Galanin within the normal and hyperplastic anterior pituitary gland: localization, secretion, and functional analysis in normal and human growth hormone-releasing hormone transgenic mice

Studies evaluating estrogen-induced anterior pituitary tumors revealed a strong direct correlation between expression of the peptide galanin and tumor growth. To evaluate further the potential roles of galanin in the hyperplastic pituitary, we used a model of estrogen-independent anterior pituitary tumor formation, the male human GH-releasing hormone (hGHRH) transgenic mouse. Pituitaries of hGHRH transgenic mice are characterized by a hyperplasia of somatotrophs and contain markedly elevated levels of galanin. We examined the population of galanin-producing pituitary cells in 4- to 6-month-old male hGHRH transgenic mice and their nontransgenic siblings. The percentage of galanin-containing pituitary cells was significantly increased within the anterior pituitaries of hGHRH transgenic mice. By using the cell immunoblot assay we found that the basal secretion of galanin and GH from individual pituitary cells of hGHRH transgenic mice was significantly greater than that from pituitary cells of nontransgenic mice. By modifying the cell immunoblot assay, we determined that somatotrophs from both hGHRH transgenic and normal mice that were positive for galanin immunoreactivity secreted significantly greater amounts of GH than those somatotrophs devoid of galanin immunoreactivity. Moreover, immunoneutralization of galanin significantly decreased GH secretion from pituitary cells obtained from hGHRH transgenic mice. Thus, we now show that the increased levels of galanin peptide within the hyperplastic pituitaries of hGHRH transgenic mice are due to an increase in the population of cells containing galanin, and that galanin participates in the augmented secretion of GH from hyperplastic proliferating pituitary cells.     

Regulatory role of galanin in control of hypothalamic-anterior pituitary function.

The role of the neuropeptide galanin in the regulation of anterior pituitary function was studied in vivo in conscious male rats and in vitro with cultured anterior pituitary cells. Galanin (50-200 ng; 15-60 pmol) injected into the third cerebral ventricle of rats produced highly significant, dose-related increases of plasma growth hormone (GH) concentrations, whereas galanin increased prolactin (PRL) and decreased thyroid-stimulating hormone (TSH) levels only at the highest dose (60 pmol) tested. Intravenous galanin failed to alter PRL and TSH levels in these rats. In contrast with the results with intraventricular injection of the peptide, intravenous injection of 30 or 300 pmol of galanin produced small, brief, dose-related increases in plasma GH. The response to the 300-pmol dose was less than that induced by a factor-of-20-lower intraventricular dose, which establishes a central action of galanin. Galanin in concentrations ranging from 1 nM to 1 microM failed to alter significantly GH, PRL, or TSH release from dispersed anterior pituitary cells. It also failed to alter GH secretion in response to 100 nM GH-releasing hormone; however, at this dose galanin did potentiate the effect of 100 nM TSH-releasing hormone on TSH and PRL release. Thus, the effects of third-ventricular injection of the peptide are mediated by the hypothalamus. To determine the physiological significance of galanin in control of pituitary hormone release, highly specific antiserum against galanin was injected intraventricularly. Third-ventricular injection of 3 microliter of galanin antiserum resulted in a dramatic decrease in plasma GH values as compared with those of normal rabbit serum-injected controls within 15 min, which persisted until the end of the experiment (5 hr postinjection). Galanin antiserum did not decrease plasma PRL or TSH levels at any time period after its third-ventricular injection; however, a transient increase of plasma TSH levels occurred after 30 and 60 min in comparison with TSH levels in normal rabbit serum-injected controls. Since there was no effect of the antiserum on plasma PRL and only a transient elevation in TSH, galanin may not be physiologically significant enough during resting conditions to alter PRL and TSH release in the male rat. The results of the experiments with galanin antiserum indicate that endogenous galanin has a tonic action within the hypothalamus to stimulate GH release. The rapidity of onset of the effects of galanin and the antiserum directed against it suggest that it acts to stimulate release of GH-releasing hormone from periventricular structures, which then stimulates the release of GH.     

