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Original Studies |
Institute of Endocrine Sciences, Ospedale Maggiore, University of Milan, and Italian Auxologic Center (L.P.), IRCCS, 20122 Milan, Italy
Address all correspondence and requests for reprints to: Anna Spada, M.D., Istituto di Scienze Endocrine Ospedale Maggiore, IRCCS, Via Francesco Sforza 35, 20122 Milan, Italy. E-mail: endosci{at}imiucca.csi.unimi.it
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, a Ca2+-sensing receptor (CaSR) that mediates the inhibition of PTH secretion has been identified and cloned from bovine parathyroid gland (6). CaSR is a G protein-coupled receptor, and its activation results in increases in inositol phosphate turnover and [Ca2+]i (7, 8). Transcripts that hybridize with probes of the cloned CaSR are expressed in a number of tissues of different species (7, 8, 9). Recently, CaSR expression has been documented in mouse and rat anterior pituitary, whereas the intracellular signaling elicited by CaSR activation has been investigated in mouse corticotroph AtT-20 cells (10, 11). We show here that a similar CaSR receptor is also expressed in human pituitary cells, and its activation modulates [Ca2+]i and cAMP levels.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The study includes 13 human pituitary adenomas [7
nonfunctioning adenomas (NFPA) and 6 GH-secreting adenomas (GH-omas)
diagnosed on the basis of clinical features and standard hormonal
criteria], a pool of autoptic normal pituitaries obtained from
postmortem proceedings carried out within 24 h after death, and a
pool of normal parathyroid biopsies from patients operated on for
primary hyperparathyroidism. The presence of
Gs
gene mutations in GH-omas was ruled out as
previously described (12). No patient had previously undergone
pituitary irradiation. Immunohistochemistry, carried out as previously
described (13), showed a high percentage of cells positive for GH in
GH-omas, whereas the large majority of cells from NFPA were positive
for glycoprotein hormone subunits. Part of the tissue was quickly
frozen at -80 C, and part was placed in sterile culture medium for
cell culture. The study was approved by the local ethical committee.
Informed consent was obtained from all patients.
RT-PCR
Total ribonucleic acid (RNA) was extracted from tissues according to the method of Chomczynski and Sacchi (14). To avoid any amplification from genomic DNA contamination, the samples were pretreated with deoxyribonuclease. Total RNA was used to perform RT as previously described (15). Briefly, 1 µg total RNA was incubated for 1 h at 42 C in the presence of 2.5 U reverse transcriptase; 1 U ribonuclease inhibitor; 2.5 µmol/L random hexamers; 1 mmol/L each of deoxy (d)-ATP, dCTP, dGTP, and dTTP; 5 mmol/L of MgCl2; 50 mmol/L KCl; and 50 mmol/L Tris-HCl (pH 8.3) in a total volume of 20 µL (GeneAmp, Perkin Elmer Corp., Norwalk, CT). Subsequently, the reaction mixture was heated at 96 C for 5 min and quick-chilled on ice. The total volume of the reactions was subjected to PCR amplification using specific primers amplifying a fragment of 330 bp. The sense primer corresponds to nucleotides 385–405 (5'-AGCTGCTGCTGGGTCCTCTTGG-3'), and the antisense primer is complementary to nucleotides 693–715 (5'-CAAAACTCAGGGTGGCTTCCAAG-3') of the human CaSR complementary DNA (16). PCR amplification was performed adding to the product of RT: 50 mmol/L KCl, 50 mmol/L Tris-HCl (pH 8.3), 2 mmol/L MgCl2, 15 pmol of each primer, and 2.5 U Taq DNA polymerase (AmpliTaq). The reaction mix was subjected to denaturation at 94 C for 3 min, followed by 35 cycles of 94 C for 1 min, 66 C for 45 s, and 72 C for 1 min. A final cycle at 72 C for 10 min was carried out to allow complete extension of the amplified fragments. The amplified products were visualized on a 3% agarose gel stained with ethidium bromide.
