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Effect of galanin on growth hormone (GH) response to thyrotropin releasing hormone of rat pituitary gh-secreting adenomatous cells (GH1) in culture
Life Sciences, Vol. 58, No. 1, pp. KHO, 19% Copyright a 1995 Elsevier Science Inc. Printed in the USA. All rights reserved @X4-3205/% S15.00 + [email protected]
0024-3205(95)02258-9
ELSEVIER
EFFECT OF GALANIN ON GROWTH HORMONE (GH) RESPONSE TO THYROTROPIN RELEASING HORMONE OF RAT PITUITARY GH-SECRETING ADENOMATOUS CELLS (GH,) IN CULTURE Andrea Giustina, Carlo Bonfanti#, Massimo Licini, Beatrice Stefana, Giorgio Ragni, Adolf0 Turano# Sezione di Endocrinologia, Clinica Medica and #Institute of Microbiology, School of Medicine, University of Brescia, 25123 Brescia, Italy. (Received in final form October 19, 1995)
The growth hormone (GH) releasing effect of thyrotropin-releasing hormone (TRH) and galanin, a 29-amino acid peptide widely distributed in mammalian CNS, alone or in combination was investigated in cultured rat pituitary tumor cells (GH,). TRH stimulated GH secretion in GH, cells (maximal stimulation at the dose of 0.1 +I). Galanin alone had a significant GH inhibitory effect in GH, cells at all the doses used. When the two peptides were administered in combination, no significant changes as compared to baseline levels were observed. The results of this study indicate that galanin has potent direct inhibitory effects on baseline and TRH-stimulated GH release from rat tumor cells. Key Words: galanin, TRH, GH, cells
Galanin is a 29-amino acid, straight chain, biologically active peptide, derived from a 123-amino acid precursor protein, preprogalanin (1). Galanin was originally isolated from porcine intestine (2), but galanin-like immunoreactivity is widely distributed in central and peripheral neurons of several mammalian species, including humans (3). Galanin plays a significant role in the regulation of GH secretion in man. Human galanin is able to elicit GH secretion when given alone and to increase the GH response to GH-releasing hormone (GHRH) in normal man (4). The mechanism underlying this action of galanin is unknown; some experiments suggest that galanin may act at the hypothalamic level (5,6). However, recent data suggest that galanin may also have a small but significant direct stimulating effect on GH release from monolayer cultures of rat anterior pituitary cells (7). We have recently reported that the same dose of galanin which is able to stimulate GH secretion in normal somatotrophs exerts, on the contrary, a quite potent inhibitory effect on GH release from a cultured rat adenomatous celI line (GH,) (8). We have also previously shown that the same dose of galanin that results in viva in the stimulation of GH secretion in normaI man (4) is, on the contrary, able to induce a significant GH inhibition in acromegalic patients (9). Thyrotropin releasing hormone (TRH) is a well known stimulus of GH secretion in normal and Corresponding Author: Andrea Giustina, MD; CIinica Medica c/o 2” Medicina - Spedali Civili, 25123 Brescia, Italy
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adenomatous rat anterior pituitary cells (10,ll). Moreover, TRH administration is also able to cause paradoxical increases in serum GH in patients with acromegaly (12). Aim of the study was to investigate the effect galanin on TN-I-mediated GH secretion in tumoral cultured rat pituitary cells (GH,).