Targeted overexpression of galanin in lactotrophs of transgenic mice induces hyperprolactinemia and pituitary hyperplasia.

We generated transgenic mice that carry 4.6 kb of the mouse galanin gene fused to 2.5 kb of the rat PRL promoter. In all transgenic lines that carried and transmitted the transgene, there were significant increases in galanin messenger RNA and peptide levels in the anterior pituitary in both male and female transgenic mice, and the elevation of galanin was restricted to the anterior lobe. Furthermore, galanin release from pituitary cells in vitro of both male and female transgenic mice was dramatically increased compared with that in control mice. At 2-4 months of age, pituitary PRL contents in female transgenic mice were increased compared with those in normal controls. Moreover, PRL messenger RNA levels were increased in female transgenic mice. However, plasma levels of PRL in female transgenic mice were not significantly higher until 6 months of age. By 11 months of age, cell numbers in the anterior pituitary were increased in female, but not male, transgenic mice. The percentage of lactotrophs in female transgenic mice as well as PRL gene expression per cell were significantly higher. No differences were detected in PRL content, gene expression, or release between normal and transgenic male mice. Six weeks of estrogen treatment significantly increased anterior pituitary weights and PRL secretion in male transgenic mice compared with that in normal male mice. In addition, anterior pituitary weights and PRL secretion were decreased in female transgenic mice compared with controls 6 weeks after ovariectomy. We conclude that overexpression of galanin in lactotrophs stimulates PRL synthesis and secretion and acts as a growth factor resulting in the formation of pituitary hyperplasia and hyperprolactinemia. Furthermore, estrogen appears critical for these galanin-mediated events.     

Effect of endocrine manipulation on anterior pituitary galanin in the rat.

Galanin is widely distributed throughout the rat neural and endocrine system. The highest concentrations are found in the anterior pituitary, and it can influence classical pituitary hormone secretion. The effects of endocrine manipulation on pituitary galanin content, mRNA, and immunostaining have been investigated in the rat. In females, medical (39 +/- 4 fmol/gland), surgical (33 +/- 2), or combined (28 +/- 6) castration resulted in a highly significant decrease in galanin content (control, 223 +/- 14; P less than 0.0001). Estrogen in physiological and pharmacological doses produced a significant increase in galanin content (368 +/- 14 and 373 +/- 13, respectively; P less than 0.01) associated with an increase in galanin mRNA content. In the male, high dose dexamethasone and thyroidectomy caused a fall in galanin content, while galanin mRNA levels showed a rise and fall, respectively. Adrenalectomy caused a rise in galanin content, while adrenalectomy and castration produced a dramatic decrease in tissue galanin content. No change in galanin mRNA was observed in these groups. Galanin immunostaining paralleled the results of tissue content in all groups examined, except in the medically castrated group, in which there was some intragroup variation in staining patterns. In normal and high-dose estrogen-treated females, galanin expression was seen mainly in lactotrophs, with a small number of somatotrophs and thyrotrophs staining. In the male, galanin expression was confined to somatotrophs and thyrotrophs. Galanin mRNA was localized at the cellular level by in situ hybridization. In the normal pituitary only scattered lactotrophs contained message, while in high-dose estrogen-treated animals the number of positive cells, mostly lactotrophs, was vastly increased. Thus, the cellular localization of galanin immunostaining varies between the sexes. Galanin peptide and mRNA levels in the pituitary are powerfully influenced by endocrine status.     

Estrogen increases galanin immunoreactivity in hyperplastic prolactin-secreting cells in Fisher 344 rats.

