0021-972X/91/7304-0913$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright {O 1991 by The Endocrine Society
Vol. 73, No. 4 Printed in U.S.A.
Hypothalamic Peptides Modulate Cytosolic Free Ca2+ Levels and Adenylyl Cyclase Activity in Human Nonfunctioning Pituitary Adenomas* ANNA SPADA, FARZIN REZA-ELAHI, ANDREA LANIA, PALOMA GIL-DELALAMO, MONIQUE BASSETTI, AND GIOVANNI FAGLIA Institute of Endocrine Sciences, Ospedale Maggiore IRCCS (A.S., F.R-E., A.L., P.G.d.A., F.G.) and CNR Center of Cytopharmacology, Department of Pharmacology (M.B.), University of Milan, and Italian Auxological Center (F.R-E.), Milan, Italy
ABSTRACT. The effects of hypothalamic peptides (TRH, GnRH, arginine vasopressin, vasoactive intestinal peptide, GHRH, CRH, and SRIH) on cytosolic free calcium concentrations ([Ca2+]i) and adenylyl cyclase (AC) activity were evaluated in 12 nonfunctioning pituitary adenomas. TRH, GnRH, and arginine vasopressin induced a marked [Ca2+]i rise in 10/12, 4/ 12, and 2/5 tumors, respectively. The transients induced by these peptides were due to both Ca2+ mobilization from the intracellular stores and Ca2+ influx from the extracellular medium. AC activity was evaluated in 10 adenomas; 1 nM vasoactive intestinal peptide induced a 2- to 6-fold stimulation of the
A
enzyme activity in all tumors, while neither GHRH nor CRH were effective. Moreover, in 5/10 tumors 1 MM SRIH reduced both AC activity and [Ca2+]i, while in 2/10 the peptide caused a significant rise in [Ca2+]i despite the AC inhibition and in 3/10 SRIH did not modify either AC activity or [Ca2+]i. This study indicates that in nonfunctioning pituitary adenomas a wide spectrum of hypothalamic peptides modulate [Ca2+]i and AC activity. Moreover, the presence of biologically active receptors may offer a possible target for therapeutic intervention. (J Clin Endocrinol Metab 73: 913-918, 1991)
BOUT a quarter of human pituitary tumors are not associated with clinical or biochemical evidence of hypersecretion of known pituitary hormones. Although they are not a unique biological entity, as a whole they are currently defined nonfunctioning, nonsecreting, silent, or chromophobe adenomas (1-6). Recently, several studies have been undertaken in order to investigate the secretory potential of these tumors. In vitro studies including immunocytochemistry, cell culture, messenger RNA analysis have demonstrated that these tumors frequently contain and release pituitary hormones, particularly gonadotropins and/or free a-subunit of pituitary glycoprotein hormones («-SU) (7-10), despite the fact that they do not sustain in vivo hormone hypersecretion, with the exception of a minority of cases causing high levels of circulating a-SU (2-6). Receptors for hypothalamic peptides have been iden-
tified in nonfunctioning adenomas. In particular, high affinity TRH (11) and dopamine (12) binding sites have been found in the majority of nonfunctioning tumors, while SRIH receptors have been identified in a variable proportion of cases by either in vitro binding studies or autoradiographic visualization (13, 14). However, the possibility that receptor occupancy may activate cellular responses has been, so far, poorly investigated due to the low secretory activity of these tumors. Therefore, we assessed the presence of biologically active receptors in nonfunctioning adenomas by examining the generation of intracellular effectors induced by hypothalamic peptides. In this study, we report evidence of activation of the second messenger systems, in particular cytosolic free calcium and adenylyl cyclase activity, triggered by hypothalamic peptides in these tumors.
Received January 16,1991. Address all correspondence and requests for reprints to: Anna Spada, M.D., Institute of Endocrine Sciences, University of Milan, Ospedale Maggiore IRCCS, Padiglione Sacco, Via F. Sforza 35,1-20122-Milano, Italy. * This work was supported in part by the Consiglio Nazionale delle Ricerche (Grant 89.03407.14.115.04912) and Ministero Pubblica Istruzione (Rome, Italy).
Patients and adenomas
Materials and Methods Twelve patients affected with nonfunctioning pituitary adenomas (four men and eight women; aged 35-68 yr) and requiring adenomectomy were included in this study. Before surgery, no patient had elevated serum GH, PRL, ACTH, LH, FSH, TSH, and «-SU levels as determined by specific RIA, immu913
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noradiometric assay (IRMA) or time-resolved immunofluorimetric assays. No patient had previously undergone pituitary irradiation or medical treatment including drugs known to modify pituitary function. The adenomas were surgically removed by the transphenoidal route. For electron microscopy, small tissue fragments were fixed, postfixed, and embedded in Epon 812 as previously described (15). Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Philips CM10 electron microscope (Philips Industries, Eindhoven, The Netherlands). Based on electron microscopy, five adenomas were null cell adenomas and seven were oncocytomas. The presence of pituitary hormones was assessed by the use of specific antibodies and protein A-gold immunotechnique (15). Briefly, ultrathin sections were mounted on nickel grids with Formvar and treated with ethanol saturated with NaOH and then with 10% H 2 0 2 . The antisera used were an anti-hLH/3-serum, an anti-hTSH/3 serum, an anti-hFSH/3-serum (from NIDDK National Hormone and Pituitary Program), an anti-hLH a-serum, an anti-hACTH serum (from UCB, Brussels, Belgium), an antihPRL serum (gift of Dr. H. Friesen, Winnipeg, Manitoba, Canada, purified by affinity chromatography), and an antihGH serum developed and purified in our laboratory (15). Incubations were carried out at room temperature for 2 h (antisera) and 60 min (protein A-gold complexes). The cell composition of the adenomas was determined by examining 200-300 cells in sections from three random tissue blocks of each adenoma. No tumor showed cells immunoreactive for GH, PRL, or ACTH, while tumors 2 and 5 were positive for all glycoprotein subunits and tumor 1 for a-SU, LH/3, and TSH/3. Measurement of cytosolic free Ca2+ concentrations ([Ca2+Ji)
Cells were enzymatically dispersed as previously described (16). After 24 h in Dulbecco's modified Eagle's medium, cells were resuspended at 4-5 x 106 cells/mL in Krebs-Ringer Hepes incubation medium (KRH) that contained: 125 mM NaCl, 5 mM KC1,1.2 mM KH2PO4,1.2 mM MgSO4, 2 mM CaCl2, 25 mM HEPES-NaOH (pH 7.4), and 6 mM glucose. Cells were loaded with Ca2+ indicator fura-2 by incubating the cells with 5 nM fura-2-acetoxymethylester for 30 min at 37 C. Thereafter the cell suspensions were diluted 5-fold with warm KRH, washed, and resuspended in 1.5 mL KRH. Fluorescence recordings were carried out with a cell concentration of 3-4 X 105/mL in a Perkin-Elmer LS5 spectrofluorimeter (Perkin Elmer, Norwalk, CT) at 345 nm excitation and 490 nm emission, with slits of 5 and 10 nm, respectively. Cytosolic free Ca2+ concentration ([Ca2+]i) was calculated according to Grynkiewicz et al. (17). All values were corrected for changes in cellular autofluorescence (18). Adenylyl cyclase (AC) assay AC assay was carried out as previously described on crude membrane preparations sedimented from tumor homogenates by centrifugation at 20,000 X g for 10 min (19). The assay mixture contained 25 mM Tris-HCl (pH 7.4), 10 mM theophylline, 1 mM cAMP, 0.2 mM EGTA, 0.15 mM [8-14C]ATP (40 dpm/pmol), 7 mM phosphocreatine, and creatinphospho-kinase
JCE & M • 1991 Vol 73 • No 4
(20 U/mL). The reaction was initiated by the addition of membranes (0.5 mg protein/mL) and incubated at 30 C for 8 min. Isolation and estimation of the amount of [8-14C]cAMP formed were as previously described (19). Statistical analyses The results are expressed as means ± SD. Unpaired two tailed Student's t test was used to detect the significance of the differences between two means. A value of P < 0.05 was accepted as statistically significant. Materials ATP, cAMP, creatine phosphate, creatin phosphokinase, guanosine triphosphate (GTP), EGTA, trypsin, soybean trypsin inhibitor, TRH, GnRH, arginine vasopressin (AVP), SRIH, CRH, GHRH, vasoactive intestinal peptide (VIP), and verapamil were obtained through Sigma (St. Louis, MO). Fura-2AM was purchased from Molecular Probes (Junction City, OR). Culture media were purchased from Flow Laboratories (Mackenheim, West Germany); all other chemicals were reagent grade.
