCAAGATGCGCATG-3' and
5'-TGACCACTCTCCGCTCCGGTTAGC-3'; sst2A (7):
5'-CGGAGCAACCAGTGGGGTAGGAGC-3' and 5'-TCAGATACTGGTTTGGAGGTCTCC-3';
sst3 (8): 5'-GTGAAGGTGCGGTCGACCACACGG-3' and
5'-TTCATCTCCTGCCCT-CTCTGCATC-3'; sst4
(9): 5'-CTGGCTGGCAACAACGGAGACGCT-3' and 5'-TCCTGAGGCTCCTAGGGACAGAAC-3';
sst5 (10): 5'-GTCAAGGTGAAGGCTGCAGGCATG-3' and
5'-GGCATTCAAATCCTGCTGGTCTGC-3'; GAPDH: 5'-TGAAGGTCGGTGTCAACGGATTTGGC-3'
and 5'-CATGTAGGCCATGAGGTCCACCAC-3'; sst2A and
sst2B isoforms (7, 11): 5'-CGGAGCAACCAGTGGGGTAGGAGC-3',
5'-TCCGGTTTCTGCCGGGTAGCTG-3'. PCR products were confirmed by their
nucleotide sequencing and restriction digestion using restriction
enzymes: sst1 PCR product (473 bp): HaeII,
PvuII, Sau3AI; sst2A (564 bp):KpnI, BstXI, BamHI; sst3 (455
bp): BamHI, Cfrl3I, BanII; sst4 (572
bp): HincII, NdeI, PvuII;
sst5 (424 bp): HincII, AvaI,
HinfI.
Quantitative analysis by competitive PCR
Competitors for all subtypes and GAPDH were constructed using
the PCR MIMIC construction kit (27). The absorbance of these
competitors was measured at 260 nm to determine the molar quantity,
after which competitors were diluted using 1xPCR buffer without Ficoll
and dye. The size (base pairs) of each competitor was 248 bp for
sst1 and sst3, 348 bp for sst2A and
sst4, 604 bp for sst5, and 606 bp for GAPDH. An
appropriate amount of competitors and 0.1–0.25 µCi
[32P]dCTP were added to a PCR reaction mixture
containing sample cDNA. After PCR amplification, 6-µl aliquots of PCR
products were resolved on a 2% ethydium bromide-agarose gel in 1x
Tris-borate-EDTA buffer. Agarose gels were wiped with filter paper no.
2 (Toyo Roshi Kaisha Ltd., Tokyo, Japan) thoroughly, placed on 10
sheets of the filter paper, and dehydrated using a gel dryer (Bio-Rad,
Richmond, CA; model 583) without heating. After suction for 15 min,
gels were transferred to six new sheets of filter paper and further
dehydrated for about 15 min. Agarose gels were exposed on an imaging
plate (IP) of Fuji Photo Film Co., Ltd. (Tokyo, Japan). Radioactivity
[photo-stimulated luminescence intensity (PSL)] of the IP
autoradiogram was determined using the FUJIX Bio-Imaging Analyzer,
BAS-2000II (Fuji Photo Film Co., Ltd.). Competitive PCR was usually
performed in duplicate for cDNA synthesized from each RNA sample. The
results obtained from RNA samples indicated in each experiment are
expressed as the mean ± SE, and statistical analysis
was performed by Student’s t test.
Binding assay
Binding assay of [125I]Tyr11-SRIF to
the pituitary plasma membranes was performed in duplicate for each
membrane preparation using either crude membranes or purified membranes
from rat pituitaries as previously described (19, 20). The results are
calculated using data obtained from three to five separate experiments
with different membrane preparations and expressed as the mean ±
SE with the statistical analysis by Student’s t
test. Hill coefficients were calculated from displacement curves of
analogs and obtained graphically (28).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). RT-PCR products were specific and
were verified by their absence without RT. After a known amount of each
SRIF receptor subtype cDNA from the total RNA was amplified in the
presence of 2-fold dilutions of a corresponding competitor at a given
cycle, the ratio of the PCR product for each subtype (target) to its
corresponding competitor was obtained. This is given as the ratio of
PSL as shown in Fig. 1B. When the ratio was plotted against the
reciprocal of the amount of competitor, the plot was linear. The ratio
of 1.0 indicates that the amount of target cDNA was equal to the amount
of corresponding competitor, as previously described (27).
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7.0 vs. 4.2) (Fig. 2B). Therefore, preestimation of the
ratio between sst2A and sst2B PCR products in a
given sample allowed us to calculate the amount of sst2B.