Metho&
GH, cells were kindly provided by Dr. Ferrari (Istituto Zooprofilattico Sperimentale della Lombardia e dell’ Emilia, Brescia, Italy). GH, cells were grown in monolayer culture using multiwell plates. The cells were inoculated at initial density of 5x10’ cells per well with growth medium (Ham’s F-10 medium containing 2.5% fetal calf serum and 15% horse serum) and were incubated at 37 C in an atmosphere of 95% air and and 5% C& for 48 to 72 h before the experiments were performed. At the time of the experiments, cells were washed twice with PBS in each experiment. Groups of 3 wells were incubated for 4 hours with growth medium alone (control incubations), or growth medium containing human galanin or TBH at the concentrations indicated below. All the experiments were repeated twice (at 48-72 h intervals) and data from the six wells undergoing the same experimental condition were pooled together. At the end of the experiments, culture media were collected and stored frozen at -70-C until being assayed for rat GH. -Peptides Human TBH (Serono, Milano, Italy) and human galanin (Peninsula Laboratories, Belmont, CA) were used. Control em: TN-I was added’ to the culture media at the following concentrations: 0.01,O.l and 1gM; galanin was added at concentrations of 0.1, 1 and 10 PM. m: a) the effect of TBH 0.1 PM was tested on the GH dose- response curve to galanin 0. 1, 1 and 10 PM; b) the effect of galanin 1 PM was tested on the GH dose-response to TRH 0.01, 0.1 and 1 PM. -Assays Bat (r) GH was assayed using a commercial IUA kit (Amersham Italia, Milano, Italy). The assay has a sensitivity of 0.16 ng/tube, the i&a-assay coefficient of variation (CV) is 3% and the interassay CV is 10.5 % . Briefly, the assay is based on the competition between u&belled rGH and a fixed quantity of izsI -1abelled rGH for a limited number of binding sites on a rGH specific antibody. With fixed amounts of antibody and radioactive ligand, the amount of radioactive ligand bound is inversely proportional to the concentration of added non-radioactive ligand. The antibody bound rGH is then reacted with a second antibody that is bound to magnetizable polimer particles. Separation of the antibody bound fraction is obtained by using a magnet and decanting the supernatant. Measurement of the radioactivity in the pellet enables the amount of labelled rGH in the bound fraction to be calculated. The concentration of unlabelled rGH in the sample is then determined by interpolation from a standard curve. GH data are presented as means + SEM of the values obtained from wells which received the same treatment and are expressed as percent of control wells. The statistical analysis of the effects ofthedifferentdosesofeitberTRHorgalaninonGHsecretionhas~performedwiththe Kruskall-Wallis test. The Friedman’s test has been used to analyze the interaction of the two peptides on GH secretion. A pcO.05 was considered statistically significant.
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Fig. 1 shows the effect of graded concentrations of human TRH and galanin on GH release from GH, cells. TRH significantly stimulated GH release expressed as percent change as compared to the control GH values. However, no clear dose-response relationship was observed. In fact, the maximal GH increase in response to TRH was found in GH, cells at the dose of 0.1 PM of the peptide. Conversely, all the doses of galanin were able to decrease baseline GH release in GH, cells. The maximal GH inhibitory effect of galanin in GH, cells was obtained already at the dose of 0.1 PM.
_ -v) D
120
1 TRH
r;r
GAL
Cl L
c Q
+0
60 /
I
I
Peptide
I
dose.
I
(PM)
Fig. 1 Growth hormone (GH) concentrations (mean f SEM) expressed as percent (%) of control wells in cultured GH, cells after 4 h exposure to TFtH alone (TRH) (v) at the following doses: 0.01, 0.1 and 1 PM, and after 4 h exposure to galanin alone (0) at the following doses: 0.1, 1 and 10 FM. * pcO.05 vs baseline.
Gala&,
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Fig. 2 shows, interestingly, that human galanin (1 ,uM) reduced the TRH-stimulated GH release from cultured GH, cells at all the doses studied (0.01 to 1 PM). However, only the combination of galanin 1 ,LLM+ TRH 0.1 PM resulted in a significantly lower GH secretion as compared to TRH 0.1 pM alone. In a further series of experiments (Fig.3) we evaluated the effect of increasing doses of human galanin (0.1, 1 and 10 PM) on GH release at baseline or stimulated by a single dose of TRH (0.1 PM) in GH, rat cells. Addition of TRH in the culture medium resulted in a block of the GH inhibitory effect of galanin in GHI cells.
_ -ul a B z L + c :: V-
140-
120.
0
ik
80
I
I
I
I
0.1
0.01
0
TRH
dose
I
1
(PM)
Fig.2
Growth hormone (GH) concentrations (mean + SEM) expressed as percent (X) of control wells in cultured GH, cells after 4 h exposure to TRH alone (0) (0.01, 0.1, 1 PM) and in combination with galanin (v) 1 pM. * p < 0.05 vs baseline. 0 p < 0.05 vs TRH alone.