Results [Ca2+]i modulation by hypothalamic peptides Resting [Ca2+]i levels of tumoral cells were variable, ranging from 69 to 282 nM (132 ± 32 nM, n = 54). TRH, GnRH, and AVP were effective in elevating [Ca2+]i in a variable proportion of nonfunctioning adenomas. The addition of TRH (100 nM) caused a marked [Ca2+]i rise in 10/12 tumors, while GnRH (100 nM) was effective in 4/12 (Fig. 1). The stimulatory effects induced by TRH and GnRH were detected at concentrations higher than 1 nM and the maximal effect occurred at 100 nM. The effects of AVP (100 nM) on [Ca2+]i were evaluated in five tumors (3-6,8). A significant rise in [Ca2+]i was observed only in two adenomas (from 119-196 nM in tumor 3 and from 165-465 in tumor 4) while in the remaining tumors AVP was completely ineffective. Both TRH and GnRH caused biphasic transients composed by a rapid increase, followed within few seconds by a decrease to a lower plateau that was maintained for many minutes (Fig. 2). When the peptides were applied in Ca2+-free medium, the initial peak was maintained in part, suggesting that this component of [Ca2+]i transients was due to Ca2+ mobilization from the intracellular stores. When verapamil was applied after the peptide addition, the plateau phase was rapidly interrupted indicating that this component of [Ca2+]i transients was due to Ca2+ influx from the extracellular medium (Fig. 2). Similar results were obtained after AVP addition (data not shown). In cell preparations obtained from three adenomas (2,6, 7) both GnRH and TRH induced [Ca2+]i rises. When the two peptides were added one after the other to the cells,
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915
PEPTIDES IN NONFUNCTIONING PITUITARY ADENOMAS 8OO-1
D
Basal
•
TRH
600*
[Ca2+]i,nM TRH TRH
485
I
290
7 400 M (0
EGTA
O 200-
201
158 GnRH
1
2
3
4
5
6
7
8
9101112
800
D Basal
•
GnRH
600-
400 i O
200-
I
.kdi 1
2
3
lard
1 4
5 6 7 8 9 adenomas (n.)
10 11 12
FIG. 1. Effect of TRH (0.1 nM) {upper part) and GnRH (0.1 fiM) (lower part) on [Ca2+]i in cell suspensions obtained from 12 human nonfunctioning pituitary adenomas. Values given are means ± SD of three determinations. *,P< 0.01.
[Ca2+]i values showed a progressive increase, although the response to the second agent was in part attenuated (Fig. 3). In the presence of agents able to either chelate extracellular Ca2+ (3 mM EGTA) or block voltage-dependent channels (verapamil), [Ca2+]i rise occurred only after the addition of the first peptide, while the second one was completely ineffective, thus indicating that in these cells both TRH and GnRH increase [Ca2+]i by mobilizing the same intracellular pool (Fig. 3). SRIH caused a wide spectrum of responses. In fact, SRIH lowered resting [Ca2+]i in cells obtained from three adenomas (tumors 1, 3, and 9, Table 1 and Fig. 4) while in two tumors (tumors 7 and 10) the inhibitory effect was only detected on [Ca2+]i increases induced by either GnRH or TRH (Fig. 4 and data not shown). The lowering of either resting or stimulated [Ca2+]i caused by SRIH in these five adenomas was completely abolished when Ca2+ influx was prevented by either EGTA in excess or verapamil (data not shown). As shown in Table 1, in two adenomas (cases 5 and 12, Fig. 4) SRIH caused a significant increase in [Ca2+]i that was essentially due to intracellular Ca2+ mobilization, whereas in three adenomas (tumors 6, 8, and 11) SRIH did not modify either resting or stimulated [Ca2+]i.
228
167
«*r
1 min.
FIG. 2. Effects of different peptides on [Ca2+]i in cells obtained from tumor 12 (TRH-induced transients, upper part) and tumor 4 (GnRHand AVP-induced transients, central and lower parts). Two to 4 X 106 fura 2-loaded cells were inserted into a thermostatically controlled cuvette and the suspensions were maintained under continuous stirring. Agents were added to the cell suspensions in the sequence showed in the tracings at the following final concentrations: TRH, 0.1 MM; verapamil (Vp), 10 MM; EGTA, 3 mM; GnRH, 0.1 MM; AVP, 10 nM. The arrows correspond to the addition of the drugs.
Modulation of AC activity by hypothalamic peptides Basal and stimulated AC activity was evaluated in membrane preparations obtained from 10 nonfunctioning adenomas. As shown in Fig. 5, neither GHRH (1 ^M) nor CRH (1 ixM) were effective in stimulating AC activity while VIP (1 JUM) induced a marked increase of the enzyme activity in all tumors. The dose-response curves obtained in one adenoma showed that GHRH and CRH were ineffective at concentrations as high as 10 iiM, while the stimulation by VIP was already evident at 0.1 ^M, reaching the plateau at 1 /uM (Fig. 6). As shown in Table 1, SRIH (1 nu) caused a clear reduction of AC activity in membrane preparations obtained from 7 tumors, while it was ineffective in 3. The inhibitory effect induced by SRIH was detected at concentrations higher than 10 nM and the maximal effect occurred at 1 ^u.
Discussion The present study clearly indicates that hypothalamic peptides may generate intracellular effectors, particularly calcium and cAMP, in a significant number of human nonfunctioning pituitary adenomas. As far as the
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GnRH
390
I
EGTA
TRH
I
F
199
TRH
336
EGTA
I»
GnRH
I
I
158 .1 min,
FlG. 3. Effects of TRH (0.1 ^M) and GnRH (0.1 nM) on [Ca2+]i in cells obtained from tumor 7 in the absence and presence of EGTA (3 mM).