Galanin is a widely distributed regulatory peptide which modulates the pituitary secretion of PRL and GH. Estrogen administration strongly stimulates galanin gene expression in the rat anterior pituitary. In adult female Fischer 344 rats, estrogen also induces hyperplasia of lactotropes. We used immunocytochemical analysis to assess the effects of estrogen on galanin-like immunoreactivity (Gal-IR) in the rat pituitary and hypothalamus during sc diethylstilbestrol (DES) implantation and after its removal at 30 days. In the anterior pituitary, DES implantation increased the portion of Gal-IR-containing cells from less than 2% in the control rats to 18.3% after 3 days of DES and 36% after 30 days. These changes paralleled the lactotrope hyperplasia exhibited in response to DES exposure. Ten and 30 days after removal of the DES capsules, the percentage of Gal-IR-containing cells in the anterior pituitary decreased to 6.3% and 1.5%, respectively. Colocalization studies revealed that Gal-IR-containing cells were predominantly lactotropes. Immunoelectron microscopy demonstrated that Gal-IR was concentrated in the Golgi region of these hyperplastic lactotropes and suggests that little of the synthesized galanin is secreted. The distribution of Gal-IR in the hypothalamus, median eminence, and neurohypophysis was unaffected by DES treatment. These data demonstrate that galanin is synthesized by hyperplastic pituitary lactotropes of Fischer 344 rats and that peptide accumulation is dependent on the presence of circulating estrogens. In contrast, neuronal galanin synthesis in the hypothalamus does not appear to be regulated by estrogen.     

Galanin regulates prolactin release and lactotroph proliferation

The neuropeptide galanin is predominantly expressed by the lactotrophs (the prolactin secreting cell type) in the rodent anterior pituitary and in the median eminence and paraventricular nucleus of the hypothalamus. Prolactin and galanin colocalize in the same secretory granule, the expression of both proteins is extremely sensitive to the estrogen status of the animal. The administration of estradiol-17β induces pituitary hyperplasia followed by adenoma formation and causes a 3,000-fold increase in the galanin mRNA content of the lactotroph. To further study the role of galanin in prolactin release and lactotroph growth we now report the generation of mice carrying a loss-of-function mutation of the endogenous galanin gene. There is no evidence of embryonic lethality and the mutant mice grow normally. The specific endocrine abnormalities identified to date, relate to the expression of prolactin. Pituitary prolactin message levels and protein content of adult female mutant mice are reduced by 30–40% compared with wild-type controls. Mutant females fail to lactate and pups die of starvation/dehydration unless fostered onto wild-type mothers. Prolactin secretion in mutant females is markedly reduced at 7 days postpartum compared with wild-type controls with an associated failure in mammary gland maturation. There is an almost complete abrogation of the proliferative response of the lactotroph to high doses of estrogen, with a failure to up-regulate prolactin release, STAT5 expression or to increase pituitary cell number. These data further support the hypothesis that galanin acts as a paracrine regulator of prolactin expression and as a growth factor to the lactotroph.     

On the role of the peptide galanin in regulation of growth hormone secretion.

The effects of the peptide galanin on growth hormone secretion were studied in vitro using cultured rat and human anterior pituitary cells, and in vivo by iv administration of galanin in both rats and humans. Galanin in concentrations from 10 nmol/l to 1 mumol/l did not alter basal GH release, but slightly inhibited GHRH-stimulated GH release from cultured rat anterior pituitary cells. Galanin (1 mumol/l) did not significantly change basal or GHRH-stimulated GH secretion from cultured human anterior pituitary cells. In contrast, iv injection of 1 microgram (300 pmol) galanin to rats induced an increase in plasma GH that was reproducible at repetitive injections. The galanin-induced GH release in rats was of a lower magnitude than the increase in plasma GH after iv injections of GHRH, and was seen with a 5-15 min delay in comparison to iv administered GHRH. In man, iv infusions of galanin (40 pmol.kg-1.min-1.(40 min)) also caused a significant increase in plasma GH, but it occurred 25-30 min after the beginning of the infusion. These results suggest an indirect action of galanin on GH release in both rats and humans, i.e. galanin does not directly affect the somatotropes. In agreement with a central action, no binding sites for galanin could be demonstrated in the rat anterior pituitary by autoradiography. Since galanin did not affect somatostatin release from fragments of rat mediobasal hypothalamus, the stimulatory effects of galanin on GH release are most likely mediated via a stimulatory effect on GHRH neurons.     