Vol.58, No. 1, 1996
Galanin, TRH and Rat GH Adenoma Cells
87
60 -
40
-
I
0
I
I
0.1 GAL
1 dose
I
10
(phi)
Fig.3 Growth hormone (GH) concentrations (mean + SEM) expressed as percent (%) of control wells in cultured GH, cells after 4 h exposure to galanin at the following doses: 0.1, 1 and 10 PM, alone (0) and in combination with TRH (v) 0.1 FM. * ~~0.05 vs baseline. 0 p < 0.05 vs galanin alone.
Discussion
Our data confirm that human galanin inhibits GH release from GH, cells in monolayer cultures. Moreover, our data show that TRH is a weak but significant GH stimulus in GH, cells. We also demonstrate that galanin is able to completely abolish the GH response to TRH in GH, cells. GH, cells are an established cloned tissue culture line derived from a rat pituitary tumor which have become an important source for studying the biology of GH secretion. These cells secrete GH in the basal state and have been reported to have significant GH responses to TRH, cortisol,
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bromocryptine and derivatives of CAMP ( 13). However, we have previously shown that GH, cells differ from normal rat anterior pituitary cells due to their completely absent response to GHRH (8). We have also recently reported that galanin has a quite potent paradoxical inhibitory effect on GH release from the GH, cell line (8). Moreover, we have previously shown that the inhibitory in vitro effect of galanin could also be observed in human GH-secreting adenoma cells obtained from acromegalic patients undergoing neurosurgery (9,14). At present, the mechanism by which galanin can paradoxically decrease GH levels in both human and rat GH-secreting adenoma cells remains to be explained. In keeping with recent in vitro studies, it may be hypothesized that galanin, as well as dopamine agonist drugs (15), interacting at the pituitary level with its own receptors expressed by the adenomatous cells, may be able to directly inhibit pituitary GH secretion. This hypothesis is also supported by the observation that galanin reduces GH levels in acromegalic patients with persistently elevated GH levels after pitutary surgery (14). Using autoradiographic methods, it has been possible to demonstrate the presence of galanin binding sites in the rat central nervous system (CNS) . Extensive binding has been observed in the terminal areas of primary sensory neurons in the medulla obhmgata and in the spinal cord; therefore, it has been suggested that galanin may be involved in the generation or in the modulation of neuroendocrine messages (16). High affinity galanin binding sites have been reported in the rat CNS (16), whereas there are only a few available reports about galanin receptors in rat pituitaries (17). Galanin receptors have been characterized on membranes from a hamster pancreatic beta cell tumor (18), in a rat insulinoma cell line (19) and cloned in a human melanoma cell line (20). These studies have shown that galanin receptor is a glycoprotein of 54 kDa coupled to the inhibitory guanine nucleotide binding protein Gi (21). Depending on the target tissue, different pathways for intracellular signalling by galanin are involved: inhibition of adenylate cyclase (22), blockage of voltage-dependent calcium channels (23) and activation of ATP-sensitive K+ channels (24). Galanin does not stimulate CAMP accumulation in rat somatotrophs (25); conversely, it has been suggested that somatotroph calcium activity may be affected by galanin (26). We have hypothesized that GH, cells, as well as human GH-secreting adenoma cells, may express a mutant galanin receptor which is able to interact with the peptide but may either be linked to different intracellular signal transduction pathways as in other cell types, e.g. CAMP (22) or cause opposite changes in the same signalling system used in normal rat somatotrophs, e.g. decrease