hypothalamic peptides known to activate receptors coupled to Ca2+ rise in normal pituitary cells are concerned, TRH was effective in increasing [Ca2+]i in the majority of tumors, while GnRH and AVP were effective in a lower proportion of tumors. As observed in normal and clonal cells (20-27), in cells obtained from nonfunctioning adenomas [Ca2+]i elevations induced by TRH, GnRH, and AVP were found to have a dual origin, mobilization from intracellular stores, and influx from
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the extracellular spaces. Taking into account that the increase in intracellular Ca2+ mobilization is due to inositol 1,4,5-trisphosphate formation during receptor activation (22, 24, 28, 29), the observation that TRH and GnRH increase intracellular Ca2+ mobilization in nonfunctioning pituitary adenomas well correlates with the findings that these two peptides increase inositol phospholipid turnover in a good proportion of these tumors (30). Although Ca2+ values obtained in cell suspensions represent the average over a large number of cells that may be heterogeneous, some experimental data seem to indicate that the responsiveness to two peptides observed in some nonfunctioning tumors is attributable to the coexistence of receptors for both agents in a good proportion of cells. In fact, when Ca2+ influx was prevented by EGTA or verapamil, it was clear that the different peptides use the same intracellular store to raise [Ca2+] i. These data suggest that tumoral cells possess receptors for multiple agents; each peptide increases both Ca2+ influx by activating specific receptor-operating channels (and this phenomenon may account for the increase in [Ca2+]i after each agent in the presence of extracellular Ca2+) and intracellular Ca2+ redistribution by mobilizing the same intracellular Ca2+ pool (and this may account for the [Ca2+]i rise after the first peptide and the lack of effect after the second one in the absence of extracellular Ca2+). As far as the hypothalamic peptides that activate receptors coupled to AC stimulation in the normal pituitary are concerned, VIP caused a marked increase of AC activity in all tumors. The stimulation of cAMP production induced by VIP in nonfunctioning tumors was similar to that observed in the normal counterpart, i.e. lactotrophs, and it occurred at a similar range of peptide concentrations (31). By contrast, the other hypothalamic hormones known to activate AC activity in pituitary
TABLE 1. Effects of SRIH on AC activity and cytosolic-free Ca2+ concentrations in human nonfunctioning pituitary adenomas [Ca2+]i, nM
AC (pmol cAMP/mg prot x min) Adenoma Basal 1 3 9 7 10 5 12 6 8 11
44.5 ± 30.2 ± 18.5 ± 53.2 ± 30.4 ± 32.0 ± 28.0 ± 24.6 ± 29.5 ± 18.2 ±
1.2 0.5 1.5 2.5 2.2 1.5 1.7 1.2 2 1.2
SRIH
% variation
Basal
SRIH
% variation
30.8 ± 2.6 23.4 ± 0.5 10.4 ± 1.6 39.2 ± 2.0 20.2 ± 1.4 25.2 ± 1.2 21.0 ±1.1 25.0 ± 0.8 26.0 ± 1.5 16.6 ± 0.9
-31° -22C -44° -26C -33° -21° -25 6 +1 -11 -9
105 ± 11 128 ± 13 281 ± 13 145 ± 12 194 ± 15 82 ±14 151 ± 11 95 ± 8 103 ± 10 143 ± 12
75 ± 9 79 ± 10 189 ± 18 140 ± 11 177 ± 13 128 ± 10 228 ± 24 100 ± 11 94 ± 9 143 ± 16
-28 6 -38 6 -33* -4 -9 +45° +34* +5 -9 0
" P < 0.01 us. basal. * P < 0.05 us. basal. c P < 0.005 us. basal.
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PEPTIDES IN NONFUNCTIONING PITUITARY ADENOMAS Ca2+]i,nM
917
2001
SRIH
150-
100-
E 50 i GnRH
SRIH
654
0.1
SRIH
10
314 205
143 1 min. 2+
FIG. 4. Effect of SRIH on [Ca ]i in cells obtained from tumor 9 {upperpart, left), tumor 5 (upperpart, right), tumor 7 (lower part, left), and tumor 11 (lower part, right). The final concentrations were; SRIH, 1 nu; GnRH, 0.1 ^M.
•
~ 200 T
basal GHRH
m CRH m VIP
o Q.
cn | "o
I 100-
1 2
3
4 5 6 7 adenomas (n.)
8
10
FIG. 5. Effects of GHRH (1 MM), CRH (1 pM), and VIP (1 /IM) on adenylyl cyclase activity in membrane preparations obtained from 10 nonfunctioning adenomas. Values given are the means of three determinations; SD values were less than 5%.
cells—CRH and GHRH (26,32)—did not modify enzyme activity in any tumor studied. In agreement with the previous identification of SRIH receptors in a good proportion of nonfunctioning tumors (13,14), SRIH induced important modifications of intracellular signals in the majority of the adenomas of the present series. As it has been reported to occur in several cell types (33, 34), SRIH reduced both cAMP accumulation, via inhibition of membrane AC activity, and resting and/or stimulated [Ca2+]i, via reduction of Ca2+ influx in a subset of nonfunctioning adenomas. In a minority of tumors, SRIH triggered a different pattern of intracellular events; inhibition of AC activity and increase in intracellular Ca2+ mobilization. The different action of SRIH might be due to the appearance of different SRIH receptor subtypes, coupled to the stimulation
FIG. 6. Effect of increasing concentrations of VIP, GHRH, and CRH on adenylyl cyclase activity on membrane preparations obtained from tumor 3. Values given are the means ± SD of three determinations.
of Ca2+ mobilization, during tumoral transformation. Alternatively, modifications might occur at postreceptor level; in particular, the quantity and quality of G proteins available to receptor activation might be different in tumoral cells. On this line, it is worth noting that dopaminergic D2 receptors expressed in fibroblasts are coupled to the stimulation of Ca2+ mobilization whereas the same receptors expressed in GH cell lines are coupled to [Ca2+]i decrease (35). The finding that, in some nonfunctioning pituitary adenomas, modifications at receptor and/or postreceptor step may give stimulatory properties to SRIH signal may have important implications in view of therapeutical trials with SRIH analogs proposed by several authors (for review see Ref. 36). Taken together these data are consistent with the idea that nonfuctioning pituitary adenomas are not a unique biological entity; in fact, these tumors differ not only in the morphological characteristics and biosynthetic capacity but also in the responsiveness to hypothalamic peptides. Moreover, the finding that hypothalamic peptides interact with biologically active receptors leading to the generation of intracellular effectors may be of interest considering that calcium and cAMP are involved in the control of multiple cell functions, not exclusively related to hormone secretion, such as replication and differentiation (37, 38). Finally, the presence of multiple functioning receptors may offer a possible target for therapeutic intervention in nonfunctioning pituitary adenomas, either by the use of appropriate agonists or antagonists, or by using the ligands as carriers to convey therapeutically useful amounts of radiation or toxins to the tumor (39). Acknowledgments We are indebted to National Hormone and Pituitary Program (NIDDK, Baltimore, MD) for the gift of pituitary glycoprotein hormone antibodies. We are grateful to Dr. M. Giovanelli (Department of Neurosurgery, University of Milan, Italy) and Dr. G. Nicola (Department
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of Neurosurgery, General Hospital of Legnano, Italy) for supplying pituitary adenomas.