Effect of galanin on the growth hormone response to growth hormone-releasing hormone in acromegaly.

Galanin enhances growth hormone (GH)-releasing hormone (GHRH)-stimulated GH secretion in normal man. In acromegaly, circulating GH levels are increased and the GH response to GHRH may be exaggerated. Galanin has been recently shown to decrease circulating GH levels in acromegaly. The aim of our study was to investigate the effects of galanin on the GH response to GHRH in acromegalic subjects. Five acromegalic patients (three men and two women) and seven healthy adult subjects (five men and two women) were studied. GHRH-induced GH secretion was evaluated during a 40-minute intravenous (IV) infusion of saline (100 mL) or porcine galanin (12.5 micrograms/min in 100 mL saline). In normal subjects, delta GH levels after GHRH+porcine galanin administration (47 +/- 7.5 micrograms/L) were significantly higher in comparison to levels obtained with GHRH+saline (21.7 +/- 3.5 micrograms/L, P < .05). In acromegalic patients, GH responses to GHRH (delta GH, 18.8 +/- 8.6 micrograms/L) were not altered by galanin infusion (delta GH, 17.6 +/- 5 micrograms/L). Our results give the first evidence that the same dose of galanin that induces a significant enhancement of the GH response to GHRH in normal subjects has no effect on the GH response to GHRH in acromegalic patients. It can be hypothesized that galanin may interact at the pituitary level with its own receptors expressed by somatotropes independent of GHRH. Failure of galanin to enhance GH response to GHRH in acromegalic patients could be due to a change in function of the galanin receptor on GH-secreting adenomatous cells.     

Characterization of a high-affinity galanin receptor in the rat anterior pituitary: absence of biological effect and reduced membrane binding of the antagonist M15 differentiate it from the brain/gut receptor.

Structure-activity studies demonstrate that galanin fragments 1-15 and 2-29 are fully active, whereas fragment 3-29 has been reported to be inactive, in a number of different in vivo models. M15, a chimeric peptide comprising galanin 1-13 and substance P5-11, has recently been found to be a potent galanin antagonist. Direct effects of galanin at the level of the pituitary have been defined, yet, paradoxically, a number of studies have been unable to demonstrate galanin binding to an anterior pituitary receptor. Porcine galanin stimulated prolactin release from dispersed rat anterior pituitary cells up to 180% +/- 12% (mean +/- SEM) of control secretion. The addition of a specific galanin antiserum caused a profound inhibition of basal prolactin release, maximal inhibition being 12% +/- 0.5% of control secretion. Addition of M15 produced no effect on basal or galanin-stimulated prolactin release. Galanin fragment 3-29 was fully active when compared to galanin 1-29. Fragments 5-29 and 8-29 stimulated prolactin release to a lesser extent and galanin 1-15, 10-29, and 20-29 had no significant prolactin-releasing activity. Using [mono(125I)iodo-Tyr26]galanin or porcine 125I-labeled Bolton-Hunter [mono(125I)iodo-Lys25]galanin, no anterior pituitary membrane binding was observed. In contrast, 125I-labeled Bolton-Hunter N-terminally labeled galanin allowed characterization of a single high-affinity anterior pituitary galanin receptor with a Kd of 4.4 +/- 0.34 nM and a Bmax of 79 +/- 8.3 fmol/mg of protein. The IC50 for porcine galanin was 0.51 +/- 0.04 nM but for M15 was in excess of 10 microM. Galanin 3-29 fully displaced the label with an IC50 of 0.96 +/- 0.7 nM. The IC50 for galanin 5-29 was 200 nM, whereas 8-29 and 1-15 were > 1 microM. Galanin 10-29 and 20-29 failed to displace the label. These data suggest the presence of a high-affinity pituitary galanin receptor, designated GAL-R2, in which region 3-10 and amino acid 25 are crucial for membrane binding and biological activity, in contrast to the known gut/brain galanin receptor (designated GAL-R1). A number of tissues known to bind or respond to galanin were screened. GAL-R2 would appear to be expressed only in the anterior pituitary and hypothalamus.     