intracellular calcium. Much of the work on the secondary message events involved in TRH action has been performed on GH3 cells and mouse thyrotropic tumor cells. TRH stimulates the activity of phospholipase C, which causes rapid hydrolisis of phosphatidyl-inositol 4,5biphosphate to inositol 1,4,5 biphosphate and 1,2 diacylglycerol (27). The latter, in turn, activates intracellular protein kinase C. TRH also causes an immediate, rapid increase in intracellular free calcium (which decays rapidly), followed by an extended plateau of elevated intracellular free calcium (28). This biphasic action correlates with secretory acvtivity, electrical changes, and the induction of calcium fluxes in GHr cells (29). The first phase reflects increased release from intracellular stores, whereas the second phase represents calcium influx. TRH also increases CAMP levels in pituitary tissues, but the increase in probably secondary to stimulation of phosphatidylinositol turnover by TRH. In different brain regions, TRH selectively stimulates either CAMP generation or phosphatidylinositol turnover (30). On the basis of our data, it can be hypothesized that galanin and TRH may interact with different
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TRH and Rat GH Adenoma Cells
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receptors on the surface of GH, rat adenomatous cells, but may have a common second messenger, e. g., intracellular calcium. In fact, the two peptides can be hypothesized to cause opposite changes of this second messenger at the intracellular level. Therefore, in the presence of galanin the stimulatory effect of TRH on GH secretion in GHi cells may be abolished. Conversely, the addition of TRH to the culture medium blocks the inhibitory effect of galanin on GH secretion in GH, cells. In conclusion, we have shown that galanin not only decreases baseline GH secretion in GH, cells, but is also able to inhibit the TRH-evoked GH response in this in model. Galanin and TRH may be hypothesized to act on the GH, cells causing opposite changes in the same intracellular second messenger.
The authors wish to thank Dr. A. Negro-V&u for kindly providing human galanin and Ms. Patrizia Beccalossi for her precious technical assistance. This research was partially supported by the Centro Studi e Ricerche di Neuroendocrinologia (Brescia, Italy).
1. 2. 3. 4. 5.
6. 7. 8. 9. 10.
11. 12.
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A. ROKAEUS and M. BROWNSTEIN, Proc. Natl. Acad. Sci. a 6287-6291 (1986). K. TATEMOTO, A. ROKAEUS, H. JORNVALL, T.J. MCDONALD and V. MUTT, Febs. L&t. 164 124-128 (1983). A. ROKAEUS, Trends Neurosci. u 158-164 (1987). A. GIUSTINA, M. LICINI, M. SCHETTINO, M. DOGA, G. PIZZOCOLO and A. NEGRO-VILAR, Am. 3. Physiol. 264 E57-E61 (1994). A. GIUSTINA, A.R. BUSSI, E. BRESCIANI, M. DOGA, M. SCHETTINO, G. GIUSTINA, Neuroendocrinol. Lett. 16 151-158 (1994). A. GIUSTINA, A. GIRELLI, S. BOSSONI, F. LEGATI, M. SCHETTINO, W.B. WEHRENBERG, Metabolism Q 548-551 (1992). S.M. GABRIEL, C.M. MILBURY, J.A. NATHANSON and J.B. MARTIN, Life Sci. Q 1981-1986 (1988). A. GIUSTINA, C. BONFANTI, M. LICINI, C. DE RANGO, G. MILANI, Life Sci. Xj 1845-1851 (1994). A. GIUSTINA, C. BODINI, M. DOGA, M. SCHETTINO, G. PIZZOCOLO and G. GIUSTINA, J. Clin. Endocrinol. Metab. 24 1296-1300 (1992). Y. YAJIMA, T. SAITO, Acta Endicrinol. (Copenh). l&l 287-294 (1983). R.E. OSLUND Jr., J.T. LEUNG, S.V. HAJEC, T. WINOKUR, M. MELMAN, Endocrinology m 1245-1252 (1978). G. GIUSTINA, E. RESCHINI, M. PERACCHI, L.CANTALAMESSA, F. CAVAGNINI, M. PINTO, P. BULGHERONI, J. Chn. Endocrinol. Metab. 38 906909 (1974). KM. GAUTVIK, R.F. HOYT, A.H. TASHJIAN Jr., J. Cell. Physiol. 82 401-409 (1973). A. GIUSTINA, E. BRESCIANI, A.R. BUSSI, A. BOLLATI. C. BONFANTI, G. BUGARI, L. CHIESA, G. GIUSTINA, 1. Clin. Endocrinol. Metab. SQ 1333-1340 (1995).