References 1. Crichton M, Christy NP, Damon A. Host factors in "cromophobe" adenoma of the anterior pituitary: a retrospective study of 464 patients. Metabolism. 1981;30:248-67. 2. MacFarlane IA, Beardwell CG, Shalet SM, Ainslie G, Rankin E. Glycoprotein hormone a-subunit secretion in patients with pituitary adenomas: influence of TRH, LRH and bromocriptine. Acta Endocrinol (Copenh). 1982;99:487-92. 3. Ridgway EC, Klibanski A, Ladenson PW, et al. Pure alpha-secreting pituitary adenomas. N Engl J Med. 1981;304:1254-9. 4. Ridway EC. Glycoprotein hormone production by pituitary tumors. In: Black PMcL, ed. Secretory tumors of the pituitary gland. New York: Raven Press; 1984:343-63. 5. Snyder PJ. Gonadotroph cell adenomas of the pituitary. Endocr Rev. 1985;6:552-63. 6. Beck-Peccoz P, Persani L, Medri G, Iglesias Guerrero M, Spada A, Faglia G. New aspects in non functioning pituitary tumors. In: Casanueva FF, Dieguez C, eds. Recent advances in basic and clinical neuroendocrinology. International Congress Series 864. Amsterdam: Excerpta Medica; 1989:295-302. 7. Surmount DWA, Winslow CLJ, Loizou M, White MC, Adams EF, Mashiter K. Gonadotropin and alpha subunit secretion by human functionless pituitary adenomas in cell culture; long term effects of luteinizing hormone releasing hormone and thyrotrophin releasing hormone. Clin Endocrinol (Oxf). 1983;19:325-36. 8. Asa SL, Gerrie BM, Singer W, Horvath E, Kovacs K, Smyth HS. Gonadotropin secretion in vitro by human pituitary null cell adenomas and oncocytomas. J Clin Endocrinol Metab. 1986;62:1()1119. 9. Yamada S, Asa SL, Kovacs K, Muller P, Smyth HS. Analysis of hormone secretion by clinically nonfunctioning human pituitary adenomas using the reverse hemolytic plaque assay. J Clin Endocrinol Metab. 1989;68:73-80. 10. Kwekkeboom DJ, De Jong FH, Lamberts SWJ. Gonadotropin release by clinically nonfunctioning and gonadotroph pituitary adenomas in vivo and in vitro; relation to sex and effects of thyrotropin-releasing hormone, gonadotropin-releasing hormone and bromocriptine. J Clin Endocrinol Metab. 1989;68:1128-35. 11. Le Dafniet M, Grouselle D, Li JY, et al. Evidence of thyrotropinreleasing hormone (TRH) and TRH-binding sites in human nonsecreting pituitary adenomas. J Clin Endocrinol Metab. 1987;65:1014-19. 12. Bevan JS, Burke CW. Non-functioning pituitary adenomas do not regress during bromocroptine therapy but possess membranebound dopamine receptors which bind bromocriptine. Clin Endocrinol (Oxf). 1986;25:561-72. 13. Ikuyama S, Nawata H, Kato K, Karashima T, Ibayashi H, Nagkagaki H. Specific somatostatin receptors on human pituitary adenoma cell membranes. J Clin Endocrinol Metab. 1985;61:66671. 14. Reubi JC, Heitz PU, Landolt AM. Visualization of somatostatin receptors and correlation with immunoreactive growth hormone and prolactin in human pituitary adenomas: evidence for different tumor subclasses. J Clin Endocrinol Metab. 1987;65:65-73. 15. Bassetti M, Spada A, Arosio M, Vallar L, Brina M, Giannattasio G. Morphological studies on mixed growth hormone (GH)- and prolactin (PRL)-secreting human pituitary adenomas. Coexistence of GH and PRL in the same secretory granule. J Clin Endocrinol Metab. 1986;62:1093-100. 16. Spada A, Sartorio A, Bassetti M, Pezzo G, Giannattasio G. In vitro effect of dopamine on growth hormone (GH) release from human GH-secreting pituitary adenomas. J Clin Endocrinol Metab. 1982;55:734-40. 17. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985;260:3440-50. 18. Pandiella A, Reza-Elahi F, Vallar L, Spada A. Alpha 1 adrenergic stimulation of in vitro growth hormone release and cytosolic free
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Ca2+ in rat somatotrophs. Endocrinology. 1988;122:1419-25. 19. Spada A, Nicosia S, Cortelazzi L, et al. In vitro studies on prolactin relaese and adenylate cyclase activity in human prolactin-secreting pituitary adenomas. Different sensitivity of macro- and microadenomas to dopamine and vasoactive intestinal peptide. J Clin Endocrinol Metab. 1983;56:1-1O. 20. Malgaroli A, Vallar L, Reza-Elahi F, Pozzan T, Spada A, Meldolesi J. Dopamine inhibits cytosolic Ca2+ increases in rat lactotroph cells. Evidence of a dual mechanism of action. J Biol Chem. 1987;262:13920-7. 21. Gershengorn MC, Thaw C. Calcium influx is not required for TRH to elevate free cytoplasmic calcium in GH3 cells. Endocrinology. 1983;113:1522-4. 22. Ramsdell J, Tashjian Jr AH. Thyrotropin-releasing hormone (TRH) elevation of inositol trisphosphate and cytosolic free calcium is dependent on receptor number. J Biol Chem. 1986;261:5301-6. 23. Naor Z, Eli Y. Synergistic stimulation of luteinizing hormone release by protein kinase C activators and Ca2+ ionophores. Biochem Biophys Res Commun. 1985; 130:848-53. 24. Hirota K, Hirota T, Aguilera G, Catt KJ. Hormone induced redistribution of calcium activated phospholipid dependent protein kinase in pituitary gonadotrophs. J Biol Chem. 1985;260:3243-6. 25. Limor R, Ayalon D, Capponi AM, Childs GV, Naor Z. Cytosolic free calcium levels in cultured pituitary cells separated by centrifugal elutriation: effect of gonadotropin-releasing hormone. Endocrinology. 1987;120:497-503. 26. Antoni FA. Hypothalamic control of adrenocorticotropin secretion: advances since the discovery of 41-residue corticotropin-releasing factor. Endocr Rev. 1986; 7:351-78. 27. Raymond V, Leung PCK, Veilleux R, Labrie F. Vasopressin rapidly stimulates phosphatidic acid-phosphatidyl inositol turnover in rat anterior pituitary cells. FEBS Lett. 1985; 182:196-200. 28. Rebecchi MJ, Gershenghom MC. Thyroliberin stimulates rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate by a phosphodiesterase in rat mammotropic pituitary cells. Biochem J. 1983;216:287-94. 29. Berridge MJ, Irvine RF. Inositol trisphosphate, a novel second messenger in cellular signal trasduction. Nature. 1984;312:315-21. 30. Levy A, Lightman S. Effects of thyrotropin-releasing hormone and gonadotropin-releasing hormone on inositol phospholipid turnover in endocrinologically inactive pituitary adenomas and prolactinomas. J Clin Endocrinol Metab. 1989;69:122-6. 31. Borghi C, Nicosia S, Giachetti A, Said SI. Adenylate cyclase of rat pituitary gland. Stimulation by vasoactive intestinal polypeptide (VIP). FEBS Lett. 1979;108:403-7. 32. Bilezikjian LM, Vale W. Stimulation of adenosine 3', 5'-monophosphate production by growth hormone-releasing factor and its inhibition by somatostatin in anterior pituitary cells in vitro. Endocrinology. 1983;113:1726-34. 33. Schlegel W, Wuarin F, Wollhein CB, Zahnd G. Somatostatin lowers the cytosolic free Ca2+ concentration in clonal rat pituitary cells (GH3 cells). Cell Calcium. 1984;5:223-36. 34. Reisine T. Multiple mechanisms of somatostatin inhibition of adrenocorticotropin release from mouse anterior pituitary tumor cells. Endocrinology. l985;116:2249-66. 35. Vallar L, Muca C, Magni M, et al. Differential coupling of dopaminergic D2 receptors expressed in different cell types: stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis in Ltk-fibroblasts, hyperpolarization and cytosolic free Ca2+ concentration decrease in GH4C1 cells. J Biol Chem. 1990;265:10320-6. 36. Lamberts SWJ. The role of somatostatin in the regulation of anterior pituitary hormone secretion and the use of its analogs in the treatment of human pituitary tumors. Endocr Rev. 1988;9:41736. 37. Billestrup AM, Swanson LW, Vale W. Growth hormone-releasing factor stimulates cell proliferation of somatotrophs in vitro. Proc Natl Acad Sci USA. 1983;83:6854-7. 38. Billestrup N, Mitchell RL, Vale W, Verma IM. Growth hormonereleasing factor induces c-fos expression in cultured primary pituitary cells. Mol Endocrinol. 1987;l:300-5. 39. Reichlin S. Editorial: clinical application of somatostatin receptor imaging. J Clin Endocrinol Metab. 1990;71:564-5.