A role for galanin in human and experimental inflammatory demyelination

The neuropeptide galanin is widely expressed by many differing subsets of neurons in the nervous system. There is a marked upregulation in the levels of the peptide in a variety of nerve injury models and in the basal forebrain of humans with Alzheimer''s disease. Here we demonstrate that galanin expression is specifically and markedly upregulated in microglia both in multiple sclerosis (MS) lesions and shadow plaques. Galanin expression is also upregulated in the experimental autoimmune encephalomyelitis (EAE) model of MS, although solely in oligodendrocytes. To study whether the observed increase in expression of galanin in inflammatory demyelination might modulate disease activity, we applied the EAE model to a panel of galanin transgenic lines. Over-expression of galanin in transgenic mice (Gal-OE) abolishes disease in the EAE model, whilst loss-of-function mutations in galanin or galanin receptor-2 (GalR2) increase disease severity. The pronounced effects of altered endogenous galanin or GalR2 expression on EAE disease activity may reflect a direct neuroprotective effect of the neuropeptide via activation of GalR2, similar to that previously described in a number of neuronal injury paradigms. Irrespective of the mechanism(s) by which galanin alters EAE disease activity, our findings imply that galanin/GalR2 agonists may have future therapeutic implications for MS.     

Biochemical evaluation of patients with active acromegaly and type 2 diabetes mellitus: efficacy and safety of the galanin test

The oral glucose tolerance test, which is considered the gold standard for the diagnosis of active acromegaly, should not be performed in the presence of basal hyperglycemia. Moreover, false-positive responses may occur in patients with diabetes mellitus. Galanin has previously been demonstrated to induce paradoxical inhibition of growth hormone (GH) secretion in most patients with active acromegaly. In this study, we assessed GH response to galanin infusion in a series of 17 consecutive patients with active acromegaly, 7 of whom had coexistent type 2 diabetes mellitus and 10 were without either diabetes mellitus or impaired tolerance to glucose. 6 acromegalic patients with diabetes mellitus (85.7%) and 7 without diabetes (70.0%) showed a decrease in serum GH values during galanin infusion (chi2 0.9; p = 0.6). The GH nadir occurred at a comparable time in the two groups of acromegalic patients. Moreover, the two groups showed no significant difference (p = 0.45) in DeltaGH during galanin infusion. Galanin infusion did not induce any significant change in plasma glucose levels in both diabetic and non-diabetic patients with acromegaly. The results of our study provide evidence that the galanin test may be of value for the diagnosis of acromegaly in patients with type 2 diabetes mellitus.     

Short-term glucocorticoid administration decreases both hypothalamic and pituitary galanin synthesis in the adult male rat.

Galanin (GAL) is a peptide that has been implicated in the regulation of the growth axis. It is generally accepted that GAL can increase serum growth hormone (GH) levels, although the underlying mechanism for this increase is unknown. It is well known that long-term glucocorticoid treatment alters in vivo GH secretion, since there is a decrease in serum GH in response to stimuli. It has previously been shown in our laboratory that administration of GAL can overcome the effects of glucocorticoid administration on GH secretion. The aim of the present study was to determine the effects of long-term glucocorticoid administration on the regulation of hypothalamic and pituitary GAL mRNA levels. Adult male rats were treated for 72 hours with the synthetic glucocorticoid dexamethasone ([DEX] 40 microg/kg/d intraperitoneal injections). RNase protection assays were performed on both the hypothalamus and pituitary for the presence of GAL mRNA. As expected, DEX significantly decreased somatic growth, as evidenced by a decrease (50%) in the weight gain of glucocorticoid-treated versus control animals. It was also demonstrated that in both the hypothalamus and pituitary, glucocorticoid treatment reduced the level of GAL mRNA (to 11% and 6.5%, respectively) compared with the control condition. We conclude that the decrease in GAL mRNA may lead to a decrease in GAL secretion, which in turn may be involved in the glucocorticoid-induced inhibition of GH secretion.     