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F. PEILLON, F. CESSELIN, D. BRESSION, N. ZYGELMAN, A.M. BRANDI, A. NOUSBAUM and A. MAUBORGNE, J. Clin. Endocrinol. Metab. @ 737-741 (1979). G. SKOFITSH, M.A. SILLS, D.M. JACOBOWITZ, Peptides Z 1029-1042 (1986). D. WYNICK, D.M. SMITH, M. GHATEI, K. AKINSANYA, R. BHOGAL, P. PURKISS, P. BYFIELD, N. YANAIHARA, S.R. BLOOM, Proc. Natl. Acad. Sci. USA a 4231-4235 (1993). B. AMIRANOFF, A.L. SERVIN, C. ROUYER-FESSARD, A. COUVINEAU, K. TATEMOTO, Endocrinology 121284-289 (1987). G.W. SHARP, Y. LE MARCHAND BRUSTEL, T. YADA, L.L. RUSSO, C.R. BLISS, M. CORMONT, L. MONGE, E. VAN OBBERGHEN, J. Biol. Chem. m 7302-7309 (1989). E. HABERT-ORTIOLI, B. AMIRANOFF, I. LOQUET, M. LABURTHE, JF. MAYAUX, Proc. Natl. Acad. Sci. USA % 9780-9783 (1994). I. LAGNY-POUMIR, B. AMIRANOFF, A.M. LORINET, K. TATEMOTO, M. LABOURTHE, Endocrinology 124 2636-264 1 ( 1994). B. AMIRANOFF, A.M. LORINET, I. LAGNY-POURMIR and M. LABURTHE, Eur. J. B&hem. 177 147-152 (1988). F.R. HOMAIDAN, G.W.G. SHARP, L.M. NOWAK, Pot. Natl. Acad. Sci. USA 88 8744-8748 (1991). J.H. DE WEILLE, H. SCHMID-ANTOMARCHI, M. FOSSET, M. LAZDUNSKI, Proc. Natl. Acad. Sci. USA 85 1312-1316 (1988). M. SATO, J. TAKAHARA, M. NIIMI, R. TAGAWA and S. IRINO, Life Sci. t@ 1639-1644 (1991). P. LINDSTROM and L. SAVENDAHL, Acta Endocrionol.(Copenh). 129 268-272 (1993). A.H. DRUMMOND, J. Exp. Biol. 124 337-358 (1986). E.J. GERAS, MC. GERSHENGORN, Am. J. Phisiol. 242 109-113 (1981). M.C. GERSHENGORN, C. TRAW, Endocrionology 116 591-596 (1985). T. IRIUCHIJIMA, M. MORI, J. Neurochem. 2 1944-1946 (1989).
0024-3205(95)02258-9
ELSEVIER
EFFECT OF GALANIN ON GROWTH HORMONE (GH) RESPONSE TO THYROTROPIN RELEASING HORMONE OF RAT PITUITARY GH-SECRETING ADENOMATOUS CELLS (GH,) IN CULTURE Andrea Giustina, Carlo Bonfanti#, Massimo Licini, Beatrice Stefana, Giorgio Ragni, Adolf0 Turano# Sezione di Endocrinologia, Clinica Medica and #Institute of Microbiology, School of Medicine, University of Brescia, 25123 Brescia, Italy. (Received in final form October 19, 1995)
The growth hormone (GH) releasing effect of thyrotropin-releasing hormone (TRH) and galanin, a 29-amino acid peptide widely distributed in mammalian CNS, alone or in combination was investigated in cultured rat pituitary tumor cells (GH,). TRH stimulated GH secretion in GH, cells (maximal stimulation at the dose of 0.1 +I). Galanin alone had a significant GH inhibitory effect in GH, cells at all the doses used. When the two peptides were administered in combination, no significant changes as compared to baseline levels were observed. The results of this study indicate that galanin has potent direct inhibitory effects on baseline and TRH-stimulated GH release from rat tumor cells. Key Words: galanin, TRH, GH, cells
Galanin is a 29-amino acid, straight chain, biologically active peptide, derived from a 123-amino acid precursor protein, preprogalanin (1). Galanin was originally isolated from porcine intestine (2), but galanin-like immunoreactivity is widely distributed in central and peripheral neurons of several mammalian species, including humans (3). Galanin plays a significant role in the regulation of GH secretion in man. Human galanin is able to elicit GH secretion when given alone and to increase the GH response to GH-releasing hormone (GHRH) in normal man (4). The mechanism underlying this action of galanin is unknown; some experiments suggest that galanin may act at the hypothalamic level (5,6). However, recent data suggest that galanin may also have a small but significant direct stimulating effect on GH release from monolayer cultures of rat anterior pituitary cells (7). We have recently reported that the same dose of galanin which is able to stimulate GH secretion in normal somatotrophs exerts, on the contrary, a quite potent inhibitory effect on GH release from a cultured rat adenomatous celI line (GH,) (8). We have also previously shown that the same dose of galanin that results in viva in the stimulation of GH secretion in normaI man (4) is, on the contrary, able to induce a significant GH inhibition in acromegalic patients (9). Thyrotropin releasing hormone (TRH) is a well known stimulus of GH secretion in normal and Corresponding Author: Andrea Giustina, MD; CIinica Medica c/o 2” Medicina - Spedali Civili, 25123 Brescia, Italy