To normalize total RNA from each sample and the altered efficiency of
RT from the total RNA in each sample, we used GAPDH, a ubiquitously
expressed housekeeping gene, as an internal standard (27). The
mRNA levels of the SRIF receptor subtypes were described by the ratio
of target cDNA to GAPDH cDNA. In pituitaries from ovariectomized rats
treated with and without E2, mRNA expression of all
subtypes was detected, and more than 97% of the total subtype mRNA was
composed of octreotide-sensitive subtypes such as sst5 and
sst2 isoforms and sst3. In ovariectomized rat
pituitaries, most of the octreotide-sensitive subtypes were
sst5 and sst2 isoforms (Fig. 2C). The
sst5 mRNA level was approximately 3-fold higher than the
sst2A mRNA level in these pituitaries
(sst5/GAPDH = 1.4 x 10-2;
sst2A/GAPDH = 0.4 x 10-2). However,
chronic E2 treatment induced 3- and 5-fold increases of the
sst2A and sst2B mRNA levels, respectively,
compared with the ovariectomized rat pituitaries
(sst2A/GAPDH = 1.2 x 10-2). In
contrast, the treatment drastically decreased the sst5 mRNA
level to less than 1% of the total amount of SRIF receptor mRNA
(sst5/GAPDH = 0.01 x 10-2). In the
estrogenized rat pituitaries, sst2 isoforms and
sst3 comprised about 87% and 9% of the total SRIF
receptor mRNAs, respectively. Although the amounts of sst3
and sst1 mRNAs were small, these mRNA levels were increased
by chronic E2 treatment.
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). This sex-dependent difference was
ascribed mainly to differences in the expression levels of
sst2A and sst5 mRNAs, which were significantly
lower in female than in male rats (sst2A, P
< 0.01; sst5, P < 0.001). Furthermore,
the sst5 mRNA level, which was as high as the
sst2A level in the female rats, was approximately 2-fold
higher than the sst2A mRNA level in male rats. Although
there was a low level of sst3 transcript in both male and
female rat pituitaries, the level in female rats was 3-fold higher than
in males (P < 0.01). These results demonstrate that
mRNA expression of SRIF receptor subtypes is different in male and
female rat pituitaries.
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. RC160, as well as octreotide, has lower affinity for
sst1 and sst4 and higher affinity for other
subtypes (29, 30). From competition curves of
[125I]Tyr11-SRIF binding (Fig. 4), both pituitary membrane receptors
from ovariectomized rats and E2-treated rats displayed high
binding affinity for RC160, demonstrating that the presence of
sst1 and sst4 was negligible, at best, in these
pituitary membranes. To examine the possible existence of other
subtypes of SRIF receptors, we used other SRIF analogs having
subtype-preferential binding affinity, although none of these were
purely subtype selective (16, 30, 31, 32, 33, 34). RC160, BIM23060, and NC8–12
have higher affinity for sst2 than sst5,
whereas BIM23052 and BIM23056 bind more potently to sst5
than sst2. The binding characteristic of sst3
is more similar to that of sst5 than to sst2.
The membrane receptors from the E2-treated rats bound
BIM23060, NC8–12, and RC160 with higher affinity and BIM23052 with
lower affinity than did the receptors from ovariectomized rats. Higher
concentrations of BIM23056 were required to start the displacement of
the ligand binding to the pituitary receptors from
E2-treated rats compared with those from ovariectomized
rats. Thus, the results demonstrate that pituitary receptors from
E2-treated rats tended to possess characteristics of
sst2 rather than sst5. Moreover, the receptors
bound these analogs with Hill coefficients of 0.8–1.0, suggesting that
pituitary receptors from E2-treated rats have homologous
binding affinity (Table 1). On the other
hand, pituitary receptors from ovariectomized rats showed the binding
profile of sst5 rather than sst2, being in good
agreement with the large expression of sst5 mRNA, and
resembled those from male rats. Competition curves by SRIF analogs
tested for both ovariectomized and male rat pituitary receptors were
broad, with Hill coefficients of less than 1.0 (nH =
0.5–0.8). The observations that relatively low concentrations of
BIM23056 began to decrease the ligand binding and its high
concentrations were required for the complete displacement suggest that
the membrane receptors are likely to possess the characteristics of
sst5 rather than sst3 (30) and may express
those of sst2 isoform(s) as well. These results demonstrate
that membrane receptors from the E2-treated rats are
composed largely of sst2, in agreement with the predominant
expression of sst2 mRNAs, whereas the receptors from
ovariectomized rats and male rats are of heterogeneous subtypes,
probably consisting of sst5 and sst2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our finding that sst5 mRNA was most highly expressed in the pituitaries of ovariectomized rats and male rats was in agreement with previous studies on rat pituitaries (15) and human mammosomatotrope-derived adenomas (35). However, it should be noted that the sst5 mRNA level fluctuated under the influence of estrogen: the level was lowered 2 orders of magnitude by administering E2 into ovariectomized rats.