Possible mechanisms involved in growth hormone secretion induced by galanin in the rat

The mechanisms by which central or systemic administration of galanin stimulates GH secretion were investigated in either conscious or urethane-anesthetized male rats. Intracerebroventricular injection of synthetic porcine galanin, a 29-amino acid gut-brain peptide (0.12, 0.6, and 3 nmol/rat), resulted in a dose-related increase in plasma GH. The plasma GH level was increased by an N-terminal galanin fragment [galanin-(1-19)], but not by C-terminal fragments [galanin-(2-29) and -(21-29)]. Intravenous injection or infusion of galanin (0.6 and 3 nmol/100 g BW) also raised plasma GH. The plasma GH increase induced by galanin was inhibited by pretreatment with rabbit antiserum specific for rat GRF. Pretreatment with yohimbine or phenoxybenzamine, alpha-adrenergic blockers, or picrotoxin, a gamma-aminobutyric acid (GABA) antagonist, blunted the plasma GH increase induced by intracerebroventricular injection of galanin. On the other hand, the plasma GH increase induced by iv injection of galanin was suppressed by picrotoxin, but not by phenoxybenzamine. These findings suggest that 1) both central and systemic administration of galanin stimulate GH secretion in the rat; 2) the N-terminal structure of galanin is required to stimulate GH secretion; 3) the stimulating effect of galanin is mediated, at least in part, by hypothalamic GRF; and 4) central alpha-adrenergic and GABAergic mechanisms may be involved in GH release induced by central administration of galanin, whereas systemic injection of galanin stimulates GH release predominantly through GABAergic mechanisms in the rat.     

Pathology of acromegaly

This review summarizes current knowledge on pituitary changes in patients with acromegaly. The histologic, immunohistochemical and electron microscopic study provided conclusive evidence that a marked diversity exists between the tumors which secrete growth hormone (GH) in excess, such as densely and sparsely granulated GH cell adenoma, the mixed GH prolactin cell adenoma and the mammosomatotrope adenoma. The latter two tumors produce GH and prolactin simultaneously. Densely granulated GH cell tumors may produce thyrotropin and alpha subunit as well. Somatotrope carcinomas are extremely rare. GH cell hyperplasia can also be associated with acromegaly in patients with extrapituitary GH-releasing hormone secreting tumors. The medical therapy of acromegaly is reviewed briefly, including long-acting somatostatin analogs and pegvisomant, a GH receptor blocker.     

The neuropeptide galanin modulates behavioral and neurochemical signs of opiate withdrawal

Much research has focused on pathways leading to opiate addiction. Pathways opposing addiction are more difficult to study but may be critical in developing interventions to combat drug dependence and withdrawal. Galanin decreases firing of locus coeruleus neurons, an effect hypothesized to decrease signs of opiate withdrawal. The current study addresses whether galanin affects morphine withdrawal signs by using a galanin agonist, galnon, that crosses the blood-brain barrier, and mice genetically engineered to under- or overexpress galanin peptide. Galnon significantly decreased morphine withdrawal signs in C57BL/6 mice. Further, knockout mice lacking galanin showed exacerbated morphine withdrawal signs, suggesting that endogenous galanin normally counteracts opiate withdrawal. Transgenic mice overexpressing galanin in noradrenergic neurons also showed decreased morphine withdrawal signs, suggesting a possible neuroanatomical locus for these effects of galanin. Both c-fos immunoreactivity, a marker of neuronal activity, and phosphorylation of tyrosine hydroxylase at Ser-40, a marker of cAMP levels, are decreased in the locus coeruleus by galnon treatment after morphine withdrawal, suggesting a possible molecular mechanism for the behavioral effects of galanin. These studies suggest that galanin normally acts to counteract opiate withdrawal and that small molecule galanin agonists could be effective in diminishing the physical signs of withdrawal.     

