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Original Studies |
2nd Chair of Endocrinology (C.I., F.P.G., A.D., F.C.), Ospedale San Luca, Istituto Auxologico Italiano, Istituto di Ricovero e Cura a Carattere Scientifico, University of Milan, Milan 20143; Centro di Studio sulla Patologia Cellulare (P.M., R.P.), Centro Nazionale delle Ricerche, Istituto di Patologia Generale, University of Milan, Milan 20133; and Department of Neurosurgery (M.L.), Ospedale San Raffaele Istituto di Ricovero e Cura a Carattere Scientifico, Milan 20132, Italy
Address all correspondence and requests for reprints to: Prof. Francesco Cavagnini, Istituto Scientifico San Luca IRCCS, Via Spagnoletto 3, 20149 Milano, Italy. E-mail: cavagnini{at}auxologico.it
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Abstract |
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Galanin secretion by pituitary tumor cultures ranged from 30–230 pmol/4 h. Incubation with CRH induced an increase in galanin concentrations (100 pM CRH: 151 ± 32%; 1 nM CRH: 232 ± 43%; 10 nM CRH: 246 ± 35%; and 100 nM CRH: 270 ± 44% unstimulated levels at 24 h, P < 0.05). The stimulatory effect of CRH seemed to be dose-dependent. Basal and CRH-stimulated ACTH and galanin concentrations also exhibited a strong positive correlation in single tumor cultures.
At IPS sampling, mean basal plasma galanin concentrations in the dominant IPS were somewhat higher than those registered at the periphery (18.6 ± 1.94 vs. 15.8 ± 1.60 pmol/L, P = 0.05). Administration of CRH induced a modest but significant increase in galanin concentrations at all three sampling sites. No correlations were found between ACTH and galanin levels in the IPSs and at the periphery. Different from what was observed in patients with Cushing’s disease, CRH did not modify plasma galanin concentrations in normal subjects.
In conclusion, this study demonstrates that galanin is released by human tumoral corticotropes and responds to CRH. The role of locally produced galanin is, as yet, unknown but may possibly be that of a autocrine/paracrine modulator.
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Introduction |
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In the rat, galanin plays an important role in stimulating the preovulatory LH surge (11) and is a potent stimulus for GH and PRL secretion (1, 2, 4, 12). By comparison, in man, the best known action of galanin is the stimulation of GH secretion (1) whereas potentiation of PRL release seems to be more subtle (13, 14, 15, 16), and no effect on gonadotrope secretions has been observed (13, 16, 17). As regards the production of galanin by the human pituitary, galanin-like immunoreactivity and galanin messenger RNA have been detected only in normal corticotropes and in ACTH-secreting tumors (18, 19), thus linking galanin to ACTH in man. On the basis of this evidence, we had previously evaluated the response of pituitary hormones to administration of exogenous galanin in patients with Cushing’s disease (CD). In these patients, galanin induced a striking increase in PRL levels, by far greater than the one observed in normal subjects. We had therefore hypothesized that galanin produced by tumoral corticotropes might enhance the PRL response to exogenous galanin (14).
The aim of this study was to establish whether galanin is released by human ACTH-secreting pituitary tumors and responds to CRH. To this purpose, we performed an in vitro and an in vivo protocol: 1) evaluation of galanin release by pituitary ACTH-producing tumors in culture; and 2) measurement of plasma galanin concentrations in the inferior petrosal sinuses (IPSs) and at the periphery before and after administration of CRH. For comparison, we also evaluated peripheral galanin levels after CRH administration in a group of normal subjects.
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Materials and Methods |
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Five pituitary, pathologically confirmed, ACTH-secreting adenomas were collected during transsphenoidal surgery, dispersed with 0.1% collagenase in HAM-F10, plated at a density of 2–3 x 105 cells/well, and incubated at 37 C in DMEM, 10% FCS, 0.05 mg/mL ascorbic acid (Sigma Chemical Co., St. Louis, MO), 100 U/mL penicillin, 100 U/mL streptomycin, 10 mg/mL gentamycin, and 0.25 g/mL Amphotericin B in 5% CO2-95% O2 for 4 days. Except for ascorbic acid, all reagents for cell culture had been purchased from Hyclone Laboratories Inc. Ltd, Cramlington, UK. On the day of the experiment, wells were washed in serum-free medium containing 0.1% BSA (Sigma Chemical Co.) for 1 h, before incubation with 100 pM-100 nM ovine CRH. Medium samples were collected at 4 and 24 h for ACTH and galanin measurement. Control wells were incubated with serum-free medium containing 0.1% BSA. Each treatment was performed in triplicate or quadruplicate, according to cell availability.