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adenomatous rat anterior pituitary cells (10,ll). Moreover, TRH administration is also able to cause paradoxical increases in serum GH in patients with acromegaly (12). Aim of the study was to investigate the effect galanin on TN-I-mediated GH secretion in tumoral cultured rat pituitary cells (GH,).
Metho&
GH, cells were kindly provided by Dr. Ferrari (Istituto Zooprofilattico Sperimentale della Lombardia e dell’ Emilia, Brescia, Italy). GH, cells were grown in monolayer culture using multiwell plates. The cells were inoculated at initial density of 5x10’ cells per well with growth medium (Ham’s F-10 medium containing 2.5% fetal calf serum and 15% horse serum) and were incubated at 37 C in an atmosphere of 95% air and and 5% C& for 48 to 72 h before the experiments were performed. At the time of the experiments, cells were washed twice with PBS in each experiment. Groups of 3 wells were incubated for 4 hours with growth medium alone (control incubations), or growth medium containing human galanin or TBH at the concentrations indicated below. All the experiments were repeated twice (at 48-72 h intervals) and data from the six wells undergoing the same experimental condition were pooled together. At the end of the experiments, culture media were collected and stored frozen at -70-C until being assayed for rat GH. -Peptides Human TBH (Serono, Milano, Italy) and human galanin (Peninsula Laboratories, Belmont, CA) were used. Control em: TN-I was added’ to the culture media at the following concentrations: 0.01,O.l and 1gM; galanin was added at concentrations of 0.1, 1 and 10 PM. m: a) the effect of TBH 0.1 PM was tested on the GH dose- response curve to galanin 0. 1, 1 and 10 PM; b) the effect of galanin 1 PM was tested on the GH dose-response to TRH 0.01, 0.1 and 1 PM. -Assays Bat (r) GH was assayed using a commercial IUA kit (Amersham Italia, Milano, Italy). The assay has a sensitivity of 0.16 ng/tube, the i&a-assay coefficient of variation (CV) is 3% and the interassay CV is 10.5 % . Briefly, the assay is based on the competition between u&belled rGH and a fixed quantity of izsI -1abelled rGH for a limited number of binding sites on a rGH specific antibody. With fixed amounts of antibody and radioactive ligand, the amount of radioactive ligand bound is inversely proportional to the concentration of added non-radioactive ligand. The antibody bound rGH is then reacted with a second antibody that is bound to magnetizable polimer particles. Separation of the antibody bound fraction is obtained by using a magnet and decanting the supernatant. Measurement of the radioactivity in the pellet enables the amount of labelled rGH in the bound fraction to be calculated. The concentration of unlabelled rGH in the sample is then determined by interpolation from a standard curve. GH data are presented as means + SEM of the values obtained from wells which received the same treatment and are expressed as percent of control wells. The statistical analysis of the effects ofthedifferentdosesofeitberTRHorgalaninonGHsecretionhas~performedwiththe Kruskall-Wallis test. The Friedman’s test has been used to analyze the interaction of the two peptides on GH secretion. A pcO.05 was considered statistically significant.