In mammotropes, all subtype mRNAs, including both sst2 and sst5, have been reported to be expressed in mammotropes (14, 15). Nevertheless, SRIF is not capable of inhibiting PRL secretion without E2 treatment. Under the influence of E2, mammotropes acquire SRIF responsiveness (19). Because E2 induced a nearly 9-fold increase in receptor binding sites, as previously described (20), we first assumed that the total amounts of subtype mRNAs per GAPDH mRNA would increase. To our surprise, however, these total amounts did not change in estrogenized rat pituitaries compared with their ovariectomized counterparts. Thus, the SRIF responsiveness induced by E2 may result from a combined effect of increased expression of sst2 isoform genes and a decreased expression of the sst5 gene. Therefore, it is possible that sst2 may become functional in mammotropes when sst5 is not expressed or is expressed at an extremely low level. Alternatively, E2 may induce an additional essential protein(s) as well, which may enhance the signaling efficiency in association with sst2.
Chronic E2 treatment increases the population of mammotropes, whereas it decreases that of somatotropes (36). The E2 effect on SRIF receptor subtype mRNA expression, however, seems unlikely to derive from change in cell populations because we observed up-regulation of sst2A after short-term treatment (2–6 h) with E2 in pituitary primary cultured cells, as well as GH3 cells, and down-regulation of sst5 in primary cultured cells (Kimura, N., and S. Tomizawa, unpublished data). Moreover, short-term treatment of E2 reportedly up-regulates sst2 mRNA expression in human breast cancer (37). In the transplantable rat PRL-secreting pituitary tumor, sst2 and sst3 expressions were induced in vitro by chronic E2 treatment (38). Taken together, effects of E2 on subtype expression appear due to changes in their gene expression rather than those of cell populations.
In somatotropes, GH is secreted in a pulsatile pattern with sexual dimorphism in the rat. The levels of GH pulses are regulated by GRF, whereas those of GH troughs are regulated by SRIF (22, 39, 40). Regarding baseline levels of serum GH, the following observations have been reported. GH troughs are high in female compared with male rats (22). They are also high in chronic E2-treated male rats compared with their control counterparts (21). Clinically, chronic estrogen treatment increases serum GH baseline levels in postmenopausal women (41). The observations imply that E2 may suppress inhibitory regulation for GH troughs. When the data obtained in this study, i.e. that E2 up-regulates sst2 whereas it down-regulates sst5, are considered, it seems likely that these high baseline levels of GH under the influence of E2 may be related, in part, to the decreased expression of sst5 in the pituitary.
Despite the fact the sst2 expression is positively regulated by E2, sst2 expression was lower in female rats than in males, suggesting that sst2 gene expression is likely to be regulated by an unknown factor(s) in addition to E2.
[Leu8,D-Trp22,Tyr25]SRIF28 and cyclosomatostatin (SA: c[Ahep-Phe-D-Trp-Lys-Thr(Bzl)]) have lower affinity for sst2 than for sst5 (16, 30, 31, 32, 33). The previous results that the E2-dependent receptors from E2-treated rat pituitaries exhibited lower affinities for these analogs than E2-independent receptors from ovariectomized rat pituitaries (20) are in good agreement with the present observation that the membrane receptors from E2-treated rat pituitaries contained mainly sst2.
Although BIM23056 was reported initially to be a sst3-selective analog (3, 10, 33), no such selectivity has been found, at least under conditions in which [125I]Tyr11SRIF was used as a ligand. In contrast, the peptide binds to sst5 preferentially compared with sst3 and sst2 (30). In support of this, this study showed that BIM23056 at its low concentrations displaced [125I]Tyr11SRIF binding to a greater extent in male and ovariectomized rat pituitary membranes than in estrogenized rat pituitary membranes. On the other hand, the receptors present in the E2-treated rat pituitary membranes were found to exhibit characteristics of sst2, although sst3 mRNA constituted about 9% of the total SRIF receptor subtype mRNAs. Because of the lack of sst3 preferential analog, we could not demonstrate the sst3 binding activity specifically in the estrogenized rat pituitaries.