Patients and in vivo protocol
Fifteen patients with surgically proven CD (4 men, 11 women,
20–50 yr old) were submitted to IPS sampling. Before and 2, 5, 10, and
15 min after iv administration of 100 µg CRH (ovine CRH, UCB
Bioproducts, Brussels, Belgium), blood samples were collected
simultaneously from a forearm vein and from each IPS, into prechilled
polypropylene tubes containing EDTA (2 mg/mL) and aprotinine
(500 kIU/mL). Samples were kept in ice and centrifuged within
1 h. Plasma was stored at -80 C for ACTH and galanin
measurements. All patients presented a center-to-periphery ACTH
gradient 
3 after CRH, whereas in 5 of 15 the interpetrosal
gradient (1.4) agreed with the site of the adenoma, as detected by
surgery. In 8 healthy volunteers (2 men, 6 women, 18–50 yr old), a CRH
test and a placebo saline infusion were performed with sampling up to
120 min after the beginning of the test. Tests were performed in random
order, at least 3 days apart, between 0830 and 0900 h, in fasted
subjects. In 25 additional normal subjects (6 men, 19 women, 18–49 yr
old), a plasma sample for galanin measurement was collected in the
morning, after an overnight fast. The study was approved by the Ethical
Committee of our Institution, and informed consent was obtained from
all participating subjects.
Peptide assays
Galanin in plasma samples was measured by RIA, as previously described (20). Briefly, 2 mL plasma were acidified with an equal vol of 0.1% trifluoracetic acid (TFA) and centrifuged at 3000 x g. Supernatants were applied onto reverse-phase minicolumns (Sep Column, Peninsula Laboratories Inc., Belmont, CA), washed with 0.1% TFA, and eluted with 3 mL 0.1% TFA/acetonitrile (4/6, vol/vol). Using this protocol, recovery of synthetic human galanin was 66%. RIA was performed using a rabbit-antihuman galanin antiserum and human synthetic galanin as standard and tracer (Peninsula Laboratories, Inc.). The sensitivity of the assay is 12 pg/tube (5.7 pmol/L), and intra- and interassay coefficients of variation are 9.8% and 11.2%, respectively. Samples of patients with CD and controls were run together in each assay. Galanin in medium was assayed without prior extraction.
Plasma ACTH was measured by immunoradiometric assay (Allegro, Nichols Institute Diagnostics, San Juan Capistrano, CA). Sensitivity, intra- and interassay coefficients of variation are 0.22 pmol/L, 4.8%, and 8.9%, respectively. Medium ACTH concentrations were assayed by RIA, according to the manufacturer’s instructions (IgG Corporation, Nashville, TN) using tracer obtained from Amersham International (Little Chalfont, UK). Sensitivity, intra- and interassay coefficients of variation are 0.1 pmol/L, 6.4%, and 6.9%, respectively.
Statistical analysis
The results of the in vivo study were compared using Student’s t test for paired and unpaired data, as appropriate. Linear regression analysis was used to assess relationships between variables. A nonparametric ANOVA (Kruskal-Wallis test) was used to evaluate the statistical significance of percent increase over baseline. Data are expressed as mean ± SEM.
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Results |
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Unstimulated ACTH secretion differed widely among pituitary tumors
in culture but increased markedly after CRH, at both 4 h and
24 h, in all patients (Fig. 1
, left
panels). Basal galanin secretion at 4 h ranged from 30–230
pmol/well and was strongly correlated with ACTH concentrations at the
same timepoint (r = 0.85, P < 0.001). Incubation
with CRH induced an increase in galanin concentrations, which was
evident already at 4 h in some patients and more readily apparent
in all patients at 24 h (Fig. 1, middle panels). The
stimulatory effect of CRH on galanin release was dose-dependent;
indeed, log-transformed CRH concentrations and galanin levels were
strongly correlated (Fig. 1, right panels). Overall mean
galanin secretion at 24 h (expressed as percent of unstimulated
levels) was 151 ± 32% for 100 pM CRH, 232 ± 43% for 1 nM
CRH, 246 ± 35% for 10 nM CRH, and 270 ± 44% for 100 nM
CRH (P < 0.05, by Kruskal-Wallis; Fig. 2). In each tumor culture, ACTH and
galanin concentrations at 24 h exhibited a strong positive
correlation, and a representative example is shown in Fig. 3.