Regulation of galanin gene expression in the rat anterior pituitary gland by the somatostatin analog SMS 201-995.

In addition to inducing pituitary tumors in rats, estrogen (E2) markedly increases galanin and PRL gene expression. We previously showed that galanin secretion from pituitary cells in vitro is inhibited by dopamine and somatostatin and stimulated by TRH. The objectives of these in vivo studies were to assess whether the long-acting somatostatin analog SMS 201-995 alters 1) immunoreactive galanin or PRL levels in the anterior pituitary, neurointermediate lobe, hypothalamus, or plasma, 2) pituitary galanin and PRL mRNA levels, and 3) the development of E2-induced pituitary tumors. Ovariectomized Fischer 344 rats were implanted with E2-filled or empty Silastic capsules and treated with or without SMS 201-995 (1.5 mg) via Alzet miniosmotic pumps. Two or 6 weeks later, immunoreactive galanin and PRL levels were determined by RIA. In ovariectomized rats, the somatostatin analog lowered the anterior pituitary content of galanin by 50%, but had no effect on PRL concentrations. E2 increased galanin and PRL levels in the anterior pituitary by 220- and 4-fold, respectively. Concomitant E2 and SMS 201-995 treatment further increased galanin and PRL in the anterior pituitary by 60-80%, but decreased plasma galanin and PRL levels. Likewise, the administration of SMS 201-995 for 2 and 6 weeks inhibited the E2-induced growth of the anterior pituitary. Galanin and PRL mRNA levels were quantified by solution hybridization. Galanin mRNA levels were reduced to undetectable levels in ovariectomized rats treated with SMS 201-995. Furthermore, a 10-fold increase in galanin mRNA levels seen in the presence of E2 was inhibited 80% by SMS 201-995. PRL mRNA levels in E2-treated rats were unchanged by SMS 201-995. We conclude that SMS 201-995 1) lowers plasma galanin and PRL levels in E2-treated rats, 2) elevates the anterior pituitary contents of galanin and PRL in E2-exposed rats, probably through decreased secretion of the hormones, and 3) reduces galanin mRNA levels in E2-treated and untreated ovariectomized rats. Overall, these results establish the differential regulation of galanin and PRL gene expression in vivo by SMS 201-995. Moreover, the data demonstrate that somatostatin receptor agonists may have therapeutic potential for some prolactinomas.     