The splice isoforms, sst2A and sst2B, cannot be discriminated by binding properties using SRIF analogs, but sst2B is known to be more effective in inhibiting adenylate cyclase and more resistant in agonist-dependent loss of binding activity compared with sst2A (34, 42). Although the present study enabled semiquantification of both isoform mRNAs separately, the physiological relevance of these receptor functions still remains speculative.
In conclusion, chronic E2 treatment regulated the mRNA levels of sst2 positively and sst5 negatively in rat pituitaries. The pituitary membrane SRIF receptors increased by E2 were mainly composed of sst2 isoforms, whereas those from ovariectomized rats were heterogeneous and mostly consisted of sst5 and sst2 isoforms. Therefore, the sst2 isoforms may be involved in the control of PRL secretion as well as GH secretion (16). This was supported by a more recent report in which sst2 mediates SRIF regulation of PRL release in human fetal pituitary cultures (43). In the light of the recent report that both sst2 and sst5 contribute to the inhibition of GH secretion (43), high basal levels of pulsatile GH secretion induced by chronic E2 treatment (21, 41) may be explained by down-regulation of the sst5 mRNA level. Thus, not only sst2, but also sst5, may participate in the regulation of GH secretion with sex-dependent difference.
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Acknowledgments |
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Footnotes |
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Received October 8, 1997.
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N. Kimura, S. Tomizawa, K. N. Arai, R. Y. Osamura, and N. Kimura Characterization of 5'-Flanking Region of Rat Somatostatin Receptor sst2 Gene: Transcriptional Regulatory Elements and Activation by Pitx1 and Estrogen Endocrinology, April 1, 2001; 142(4): 1427 - 1441. [Abstract] [Full Text]
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S. Park, J. Kamegai, T. A. Johnson, L. A. Frohman, and R. D. Kineman Modulation of Pituitary Somatostatin Receptor Subtype (sst1-5) Messenger Ribonucleic Acid Levels by Changes in the Growth Hormone Axis Endocrinology, October 1, 2000; 141(10): 3556 - 3563. [Abstract] [Full Text] [PDF]
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) were: sst1, 40 ng from total RNA and 0.05 x
10-3 amol of sst1 competitor;
sst2A, 20 ng and 0.5 x 10-3 amol;
sst3, 20 ng and 0.5 x 10-3 amol;
sst4, 80 ng and 0.02 x 10-3 amol;
sst5, 20 ng and 2.0 x 10-3 amol; GAPDH,
15 ng and 0.2 amol. PCR products were resolved on a 2% agarose gel
after the indicated cycles of PCR. Upper panel, The
radioactivity of each target and competitor PCR products was analyzed
as described in Material and Methods. Lower
panel, IP autoradiogram of the PCR products of each target cDNA
(
) from total RNA samples with or without RT and its corresponding
competitor DNA (
). The same amounts of total RNAs and competitors
were used as described above. B, A constant amount of the cDNA
described above and 2-fold serial dilutions of an initial competitor
were coamplified with [32P]dCTP for varying cycles:
sst1, 0.2 x 10-3 amol for the initial
concentration of sst1 competitor and 30 cycles;
sst2A, 2 x10-3 amol and 27 cycles;
sst3, 2 x 10-3 amol and 27 cycles;
sst4, 0.1 x 10-3 amol and 32 cycles;
sst5, 8 x 10-3 amol and 26 cycles;
GAPDH, 0.8 amol and 18 cycles. All PCR products were resolved on 2%
agarose gels. IP autoradiogram of the gels is shown in upper
panel. The ratio of the target to competitor PSL was plotted
against the reciprocal of the molar amount of competitor in the
lower panel. The amount of target cDNA was calculated by
extrapolating from the intersection of the line with a ratio of 1.0
down to the x-axis.
) were incubated with
[125I]Tyr11SRIF (0.13 nM) in the
presence of varying concentrations of SRIF analogs. Maximal specific
binding in the absence of the indicated SRIF analog was taken as 100%.
Each point represents the mean ± SE of
the results obtained from three to five separate experiments in
duplicate determination. *, P < 0.05;
ovariectomized rat pituitary membranes vs.
E2-treated ovariectomized rat pituitary membranes. #,
P < 0.05; male rat pituitary membranes
vs. E2-treated ovariectomized rat pituitary
membranes.