Southern blot analysis
Southern blot analysis was performed using a 26-oligonucleotide
probe corresponding to nucleotides 554–579
(5'-TCAGGTATAATTTCCGTGGGTTTCGC-3') of human CaSR complementary DNA
(16). PCR products were fractionated on a 3% agarose gel in 1 x
Tris-borate-ethylenediamine tetraacetate and then transferred to a
Hybond - N+ (Amersham Pharmacia Biotech Buckinghamshire,
UK) Nytran membrane. The probe was 
-32P 5'-end
labeled using T4 polymerase kinase for 1 h at 37 C. The blot was
hybridized with the -32P-labeled internal probe for
12 h in 6 x SSC (standard saline citrate), 5 x
Denhart’s solution, 0.1% SDS, and 0.1 mg/mL salmon sperm. After
hybridization, the blot was washed twice in 2 x SSC and 0.1% SDS
for 15 min at room temperature and subsequently in 0.1 x SSC and
0.1% SDS at 65 C for 15 min. The filter was then exposed to an x-ray
film (X-Omat, Eastman Kodak Co., Rochester, NY) for
12 h.
Sequencing of the RT-PCR fragment of CaR
Sequencing of the RT-PCR products using both sense and antisense specific primers was performed using the AmpliTaq BigDye Terminator kit and 310 Genetic Analyzer (Perkin Elmer Corp., Applied Biosystems, Foster City, CA).
Western blot analysis
Western analysis of CaSR protein was performed using antiserum raised in rabbits (Primm srl, Milan, Italy) against a peptide corresponding to amino acids 345–359 within the predicted extracellular domain of the deduced amino acids sequence of the bovine parathyroid CaSR (GenBank accession no. 67307) and identical to corresponding peptide in the human CaSR (17). The protein concentration of parathyroid and pituitary tissues was measured using the Micro BCA protein reagent kit (Pierce Chemical Co., Rockford, IL). Total proteins (20 µg) were separated by SDS-PAGE at 7.5% and transferred electrophoretically to nitrocellulose membranes, using mol wt standards as a reference. Proteins on membranes were detected by staining with Ponceau S. The filters were then incubated with blocking solution (20 mmol/L Tris, 500 mmol/L NaCl, and 0.2% dry milk) at 4 C overnight. The filters were subsequently incubated with antiserum against CaSR at a 1:1000 dilution for 2 h at room temperature and then with goat antirabbit IgG antibody conjugated to alkaline phosphatase for 30 min. The membranes were treated with chemiluminescent substrate and enhancer (Immuno-Star Chemiluminescent Protein Detection Systems, Bio-Rad Laboratories, Inc., Richmond, CA) and exposed to x-ray film for 5–20 min. The bands were quantitated by scanning densitometry using an imaging densitometer (Bio-Rad Laboratories, Inc., GS-670). To detect the specificity of the reaction, the antiserum was preincubated with 0.5 µg/µL peptide, against which the antibody was raised for 12 h at 4 C.
Cell culture
Cells were enzymatically dispersed from tumoral tissues using trypsin and deoxyribonuclease as previously described (18). Cells were cultured at a density of 5 x 105 cell/mL in Ham’s F-10 medium (containing 0.5 mmol/L Ca2+) supplemented with 5% FCS and antibiotics for short term incubation (24–48 h) at 37 C and 5% CO2. Cells were either maintained in suspension and collected for [Ca2+]i measurements after 16–24 h or plated in 24-well plates for 24–48 h and used for cAMP and GH assays.
Measurement of cytosolic Ca2+
[Ca2+]i measurements were carried out as previously described (18). Briefly, cells were resuspended in Krebs-Ringer HEPES incubation medium and loaded with the Ca2+ indicator fura-2 by incubating the cells with 5 µmol/L fura-2/acetoxymethylester (fura-2/AM) for 30 min at 37 C. Fluorescence recordings were carried out with a cell concentration of 3–4 x 105/mL in a Perkin Elmer Corp. LS5 spectrofluorometer (Norwalk, CT) at 345 nm excitation and 490 nm emission, with slits of 5 and 10 nm, respectively. [Ca2+]i was calculated according to the method of Grynckievicz et al. (19).