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Hypothalamic Peptides Modulate Cytosolic Free Ca2+ Levels and Adenylyl Cyclase Activity in Human Nonfunctioning Pituitary Adenomas* ANNA SPADA, FARZIN REZA-ELAHI, ANDREA LANIA, PALOMA GIL-DELALAMO, MONIQUE BASSETTI, AND GIOVANNI FAGLIA Institute of Endocrine Sciences, Ospedale Maggiore IRCCS (A.S., F.R-E., A.L., P.G.d.A., F.G.) and CNR Center of Cytopharmacology, Department of Pharmacology (M.B.), University of Milan, and Italian Auxological Center (F.R-E.), Milan, Italy
ABSTRACT. The effects of hypothalamic peptides (TRH, GnRH, arginine vasopressin, vasoactive intestinal peptide, GHRH, CRH, and SRIH) on cytosolic free calcium concentrations ([Ca2+]i) and adenylyl cyclase (AC) activity were evaluated in 12 nonfunctioning pituitary adenomas. TRH, GnRH, and arginine vasopressin induced a marked [Ca2+]i rise in 10/12, 4/ 12, and 2/5 tumors, respectively. The transients induced by these peptides were due to both Ca2+ mobilization from the intracellular stores and Ca2+ influx from the extracellular medium. AC activity was evaluated in 10 adenomas; 1 nM vasoactive intestinal peptide induced a 2- to 6-fold stimulation of the
A
enzyme activity in all tumors, while neither GHRH nor CRH were effective. Moreover, in 5/10 tumors 1 MM SRIH reduced both AC activity and [Ca2+]i, while in 2/10 the peptide caused a significant rise in [Ca2+]i despite the AC inhibition and in 3/10 SRIH did not modify either AC activity or [Ca2+]i. This study indicates that in nonfunctioning pituitary adenomas a wide spectrum of hypothalamic peptides modulate [Ca2+]i and AC activity. Moreover, the presence of biologically active receptors may offer a possible target for therapeutic intervention. (J Clin Endocrinol Metab 73: 913-918, 1991)
BOUT a quarter of human pituitary tumors are not associated with clinical or biochemical evidence of hypersecretion of known pituitary hormones. Although they are not a unique biological entity, as a whole they are currently defined nonfunctioning, nonsecreting, silent, or chromophobe adenomas (1-6). Recently, several studies have been undertaken in order to investigate the secretory potential of these tumors. In vitro studies including immunocytochemistry, cell culture, messenger RNA analysis have demonstrated that these tumors frequently contain and release pituitary hormones, particularly gonadotropins and/or free a-subunit of pituitary glycoprotein hormones («-SU) (7-10), despite the fact that they do not sustain in vivo hormone hypersecretion, with the exception of a minority of cases causing high levels of circulating a-SU (2-6). Receptors for hypothalamic peptides have been iden-
tified in nonfunctioning adenomas. In particular, high affinity TRH (11) and dopamine (12) binding sites have been found in the majority of nonfunctioning tumors, while SRIH receptors have been identified in a variable proportion of cases by either in vitro binding studies or autoradiographic visualization (13, 14). However, the possibility that receptor occupancy may activate cellular responses has been, so far, poorly investigated due to the low secretory activity of these tumors. Therefore, we assessed the presence of biologically active receptors in nonfunctioning adenomas by examining the generation of intracellular effectors induced by hypothalamic peptides. In this study, we report evidence of activation of the second messenger systems, in particular cytosolic free calcium and adenylyl cyclase activity, triggered by hypothalamic peptides in these tumors.
Received January 16,1991. Address all correspondence and requests for reprints to: Anna Spada, M.D., Institute of Endocrine Sciences, University of Milan, Ospedale Maggiore IRCCS, Padiglione Sacco, Via F. Sforza 35,1-20122-Milano, Italy. * This work was supported in part by the Consiglio Nazionale delle Ricerche (Grant 89.03407.14.115.04912) and Ministero Pubblica Istruzione (Rome, Italy).
Patients and adenomas
Materials and Methods Twelve patients affected with nonfunctioning pituitary adenomas (four men and eight women; aged 35-68 yr) and requiring adenomectomy were included in this study. Before surgery, no patient had elevated serum GH, PRL, ACTH, LH, FSH, TSH, and «-SU levels as determined by specific RIA, immu913
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SPADA ET AL.
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noradiometric assay (IRMA) or time-resolved immunofluorimetric assays. No patient had previously undergone pituitary irradiation or medical treatment including drugs known to modify pituitary function. The adenomas were surgically removed by the transphenoidal route. For electron microscopy, small tissue fragments were fixed, postfixed, and embedded in Epon 812 as previously described (15). Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Philips CM10 electron microscope (Philips Industries, Eindhoven, The Netherlands). Based on electron microscopy, five adenomas were null cell adenomas and seven were oncocytomas. The presence of pituitary hormones was assessed by the use of specific antibodies and protein A-gold immunotechnique (15). Briefly, ultrathin sections were mounted on nickel grids with Formvar and treated with ethanol saturated with NaOH and then with 10% H 2 0 2 . The antisera used were an anti-hLH/3-serum, an anti-hTSH/3 serum, an anti-hFSH/3-serum (from NIDDK National Hormone and Pituitary Program), an anti-hLH a-serum, an anti-hACTH serum (from UCB, Brussels, Belgium), an antihPRL serum (gift of Dr. H. Friesen, Winnipeg, Manitoba, Canada, purified by affinity chromatography), and an antihGH serum developed and purified in our laboratory (15). Incubations were carried out at room temperature for 2 h (antisera) and 60 min (protein A-gold complexes). The cell composition of the adenomas was determined by examining 200-300 cells in sections from three random tissue blocks of each adenoma. No tumor showed cells immunoreactive for GH, PRL, or ACTH, while tumors 2 and 5 were positive for all glycoprotein subunits and tumor 1 for a-SU, LH/3, and TSH/3. Measurement of cytosolic free Ca2+ concentrations ([Ca2+Ji)
Cells were enzymatically dispersed as previously described (16). After 24 h in Dulbecco's modified Eagle's medium, cells were resuspended at 4-5 x 106 cells/mL in Krebs-Ringer Hepes incubation medium (KRH) that contained: 125 mM NaCl, 5 mM KC1,1.2 mM KH2PO4,1.2 mM MgSO4, 2 mM CaCl2, 25 mM HEPES-NaOH (pH 7.4), and 6 mM glucose. Cells were loaded with Ca2+ indicator fura-2 by incubating the cells with 5 nM fura-2-acetoxymethylester for 30 min at 37 C. Thereafter the cell suspensions were diluted 5-fold with warm KRH, washed, and resuspended in 1.5 mL KRH. Fluorescence recordings were carried out with a cell concentration of 3-4 X 105/mL in a Perkin-Elmer LS5 spectrofluorimeter (Perkin Elmer, Norwalk, CT) at 345 nm excitation and 490 nm emission, with slits of 5 and 10 nm, respectively. Cytosolic free Ca2+ concentration ([Ca2+]i) was calculated according to Grynkiewicz et al. (17). All values were corrected for changes in cellular autofluorescence (18). Adenylyl cyclase (AC) assay AC assay was carried out as previously described on crude membrane preparations sedimented from tumor homogenates by centrifugation at 20,000 X g for 10 min (19). The assay mixture contained 25 mM Tris-HCl (pH 7.4), 10 mM theophylline, 1 mM cAMP, 0.2 mM EGTA, 0.15 mM [8-14C]ATP (40 dpm/pmol), 7 mM phosphocreatine, and creatinphospho-kinase
JCE & M • 1991 Vol 73 • No 4
(20 U/mL). The reaction was initiated by the addition of membranes (0.5 mg protein/mL) and incubated at 30 C for 8 min. Isolation and estimation of the amount of [8-14C]cAMP formed were as previously described (19). Statistical analyses The results are expressed as means ± SD. Unpaired two tailed Student's t test was used to detect the significance of the differences between two means. A value of P < 0.05 was accepted as statistically significant. Materials ATP, cAMP, creatine phosphate, creatin phosphokinase, guanosine triphosphate (GTP), EGTA, trypsin, soybean trypsin inhibitor, TRH, GnRH, arginine vasopressin (AVP), SRIH, CRH, GHRH, vasoactive intestinal peptide (VIP), and verapamil were obtained through Sigma (St. Louis, MO). Fura-2AM was purchased from Molecular Probes (Junction City, OR). Culture media were purchased from Flow Laboratories (Mackenheim, West Germany); all other chemicals were reagent grade.