Vol. 58, No. 1. 1996
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Fig. 1 shows the effect of graded concentrations of human TRH and galanin on GH release from GH, cells. TRH significantly stimulated GH release expressed as percent change as compared to the control GH values. However, no clear dose-response relationship was observed. In fact, the maximal GH increase in response to TRH was found in GH, cells at the dose of 0.1 PM of the peptide. Conversely, all the doses of galanin were able to decrease baseline GH release in GH, cells. The maximal GH inhibitory effect of galanin in GH, cells was obtained already at the dose of 0.1 PM.
_ -v) D
120
1 TRH
r;r
GAL
Cl L
c Q
+0
60 /
I
I
Peptide
I
dose.
I
(PM)
Fig. 1 Growth hormone (GH) concentrations (mean f SEM) expressed as percent (%) of control wells in cultured GH, cells after 4 h exposure to TFtH alone (TRH) (v) at the following doses: 0.01, 0.1 and 1 PM, and after 4 h exposure to galanin alone (0) at the following doses: 0.1, 1 and 10 FM. * pcO.05 vs baseline.
Gala&,
86
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Fig. 2 shows, interestingly, that human galanin (1 ,uM) reduced the TRH-stimulated GH release from cultured GH, cells at all the doses studied (0.01 to 1 PM). However, only the combination of galanin 1 ,LLM+ TRH 0.1 PM resulted in a significantly lower GH secretion as compared to TRH 0.1 pM alone. In a further series of experiments (Fig.3) we evaluated the effect of increasing doses of human galanin (0.1, 1 and 10 PM) on GH release at baseline or stimulated by a single dose of TRH (0.1 PM) in GH, rat cells. Addition of TRH in the culture medium resulted in a block of the GH inhibitory effect of galanin in GHI cells.
_ -ul a B z L + c :: V-
140-
120.
0
ik
80
I
I
I
I
0.1
0.01
0
TRH
dose
I
1
(PM)
Fig.2
Growth hormone (GH) concentrations (mean + SEM) expressed as percent (X) of control wells in cultured GH, cells after 4 h exposure to TRH alone (0) (0.01, 0.1, 1 PM) and in combination with galanin (v) 1 pM. * p < 0.05 vs baseline. 0 p < 0.05 vs TRH alone.
Vol.58, No. 1, 1996
Galanin, TRH and Rat GH Adenoma Cells
87
60 -
40
-
I
0
I
I
0.1 GAL
1 dose
I
10
(phi)
Fig.3 Growth hormone (GH) concentrations (mean + SEM) expressed as percent (%) of control wells in cultured GH, cells after 4 h exposure to galanin at the following doses: 0.1, 1 and 10 PM, alone (0) and in combination with TRH (v) 0.1 FM. * ~~0.05 vs baseline. 0 p < 0.05 vs galanin alone.