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Baseline peripheral plasma levels of galanin were comparable in
patients with CD and 33 normal subjects (15.8 ± 1.60
vs. 14.8 ± 1.6 pmol/L, not significant). At IPS
sampling, mean basal plasma galanin concentrations of the dominant IPS
were slightly higher than those registered in peripheral blood
(18.6 ± 1.94 vs. 15.8 ± 1.60 pmol/L,
P = 0.05). ACTH responded to CRH in all patients with
CD (Fig. 4, upper panel),
whereas plasma galanin concentrations exhibited a variable increase
(Fig. 4, lower panel). A clear increment in galanin levels
(defined as 30% over baseline, i.e. three times the
intraassay coefficient of variation) was registered in 9 patients in
the IPS and in 5 patients at the periphery. As a whole, mean peak
galanin was significantly greater, compared with baseline, at all three
sampling sites: 20.7 ± 1.91 vs. 18.0 ± 2.00
pmol/L, P < 0.05 in the right IPS; 20.6 ± 2.06
vs. 16.4 ± 1.83 pmol/L, P < 0.05 in
the left IPS; and 20.3 ± 2.14 vs. 15.8 ± 1.60
pmol/L, P < 0.01 at the periphery. No correlations
were found between plasma ACTH and galanin levels in the IPS and at the
periphery.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In man, on the other hand, past studies had focused on the effects of galanin on pituitary hormone secretion in vitro and in vivo (1, 13, 14, 16, 27), whereas reports on the release of galanin itself are few and have been carried out only in vivo (28, 29, 30). In the human pituitary, production of galanin is confined to corticotropes, both normal and tumoral, as has been demonstrated by in situ hybridization and immunocytochemistry (18, 19). Our study tallies nicely with this evidence and, to the best of our knowledge, is the first report of galanin release by the human pituitary, specifically by tumoral corticotropes. Indeed, the results of our in vitro experiments demonstrate that galanin release by tumoral corticotropes is stimulated in a dose-dependent manner by CRH and mirrors that of ACTH, thus pointing to the corticotropes as an important source of galanin in patients with CD.
This finding is somewhat less manifest in vivo. In fact, in patients with CD, plasma galanin levels in the dominant IPS were only slightly higher, compared with those registered at the periphery. Likewise, administration of CRH induced a modest even if significant rise in galanin levels in the IPS and at the periphery. An explanation for the paucity of in vivo results could lie in the mixed origin of galanin present in petrosal and peripheral blood. Indeed, on one side, there is the contribution of extrapituitary sources [e.g. pancreas, adrenal gland, gastrointestinal and genital tract, spinal cord, and autonomic ganglia (31)] to peripheral levels. On the other hand, it is well known that galanin is produced by the hypothalamus and reaches the pituitary via portal blood vessels. Thus, the petrosal venous drainage system receives galanin derived from the median eminence and secreted by the anterior pituitary. This could also explain the absence of correlation between ACTH and galanin levels in the IPS despite the strong association observed in pituitary adenoma cultures. The dilution of the quota of pituitary-derived galanin might also explain the contrast between the small increase in galanin concentrations after CRH administration in vivo and the marked release we observed in vitro. Moreover, the amount of galanin released by the pituitary, during the 15 min of IPS sampling, may represent intracellular stores, whereas galanin, measured in culture medium after 4 and 24 h, likely included newly synthesized molecules also.
As regards the normal study group, our results agree with those recently published by Ceresini et al. (28), indicating that galanin does not seem to be influenced by CRH administration in normal subjects. In this context, the same investigators failed to detect changes in peripheral galanin levels after stimuli engendering the release of catecholamines (29, 30), notably costored with galanin in chromaffin cells (32). Here again, however, the mixing of galanin derived from different sources must not be underestimated; and we cannot exclude that a central galanin response might have occurred also in normal subjects. Further, notwithstanding the clear evidence of galanin release by the corticotrope tumor, peripheral galanin levels were comparable in normal subjects and in patients with CD. The likely inference is that peripheral plasma galanin values do not represent a suitable tool for the study of galanin release by the pituitary.
The significance of galanin released by tumoral corticotropes awaits elucidation. Galanin may act on nearby pituicytes, as previously shown in different experimental settings (10, 12). Galanin is a potent lactotrope growth factor (8) and might, as such, contribute to the hyperplasia of PRL-releasing cells frequently observed around corticotrope tumors (33). In fact, PRL levels are often elevated in patients with CD (34) but exhibit a dampened response to classic lactotrope stimuli, such as TRH and metoclopramide (14, 34). We had previously reported that PRL levels increase markedly upon infusion of galanin in patients with CD, unlike the moderate rise observed in normal subjects (14). One possible explanation for this finding had been that of a homologous up-regulation of galanin receptors situated on lactotropes, by galanin derived from tumoral corticotropes, thus potentiating the PRL response to exogenous galanin. At the time, only histochemical evidence of galanin production by tumoral corticotropes was available; but in view of the results obtained by the present study, this hypothesis seems a likely upshot. In an alternative scenario, galanin produced by tumoral corticotropes might play a role in adenoma formation. Indeed, galanin secretion and gene expression are dramatically increased in estrogen-induced prolactinomas (7, 19, 35) and in GHRH-induced somatotrope adenomas (9), whereas other studies demonstrated that galanin stimulates the proliferation of human small cell lung carcinomas (36). The growth-promoting role of galanin in ACTH-secreting adenomas is, of course, speculative but may well be worth investigating.
In conclusion, our study demonstrates that galanin is released by human tumoral corticotropes and responds like a true pituitary hormone to one of the main stimuli of ACTH secretion. The role of locally produced galanin is unknown but may possibly be that of an autocrine/paracrine modulator.
Received November 17, 1998.
Revised January 4, 1999.
Accepted January 12, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, control wells; 
, 100 pM CRH; 
, 1 nM CRH; 
, 10 nM CRH;
, 100 nM CRH.
, unstimulated; •, 100 pM CRH; 
, 10 nM
CRH; 
, 100 nM CRH.