Results [Ca2+]i modulation by hypothalamic peptides Resting [Ca2+]i levels of tumoral cells were variable, ranging from 69 to 282 nM (132 ± 32 nM, n = 54). TRH, GnRH, and AVP were effective in elevating [Ca2+]i in a variable proportion of nonfunctioning adenomas. The addition of TRH (100 nM) caused a marked [Ca2+]i rise in 10/12 tumors, while GnRH (100 nM) was effective in 4/12 (Fig. 1). The stimulatory effects induced by TRH and GnRH were detected at concentrations higher than 1 nM and the maximal effect occurred at 100 nM. The effects of AVP (100 nM) on [Ca2+]i were evaluated in five tumors (3-6,8). A significant rise in [Ca2+]i was observed only in two adenomas (from 119-196 nM in tumor 3 and from 165-465 in tumor 4) while in the remaining tumors AVP was completely ineffective. Both TRH and GnRH caused biphasic transients composed by a rapid increase, followed within few seconds by a decrease to a lower plateau that was maintained for many minutes (Fig. 2). When the peptides were applied in Ca2+-free medium, the initial peak was maintained in part, suggesting that this component of [Ca2+]i transients was due to Ca2+ mobilization from the intracellular stores. When verapamil was applied after the peptide addition, the plateau phase was rapidly interrupted indicating that this component of [Ca2+]i transients was due to Ca2+ influx from the extracellular medium (Fig. 2). Similar results were obtained after AVP addition (data not shown). In cell preparations obtained from three adenomas (2,6, 7) both GnRH and TRH induced [Ca2+]i rises. When the two peptides were added one after the other to the cells,
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915
PEPTIDES IN NONFUNCTIONING PITUITARY ADENOMAS 8OO-1
D
Basal
•
TRH
600*
[Ca2+]i,nM TRH TRH
485
I
290
7 400 M (0
EGTA
O 200-
201
158 GnRH
1
2
3
4
5
6
7
8
9101112
800
D Basal
•
GnRH
600-
400 i O
200-
I
.kdi 1
2
3
lard
1 4
5 6 7 8 9 adenomas (n.)
10 11 12
FIG. 1. Effect of TRH (0.1 nM) {upper part) and GnRH (0.1 fiM) (lower part) on [Ca2+]i in cell suspensions obtained from 12 human nonfunctioning pituitary adenomas. Values given are means ± SD of three determinations. *,P< 0.01.
[Ca2+]i values showed a progressive increase, although the response to the second agent was in part attenuated (Fig. 3). In the presence of agents able to either chelate extracellular Ca2+ (3 mM EGTA) or block voltage-dependent channels (verapamil), [Ca2+]i rise occurred only after the addition of the first peptide, while the second one was completely ineffective, thus indicating that in these cells both TRH and GnRH increase [Ca2+]i by mobilizing the same intracellular pool (Fig. 3). SRIH caused a wide spectrum of responses. In fact, SRIH lowered resting [Ca2+]i in cells obtained from three adenomas (tumors 1, 3, and 9, Table 1 and Fig. 4) while in two tumors (tumors 7 and 10) the inhibitory effect was only detected on [Ca2+]i increases induced by either GnRH or TRH (Fig. 4 and data not shown). The lowering of either resting or stimulated [Ca2+]i caused by SRIH in these five adenomas was completely abolished when Ca2+ influx was prevented by either EGTA in excess or verapamil (data not shown). As shown in Table 1, in two adenomas (cases 5 and 12, Fig. 4) SRIH caused a significant increase in [Ca2+]i that was essentially due to intracellular Ca2+ mobilization, whereas in three adenomas (tumors 6, 8, and 11) SRIH did not modify either resting or stimulated [Ca2+]i.
228
167
«*r
1 min.
FIG. 2. Effects of different peptides on [Ca2+]i in cells obtained from tumor 12 (TRH-induced transients, upper part) and tumor 4 (GnRHand AVP-induced transients, central and lower parts). Two to 4 X 106 fura 2-loaded cells were inserted into a thermostatically controlled cuvette and the suspensions were maintained under continuous stirring. Agents were added to the cell suspensions in the sequence showed in the tracings at the following final concentrations: TRH, 0.1 MM; verapamil (Vp), 10 MM; EGTA, 3 mM; GnRH, 0.1 MM; AVP, 10 nM. The arrows correspond to the addition of the drugs.
Modulation of AC activity by hypothalamic peptides Basal and stimulated AC activity was evaluated in membrane preparations obtained from 10 nonfunctioning adenomas. As shown in Fig. 5, neither GHRH (1 ^M) nor CRH (1 ixM) were effective in stimulating AC activity while VIP (1 JUM) induced a marked increase of the enzyme activity in all tumors. The dose-response curves obtained in one adenoma showed that GHRH and CRH were ineffective at concentrations as high as 10 iiM, while the stimulation by VIP was already evident at 0.1 ^M, reaching the plateau at 1 /uM (Fig. 6). As shown in Table 1, SRIH (1 nu) caused a clear reduction of AC activity in membrane preparations obtained from 7 tumors, while it was ineffective in 3. The inhibitory effect induced by SRIH was detected at concentrations higher than 10 nM and the maximal effect occurred at 1 ^u.
Discussion The present study clearly indicates that hypothalamic peptides may generate intracellular effectors, particularly calcium and cAMP, in a significant number of human nonfunctioning pituitary adenomas. As far as the
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GnRH
390
I
EGTA
TRH
I
F
199
TRH
336
EGTA
I»
GnRH
I
I
158 .1 min,
FlG. 3. Effects of TRH (0.1 ^M) and GnRH (0.1 nM) on [Ca2+]i in cells obtained from tumor 7 in the absence and presence of EGTA (3 mM).