Intracellular cAMP assay
Cells were used for intracellular cAMP assay as previously described (12). Medium was removed, and cell monolayers were washed and equilibrated for 1 h with a solution containing 125 mmol/L NaCl, 5 mmol/L KCl, 0.5 mmol/L MgSO4, 0.5 mmol/L CaCl2, 25 mmol/L HEPES (pH 7.4), 6 mmol/L glucose, and 0.1% BSA. Cells were treated for 10 min with 2 mmol/L isobutylmethylxanthine followed by a 30-min incubation with the agents to be tested. After medium removal, cells were washed, and cAMP was extracted with 1 mL ice-cold ethanol (80%) at -20 C for 24 h. The supernatants were evaporated to dryness and redissolved in cAMP kit buffer (Amersham Pharmacia Biotech, Buckinghamshire, UK) as previously described (12).
In vitro hormone release
Medium was removed by aspiration, and the cell monolayers were washed twice and preincubated for 1 h with fresh Ham’s F-10 medium supplemented with 0.1% BSA. Cells were then incubated with and without the agents to be tested for 30 min at 37 C in triplicate. At the end of incubation, the medium was removed and stored at -20 C until GH assay (Wallac Oy, Tuzku, Finland).
Materials
TRH, GHRH, trypsin, soybean trypsin inhibitor, gadolinium, neomycin, and pertussis toxin (PTX) were purchased from Sigma Chemical Co. (St. Louis, MO). Pituitary adenylate cyclase-activating peptide (PACAP) was purchased from Peninsula Laboratories, Inc. (St. Helens, UK). Nimodipine was obtained from Tocris Cookson (Bristol, UK). Culture media were purchased from Flow Laboratories (Mackenheim, Germany). Fura-2/AM was purchased from Molecular Probes, Inc. (Junction City, OR). All other reagents ware reagent grade.
Statistical analysis
The results are expressed as the mean ± SD. Paired or unpaired two-tailed Student’s t test was used to detect the significance between two series of data. P < 0.05 was accepted as statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Total RNA extracted from human parathyroid, two NFPA, and two
GH-secreting adenomas was subjected to RT-PCR using specific primers
amplifying a 330-bp fragment of the CaSR. The products of RT-PCR showed
a single fragment of the expected size in all tissues (Fig. 1
). The band was absent when RT was
omitted from reverse transcriptase reaction (Fig. 1). The Southern blot
analysis, performed in all RT-PCR products, revealed that in each of
the samples the internal probe hybridized to the 330-bp fragment,
confirming that they were products of specific amplification of CaSR
mRNA expressed in these tissues (Fig. 1). Sequence analysis of the
330-bp fragments generated by RT-PCR from pituitary (one NFPA) and
parathyroid tissues indicated a 100% homology of this portion of CaSR
with the previously reported sequence of the human parathyroid gland
(16).
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Figure 2 shows immunoblots obtained
with the antiserum directed against CaSR in normal parathyroid tissues
and in normal and tumoral pituitary, which were run simultaneously in
each experiment. Probing with this antiserum detected one single band
of approximately 150 kDa, that was absent after preabsorbing the
antiserum with the peptide against which it was raised. The protein was
expressed at high levels in the normal pituitary and in the adenomas
tested (three NFPA and three GH-omas); the amount of CaSR was similar
to that found in the parathyroid (Fig. 2).
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The effect of extracellular calcium ([Ca2+]o) on
[Ca2+]i was evaluated in cells obtained from seven NFPA
and six GH-omas. Elevation of ([Ca2+]o) increased
cytosolic Ca2+ levels ([Ca2+]i) in each cell
preparation from a resting level of 83.0 ± 8 nmol/L at 0.5 mmol/L
[Ca2+]o to a peak of 150 ± 26 nmol/L at 2.5 mmol/L
(P < 0.001). The [Ca2+]o effect was
detectable at 1.5 mmol/L in five NFPA and three GH-omas and at 2.5
mmol/L [Ca2+]o in the remaining adenomas (Fig. 3). The increases induced by
[Ca2+]o varied from one adenoma to another, but not in
the same cell preparation. The effectiveness of [Ca2+]o
to increase [Ca2+]i was similar in the different types of
adenoma (stimulation, 75 ± 40 nmol/L in NFPA and 76 ± 40
nmol/L in GH-omas; Fig. 3). A similar [Ca2+]i rise was
observed using as activators of CaSR the trivalent cation gadolinium
(Gd3+ at 30 µmol/L; Figs. 3 and 4). Similarly, neomycin (100 µmol/L)
caused stimulations of 78% and 110% in the two NFPA tested.