Discussion
Our data confirm that human galanin inhibits GH release from GH, cells in monolayer cultures. Moreover, our data show that TRH is a weak but significant GH stimulus in GH, cells. We also demonstrate that galanin is able to completely abolish the GH response to TRH in GH, cells. GH, cells are an established cloned tissue culture line derived from a rat pituitary tumor which have become an important source for studying the biology of GH secretion. These cells secrete GH in the basal state and have been reported to have significant GH responses to TRH, cortisol,
88
Cabin,
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bromocryptine and derivatives of CAMP ( 13). However, we have previously shown that GH, cells differ from normal rat anterior pituitary cells due to their completely absent response to GHRH (8). We have also recently reported that galanin has a quite potent paradoxical inhibitory effect on GH release from the GH, cell line (8). Moreover, we have previously shown that the inhibitory in vitro effect of galanin could also be observed in human GH-secreting adenoma cells obtained from acromegalic patients undergoing neurosurgery (9,14). At present, the mechanism by which galanin can paradoxically decrease GH levels in both human and rat GH-secreting adenoma cells remains to be explained. In keeping with recent in vitro studies, it may be hypothesized that galanin, as well as dopamine agonist drugs (15), interacting at the pituitary level with its own receptors expressed by the adenomatous cells, may be able to directly inhibit pituitary GH secretion. This hypothesis is also supported by the observation that galanin reduces GH levels in acromegalic patients with persistently elevated GH levels after pitutary surgery (14). Using autoradiographic methods, it has been possible to demonstrate the presence of galanin binding sites in the rat central nervous system (CNS) . Extensive binding has been observed in the terminal areas of primary sensory neurons in the medulla obhmgata and in the spinal cord; therefore, it has been suggested that galanin may be involved in the generation or in the modulation of neuroendocrine messages (16). High affinity galanin binding sites have been reported in the rat CNS (16), whereas there are only a few available reports about galanin receptors in rat pituitaries (17). Galanin receptors have been characterized on membranes from a hamster pancreatic beta cell tumor (18), in a rat insulinoma cell line (19) and cloned in a human melanoma cell line (20). These studies have shown that galanin receptor is a glycoprotein of 54 kDa coupled to the inhibitory guanine nucleotide binding protein Gi (21). Depending on the target tissue, different pathways for intracellular signalling by galanin are involved: inhibition of adenylate cyclase (22), blockage of voltage-dependent calcium channels (23) and activation of ATP-sensitive K+ channels (24). Galanin does not stimulate CAMP accumulation in rat somatotrophs (25); conversely, it has been suggested that somatotroph calcium activity may be affected by galanin (26). We have hypothesized that GH, cells, as well as human GH-secreting adenoma cells, may express a mutant galanin receptor which is able to interact with the peptide but may either be linked to different intracellular signal transduction pathways as in other cell types, e.g. CAMP (22) or cause opposite changes in the same signalling system used in normal rat somatotrophs, e.g. decrease intracellular calcium. Much of the work on the secondary message events involved in TRH action has been performed on GH3 cells and mouse thyrotropic tumor cells. TRH stimulates the activity of phospholipase C, which causes rapid hydrolisis of phosphatidyl-inositol 4,5biphosphate to inositol 1,4,5 biphosphate and 1,2 diacylglycerol (27). The latter, in turn, activates intracellular protein kinase C. TRH also causes an immediate, rapid increase in intracellular free calcium (which decays rapidly), followed by an extended plateau of elevated intracellular free calcium (28). This biphasic action correlates with secretory acvtivity, electrical changes, and the induction of calcium fluxes in GHr cells (29). The first phase reflects increased release from intracellular stores, whereas the second phase represents calcium influx. TRH also increases CAMP levels in pituitary tissues, but the increase in probably secondary to stimulation of phosphatidylinositol turnover by TRH. In different brain regions, TRH selectively stimulates either CAMP generation or phosphatidylinositol turnover (30). On the basis of our data, it can be hypothesized that galanin and TRH may interact with different
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receptors on the surface of GH, rat adenomatous cells, but may have a common second messenger, e. g., intracellular calcium. In fact, the two peptides can be hypothesized to cause opposite changes of this second messenger at the intracellular level. Therefore, in the presence of galanin the stimulatory effect of TRH on GH secretion in GHi cells may be abolished. Conversely, the addition of TRH to the culture medium blocks the inhibitory effect of galanin on GH secretion in GH, cells. In conclusion, we have shown that galanin not only decreases baseline GH secretion in GH, cells, but is also able to inhibit the TRH-evoked GH response in this in model. Galanin and TRH may be hypothesized to act on the GH, cells causing opposite changes in the same intracellular second messenger.
The authors wish to thank Dr. A. Negro-V&u for kindly providing human galanin and Ms. Patrizia Beccalossi for her precious technical assistance. This research was partially supported by the Centro Studi e Ricerche di Neuroendocrinologia (Brescia, Italy).
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