hypothalamic peptides known to activate receptors coupled to Ca2+ rise in normal pituitary cells are concerned, TRH was effective in increasing [Ca2+]i in the majority of tumors, while GnRH and AVP were effective in a lower proportion of tumors. As observed in normal and clonal cells (20-27), in cells obtained from nonfunctioning adenomas [Ca2+]i elevations induced by TRH, GnRH, and AVP were found to have a dual origin, mobilization from intracellular stores, and influx from
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the extracellular spaces. Taking into account that the increase in intracellular Ca2+ mobilization is due to inositol 1,4,5-trisphosphate formation during receptor activation (22, 24, 28, 29), the observation that TRH and GnRH increase intracellular Ca2+ mobilization in nonfunctioning pituitary adenomas well correlates with the findings that these two peptides increase inositol phospholipid turnover in a good proportion of these tumors (30). Although Ca2+ values obtained in cell suspensions represent the average over a large number of cells that may be heterogeneous, some experimental data seem to indicate that the responsiveness to two peptides observed in some nonfunctioning tumors is attributable to the coexistence of receptors for both agents in a good proportion of cells. In fact, when Ca2+ influx was prevented by EGTA or verapamil, it was clear that the different peptides use the same intracellular store to raise [Ca2+] i. These data suggest that tumoral cells possess receptors for multiple agents; each peptide increases both Ca2+ influx by activating specific receptor-operating channels (and this phenomenon may account for the increase in [Ca2+]i after each agent in the presence of extracellular Ca2+) and intracellular Ca2+ redistribution by mobilizing the same intracellular Ca2+ pool (and this may account for the [Ca2+]i rise after the first peptide and the lack of effect after the second one in the absence of extracellular Ca2+). As far as the hypothalamic peptides that activate receptors coupled to AC stimulation in the normal pituitary are concerned, VIP caused a marked increase of AC activity in all tumors. The stimulation of cAMP production induced by VIP in nonfunctioning tumors was similar to that observed in the normal counterpart, i.e. lactotrophs, and it occurred at a similar range of peptide concentrations (31). By contrast, the other hypothalamic hormones known to activate AC activity in pituitary
TABLE 1. Effects of SRIH on AC activity and cytosolic-free Ca2+ concentrations in human nonfunctioning pituitary adenomas [Ca2+]i, nM
AC (pmol cAMP/mg prot x min) Adenoma Basal 1 3 9 7 10 5 12 6 8 11
44.5 ± 30.2 ± 18.5 ± 53.2 ± 30.4 ± 32.0 ± 28.0 ± 24.6 ± 29.5 ± 18.2 ±
1.2 0.5 1.5 2.5 2.2 1.5 1.7 1.2 2 1.2
SRIH
% variation
Basal
SRIH
% variation
30.8 ± 2.6 23.4 ± 0.5 10.4 ± 1.6 39.2 ± 2.0 20.2 ± 1.4 25.2 ± 1.2 21.0 ±1.1 25.0 ± 0.8 26.0 ± 1.5 16.6 ± 0.9
-31° -22C -44° -26C -33° -21° -25 6 +1 -11 -9
105 ± 11 128 ± 13 281 ± 13 145 ± 12 194 ± 15 82 ±14 151 ± 11 95 ± 8 103 ± 10 143 ± 12
75 ± 9 79 ± 10 189 ± 18 140 ± 11 177 ± 13 128 ± 10 228 ± 24 100 ± 11 94 ± 9 143 ± 16
-28 6 -38 6 -33* -4 -9 +45° +34* +5 -9 0
" P < 0.01 us. basal. * P < 0.05 us. basal. c P < 0.005 us. basal.
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PEPTIDES IN NONFUNCTIONING PITUITARY ADENOMAS Ca2+]i,nM
917
2001
SRIH
150-
100-
E 50 i GnRH
SRIH
654
0.1
SRIH
10
314 205
143 1 min. 2+
FIG. 4. Effect of SRIH on [Ca ]i in cells obtained from tumor 9 {upperpart, left), tumor 5 (upperpart, right), tumor 7 (lower part, left), and tumor 11 (lower part, right). The final concentrations were; SRIH, 1 nu; GnRH, 0.1 ^M.
•
~ 200 T
basal GHRH
m CRH m VIP
o Q.
cn | "o
I 100-
1 2
3
4 5 6 7 adenomas (n.)
8
10
FIG. 5. Effects of GHRH (1 MM), CRH (1 pM), and VIP (1 /IM) on adenylyl cyclase activity in membrane preparations obtained from 10 nonfunctioning adenomas. Values given are the means of three determinations; SD values were less than 5%.
cells—CRH and GHRH (26,32)—did not modify enzyme activity in any tumor studied. In agreement with the previous identification of SRIH receptors in a good proportion of nonfunctioning tumors (13,14), SRIH induced important modifications of intracellular signals in the majority of the adenomas of the present series. As it has been reported to occur in several cell types (33, 34), SRIH reduced both cAMP accumulation, via inhibition of membrane AC activity, and resting and/or stimulated [Ca2+]i, via reduction of Ca2+ influx in a subset of nonfunctioning adenomas. In a minority of tumors, SRIH triggered a different pattern of intracellular events; inhibition of AC activity and increase in intracellular Ca2+ mobilization. The different action of SRIH might be due to the appearance of different SRIH receptor subtypes, coupled to the stimulation
FIG. 6. Effect of increasing concentrations of VIP, GHRH, and CRH on adenylyl cyclase activity on membrane preparations obtained from tumor 3. Values given are the means ± SD of three determinations.
of Ca2+ mobilization, during tumoral transformation. Alternatively, modifications might occur at postreceptor level; in particular, the quantity and quality of G proteins available to receptor activation might be different in tumoral cells. On this line, it is worth noting that dopaminergic D2 receptors expressed in fibroblasts are coupled to the stimulation of Ca2+ mobilization whereas the same receptors expressed in GH cell lines are coupled to [Ca2+]i decrease (35). The finding that, in some nonfunctioning pituitary adenomas, modifications at receptor and/or postreceptor step may give stimulatory properties to SRIH signal may have important implications in view of therapeutical trials with SRIH analogs proposed by several authors (for review see Ref. 36). Taken together these data are consistent with the idea that nonfuctioning pituitary adenomas are not a unique biological entity; in fact, these tumors differ not only in the morphological characteristics and biosynthetic capacity but also in the responsiveness to hypothalamic peptides. Moreover, the finding that hypothalamic peptides interact with biologically active receptors leading to the generation of intracellular effectors may be of interest considering that calcium and cAMP are involved in the control of multiple cell functions, not exclusively related to hormone secretion, such as replication and differentiation (37, 38). Finally, the presence of multiple functioning receptors may offer a possible target for therapeutic intervention in nonfunctioning pituitary adenomas, either by the use of appropriate agonists or antagonists, or by using the ligands as carriers to convey therapeutically useful amounts of radiation or toxins to the tumor (39). Acknowledgments We are indebted to National Hormone and Pituitary Program (NIDDK, Baltimore, MD) for the gift of pituitary glycoprotein hormone antibodies. We are grateful to Dr. M. Giovanelli (Department of Neurosurgery, University of Milan, Italy) and Dr. G. Nicola (Department
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of Neurosurgery, General Hospital of Legnano, Italy) for supplying pituitary adenomas.