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). The initial
peak was maintained in nominally Ca2+-free medium,
indicating that this component was due to Ca2+ release from
intracellular stores (Fig. 4). The blockade of L-type Ca2+
channels by nimodipine caused a rapid decrease in resting
[Ca2+]i levels, with a reduction from 35–54% (Fig. 4).
Moreover, this agent reduced the transient due to Ca2+
mobilization and abolished the plateau phase due to Ca2+
influx caused by CaSR activation. The effects of CaSR activators on
[Ca2+]i were not modified by PTX pretreatment (0.1
µg/mL for 16 h).
TRH (100 nmol/L) caused a 2- to 2.5-fold increase in
[Ca2+]i in all NFPA and GH-omas (Fig. 4). The increases
induced by TRH were higher than those triggered by
[Ca2+]o (stimulation, 162 ± 50% by TRH
vs. 74 ± 40% by [Ca2+]o;
P < 0.05).
Effects of CaSR agonists on intracellular cAMP levels
[Ca2+]o at 1.5 mmol/L caused a significant
stimulation of intracellular cAMP levels in six tumors (four NFPA and
two GH-omas), whereas further [Ca2+]o increases to 2.5
mmol/L were effective in the remaining four (Fig. 5). Similarly, Gd3+ at 30
µmol/L increased intracellular cAMP levels from 1.67 ± 0.29
pmol/well to 3.99 ± 1.05 (P < 0.05) in the
tumors tested (two NFPA and one GH-oma; Fig. 5). Pretreatment of cells
from two NFPA with PTX did not affect cAMP increase induced by
[Ca2+]o. Intracellular cAMP levels were significantly
increased by specific hypothalamic peptides. In particular, 10 nmol/L
GHRH was more effective than [Ca2+]o in increasing cAMP
levels in GH-omas (stimulation, 410 ± 282% by GHRH
vs. 97.8 ± 80.4% by [Ca2+]o;
P < 0.05), whereas PACAP and [Ca2+]o
elicited similar cAMP responses in NFPA (stimulation, 110 ± 82%
by PACAP vs. 97.8 ± 80.4% by
[Ca2+]o; P = NS). In the tumors
tested (two NFPA and two GH-omas), TRH at any concentration (from 10
nmol/L to 10 µmol/L) was unable to modify cAMP levels (1.27 ±
0.64 pmol/well cAMP in the absence vs. 1.30 ± 0.50
pmol/well cAMP in the presence of 10 µmol/L TRH; P =
NS).
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Hormone secretion in the presence of different
[Ca2+]o concentrations was evaluated in GH-omas.
[Ca2+]o, at concentrations of 1.5 and 2.5 mmol/L, did not
significantly increase in vitro GH release in resting
conditions (basal GH, 30.8 ± 2.1 ng/well·30 min vs.
32.1 ± 2.9 at 2.5 mmol/L [Ca2+]o;
P = NS), but caused an amplification of GHRH-induced
stimulation (Fig. 6). Similarly,
Gd3+, which was ineffective on basal GH release, increased
the GH response to GHRH (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunoblotting performed with an antiserum directed against the extracellular domain of the receptor showed that in pituitary cells, CaSR mRNA is translated into a functional protein. Although previous works reported that this antiserum recognized a doublet of about 130 and 150 kDa, representing a different degree of glycosylation of the receptor (6, 17), in our experimental conditions a single band of approximately 150 kDa was detected; the gel system we used did not allow the accurate identification of the two forms. CaSR was clearly detected in normal pituitary and pituitaries of different types, without quantitative differences from the amounts found in parathyroid tissues. These data suggest that expression of CaSR is a common feature of the pituitary cell that seems to be independent from its normal or tumoral origin as well as its secretory activity.