References 1. Crichton M, Christy NP, Damon A. Host factors in "cromophobe" adenoma of the anterior pituitary: a retrospective study of 464 patients. Metabolism. 1981;30:248-67. 2. MacFarlane IA, Beardwell CG, Shalet SM, Ainslie G, Rankin E. Glycoprotein hormone a-subunit secretion in patients with pituitary adenomas: influence of TRH, LRH and bromocriptine. Acta Endocrinol (Copenh). 1982;99:487-92. 3. Ridgway EC, Klibanski A, Ladenson PW, et al. Pure alpha-secreting pituitary adenomas. N Engl J Med. 1981;304:1254-9. 4. Ridway EC. Glycoprotein hormone production by pituitary tumors. In: Black PMcL, ed. Secretory tumors of the pituitary gland. New York: Raven Press; 1984:343-63. 5. Snyder PJ. Gonadotroph cell adenomas of the pituitary. Endocr Rev. 1985;6:552-63. 6. Beck-Peccoz P, Persani L, Medri G, Iglesias Guerrero M, Spada A, Faglia G. New aspects in non functioning pituitary tumors. In: Casanueva FF, Dieguez C, eds. Recent advances in basic and clinical neuroendocrinology. International Congress Series 864. Amsterdam: Excerpta Medica; 1989:295-302. 7. Surmount DWA, Winslow CLJ, Loizou M, White MC, Adams EF, Mashiter K. Gonadotropin and alpha subunit secretion by human functionless pituitary adenomas in cell culture; long term effects of luteinizing hormone releasing hormone and thyrotrophin releasing hormone. Clin Endocrinol (Oxf). 1983;19:325-36. 8. Asa SL, Gerrie BM, Singer W, Horvath E, Kovacs K, Smyth HS. Gonadotropin secretion in vitro by human pituitary null cell adenomas and oncocytomas. J Clin Endocrinol Metab. 1986;62:1()1119. 9. Yamada S, Asa SL, Kovacs K, Muller P, Smyth HS. Analysis of hormone secretion by clinically nonfunctioning human pituitary adenomas using the reverse hemolytic plaque assay. J Clin Endocrinol Metab. 1989;68:73-80. 10. Kwekkeboom DJ, De Jong FH, Lamberts SWJ. Gonadotropin release by clinically nonfunctioning and gonadotroph pituitary adenomas in vivo and in vitro; relation to sex and effects of thyrotropin-releasing hormone, gonadotropin-releasing hormone and bromocriptine. J Clin Endocrinol Metab. 1989;68:1128-35. 11. Le Dafniet M, Grouselle D, Li JY, et al. Evidence of thyrotropinreleasing hormone (TRH) and TRH-binding sites in human nonsecreting pituitary adenomas. J Clin Endocrinol Metab. 1987;65:1014-19. 12. Bevan JS, Burke CW. Non-functioning pituitary adenomas do not regress during bromocroptine therapy but possess membranebound dopamine receptors which bind bromocriptine. Clin Endocrinol (Oxf). 1986;25:561-72. 13. Ikuyama S, Nawata H, Kato K, Karashima T, Ibayashi H, Nagkagaki H. Specific somatostatin receptors on human pituitary adenoma cell membranes. J Clin Endocrinol Metab. 1985;61:66671. 14. Reubi JC, Heitz PU, Landolt AM. Visualization of somatostatin receptors and correlation with immunoreactive growth hormone and prolactin in human pituitary adenomas: evidence for different tumor subclasses. J Clin Endocrinol Metab. 1987;65:65-73. 15. Bassetti M, Spada A, Arosio M, Vallar L, Brina M, Giannattasio G. Morphological studies on mixed growth hormone (GH)- and prolactin (PRL)-secreting human pituitary adenomas. Coexistence of GH and PRL in the same secretory granule. J Clin Endocrinol Metab. 1986;62:1093-100. 16. Spada A, Sartorio A, Bassetti M, Pezzo G, Giannattasio G. In vitro effect of dopamine on growth hormone (GH) release from human GH-secreting pituitary adenomas. J Clin Endocrinol Metab. 1982;55:734-40. 17. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985;260:3440-50. 18. Pandiella A, Reza-Elahi F, Vallar L, Spada A. Alpha 1 adrenergic stimulation of in vitro growth hormone release and cytosolic free
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Ca2+ in rat somatotrophs. Endocrinology. 1988;122:1419-25. 19. Spada A, Nicosia S, Cortelazzi L, et al. In vitro studies on prolactin relaese and adenylate cyclase activity in human prolactin-secreting pituitary adenomas. Different sensitivity of macro- and microadenomas to dopamine and vasoactive intestinal peptide. J Clin Endocrinol Metab. 1983;56:1-1O. 20. Malgaroli A, Vallar L, Reza-Elahi F, Pozzan T, Spada A, Meldolesi J. Dopamine inhibits cytosolic Ca2+ increases in rat lactotroph cells. Evidence of a dual mechanism of action. J Biol Chem. 1987;262:13920-7. 21. Gershengorn MC, Thaw C. Calcium influx is not required for TRH to elevate free cytoplasmic calcium in GH3 cells. Endocrinology. 1983;113:1522-4. 22. Ramsdell J, Tashjian Jr AH. Thyrotropin-releasing hormone (TRH) elevation of inositol trisphosphate and cytosolic free calcium is dependent on receptor number. J Biol Chem. 1986;261:5301-6. 23. Naor Z, Eli Y. Synergistic stimulation of luteinizing hormone release by protein kinase C activators and Ca2+ ionophores. Biochem Biophys Res Commun. 1985; 130:848-53. 24. Hirota K, Hirota T, Aguilera G, Catt KJ. Hormone induced redistribution of calcium activated phospholipid dependent protein kinase in pituitary gonadotrophs. J Biol Chem. 1985;260:3243-6. 25. Limor R, Ayalon D, Capponi AM, Childs GV, Naor Z. Cytosolic free calcium levels in cultured pituitary cells separated by centrifugal elutriation: effect of gonadotropin-releasing hormone. Endocrinology. 1987;120:497-503. 26. Antoni FA. Hypothalamic control of adrenocorticotropin secretion: advances since the discovery of 41-residue corticotropin-releasing factor. Endocr Rev. 1986; 7:351-78. 27. Raymond V, Leung PCK, Veilleux R, Labrie F. Vasopressin rapidly stimulates phosphatidic acid-phosphatidyl inositol turnover in rat anterior pituitary cells. FEBS Lett. 1985; 182:196-200. 28. Rebecchi MJ, Gershenghom MC. Thyroliberin stimulates rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate by a phosphodiesterase in rat mammotropic pituitary cells. Biochem J. 1983;216:287-94. 29. Berridge MJ, Irvine RF. Inositol trisphosphate, a novel second messenger in cellular signal trasduction. Nature. 1984;312:315-21. 30. Levy A, Lightman S. Effects of thyrotropin-releasing hormone and gonadotropin-releasing hormone on inositol phospholipid turnover in endocrinologically inactive pituitary adenomas and prolactinomas. J Clin Endocrinol Metab. 1989;69:122-6. 31. Borghi C, Nicosia S, Giachetti A, Said SI. Adenylate cyclase of rat pituitary gland. Stimulation by vasoactive intestinal polypeptide (VIP). FEBS Lett. 1979;108:403-7. 32. Bilezikjian LM, Vale W. Stimulation of adenosine 3', 5'-monophosphate production by growth hormone-releasing factor and its inhibition by somatostatin in anterior pituitary cells in vitro. Endocrinology. 1983;113:1726-34. 33. Schlegel W, Wuarin F, Wollhein CB, Zahnd G. Somatostatin lowers the cytosolic free Ca2+ concentration in clonal rat pituitary cells (GH3 cells). Cell Calcium. 1984;5:223-36. 34. Reisine T. Multiple mechanisms of somatostatin inhibition of adrenocorticotropin release from mouse anterior pituitary tumor cells. Endocrinology. l985;116:2249-66. 35. Vallar L, Muca C, Magni M, et al. Differential coupling of dopaminergic D2 receptors expressed in different cell types: stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis in Ltk-fibroblasts, hyperpolarization and cytosolic free Ca2+ concentration decrease in GH4C1 cells. J Biol Chem. 1990;265:10320-6. 36. Lamberts SWJ. The role of somatostatin in the regulation of anterior pituitary hormone secretion and the use of its analogs in the treatment of human pituitary tumors. Endocr Rev. 1988;9:41736. 37. Billestrup AM, Swanson LW, Vale W. Growth hormone-releasing factor stimulates cell proliferation of somatotrophs in vitro. Proc Natl Acad Sci USA. 1983;83:6854-7. 38. Billestrup N, Mitchell RL, Vale W, Verma IM. Growth hormonereleasing factor induces c-fos expression in cultured primary pituitary cells. Mol Endocrinol. 1987;l:300-5. 39. Reichlin S. Editorial: clinical application of somatostatin receptor imaging. J Clin Endocrinol Metab. 1990;71:564-5.
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