In cells obtained from each of the tumors, CaSR agonists caused a significant increase in [Ca2+]i that was mainly due to Ca2+ mobilization, in agreement with the stimulation of phospholipase C and inositol phosphate production induced by CaSR activation in other cell systems (7, 8, 10, 11). Increases in [Ca2+]i observed in pituitary cells were similar to those observed in AtT20, but definitely lower than those reported in the parathyroid (10). As the levels of CaSR expression in pituitary and parathyroid tissues were similar, different efficiencies of CaSR coupling to endogenous G proteins might account for the differences between the two systems. Contrary to the results obtained in AtT-20 cells (10), in human pituitary cells CaSR seems to be coupled to a PTX-insensitive G protein, probably Gq and/or G11. This coupling is similar to that reported in the human parathyroid (23), suggesting the existence of species- and cell-specific differences in the pattern of G proteins available for CaSR activation. Calcium mobilization occurred between 1.5–2.5 mmol/L [Ca2+]o, which is consistent with the maximal stimulation of inositol phosphate production occurring at 3 mmol/L [Ca2+]o in AtT20 cells (10).
Although in the parathyroid, CaSR activation induces [Ca2+]i increases associated with the reduction of both intracellular cAMP levels and PTH release (8, 24), in AtT20 cells the activation of the same receptor causes a significant increase in cAMP levels, which is consistent with the marked increase in both basal and CRH-stimulated ACTH secretion induced by [Ca2+]o elevation (10, 11). The same receptor-triggered events were observed in human pituitary cells, in which a significant stimulation of cAMP levels was caused by [Ca2+]o and CaSR agonists. The molecular mechanisms involved in cAMP generation by [Ca2+]o are unknown at the present time. However, the observation that stimulation of both Ca2+ mobilization and influx induced by other agents, i.e. TRH, did not affect cAMP levels points to a direct, receptor-specific action of [Ca2+]o on cAMP production.
The relationship between hormone secretion and activation of CaSR is unclear. Indeed, in vivo and in vitro studies suggest a modulatory role of [Ca2+]o in the secretion of pituitary hormones (20, 21, 25, 26, 27, 28, 29, 30), although few and contradictory data have been obtained at Ca2+ concentrations within the normal physiological range (31, 32). Activation of CaSR did not result in hormone secretion from any of GH-omas studied, although it is worth noting that hypothalamic peptides known to stimulate in vitro GH release from cultured tumors, such as GHRH and TRH, increased cAMP production and cytosolic Ca2+ levels, respectively, at a greater extent than [Ca2+]o. However, although CaSR seems to be devoid of a direct effect on hormone secretion, one might postulate that small changes in intracellular calcium and cAMP levels by [Ca2+]o may influence the action of classical hypothalamic releasing hormones. This view is supported by our observation that the GH response to GHRH in GH-omas was amplified by activating CaSR.
In conclusion, we first demonstrate the expression of the CaSR mRNA and the protein it encodes in the human pituitary. In addition, we demonstrate that the activation of this receptor generates a series of intracellular effectors that are known to control several biological processes, including hormone secretion and cell differentiation and proliferation, thus providing evidence for an additional mechanism by which calcium might regulate pituitary cell function.
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Acknowledgments |
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Footnotes |
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2 R.R. and A.L. contributed equally to this work and should both be
considered first authors.
Received November 17, 1998.
Revised April 5, 1999.
Accepted May 11, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is associated with an
increased phosphodiesterase activity in human growth hormone-secreting
adenomas. J Clin Endocrinol Metab. 83:1624–1628. gene is associated with low levels of
Gs protein in growth hormone-secreting tumors. J
Clin Endocrinol Metab. 83:4386–4390.1-Adrenergic stimulation of in vitro growth
hormone release and cytosolic free Ca2+ in rat
somatotrophs. Endocrinology. 122:1419–1425.[Abstract]
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