Enhanced prolactin responsiveness to galanin in patients with Cushing's disease

OBJECTIVE Galanin is believed to play a role in the control of prolactin (PRL) secretion in the rat. Such a role is uncertain in humans where the neuropeptide is expressed by the corticotrophs. However, in clinical conditions of enhanced ACTH secretion, increased PRL levels are often observed. Therefore, we evaluated the effect of galanin infusion on serum PRL levels in patients with Cushing's disease and in control subjects. For comparison, the PRL responses to TRH and metoclopramide were also investigated in the same patients. DESIGN Four tests were performed: (a) 40-minute infusion of 0–3 μg/kg/min of galanin; (b) infusion of normal saline only; (c) metoclopramide test (10 mg as i.v. bolus); (d) TRH test (200 mg as i.v. bolus). PATIENTS Twenty-four normal subjects and nine patients suffering from active Cushing's disease were investigated. MEASUREMENTS Serum concentrations of PRL were measured by radioimmunoassay on blood samples collected before and for 90 minutes after drug or saline administration. RESULTS Serum baseline PRL levels were superimposable in normal subjects and in patients with Cushing's disease. In normal subjects, infusion of galanin induced a distinct PRL increase compared to saline (mean ±SEM incremental areas 6514 ±2572 vs 540 ±571 μ/l/90 min, P= 005, respectively). In patients with Cushing's disease, galanin evoked a remarkable PRL rise with hormone levels which were significantly greater (P < 0.001) than those observed in the same patients after infusion of saline (21908 ± 4180 vs 534 ± 1556 mU/l/90 min) or after galanin administration in controls (P<0.01). The PRL response to TRH and, much more so, to metoclopramide was significantly lower in patients with Cushing's disease than in normal subjects (42125± 8000 vs 73181 ± 7246 mU/l/90 min, P<0.01 after TRH and 79095 ± 27265 vs 229049 ± 10602 mU/l/90 min, P < 001 after metoclopramide). CONCLUSIONS Galanin appears to be a specific, though weak, PRL secretagogue in normal subjects. The galanin-induced PRL release was significantly increased in patients with Cushing's disease. A number of hypothetical mechanisms may underlie the enhanced PRL reactivity to galanin in Cushing's disease. This finding together with the impaired PRL responsiveness to TRH and metoclopramide, also observed in this study, is a further example of a dysregulation of PRL secretion in patients with Cushing's disease.     

Galanin secretion from anterior pituitary cells in vitro is regulated by dopamine, somatostatin, and thyrotropin-releasing hormone.

Lactotrophs, somatotrophs, and thyrotrophs have been shown to contain immunoreactive galanin. Furthermore, estrogen stimulates galanin mRNA and peptide levels in the rat anterior pituitary, particularly within lactotrophs. To determine whether galanin is released from the anterior pituitary in a regulated manner, we used cultured pituitary cells from male and ovariectomized Fischer 344 rats implanted with estrogen-containing capsules. Anterior pituitary cells (5 x 10(5) cells/well) were challenged (0.5-3 h) with hypothalamic factors known to regulate anterior pituitary hormone secretion, and medium galanin levels were measured by RIA. In female pituitary cells, galanin secretion was inhibited by dopamine (10 and 100 nM) and stimulated by TRH (20 and 100 nM). Although galanin release was significantly lower in male pituitary cells, dopamine and TRH inhibited and stimulated galanin secretion, respectively. Medium galanin levels were also significantly reduced by somatostatin (5 nM) in both female and male cells. The pattern of PRL release in response to dopamine, TRH, and somatostatin was similar to that observed for galanin, regardless of the sex of the pituitary donor. Although galanin has been localized in somatotrophs, 5 nM GH-releasing hormone (GRF) failed to alter galanin release in male as well as female pituitary cells; GH secretion was significantly increased by GRF. LHRH (5 nM) and CRF (5 nM) failed to alter galanin release in vitro. We conclude that in estrogen-exposed pituitary cells obtained from male and ovariectomized Fischer 344 rats: 1) galanin secretion is inhibited by dopamine and somatostatin, and stimulated by TRH; 2) GRF, LHRH, and CRF do not regulate galanin release in these cells; and 3) the profile of the regulated pathway for galanin release suggests that the primary location of galanin is the lactotroph, probably within secretory granules.     

Acute and chronic galanin administration decreases hypothalamic galanin synthesis in both male and female adult rats: evidence for a long-loop galanin autofeedback

The secretion of growth hormone (GH) in both male and female rats is controlled by two main neuropeptides, GH-releasing hormone (GHRH), which is stimulatory, and somatostatin, which is inhibitory. Recently, it has been shown that galanin (GAL) also stimulates GH secretion, although the underlying mechanism is still unknown. It was the aim of this study to begin to elucidate if and how GAL regulates its own production at the hypothalamic and pituitary level. Rats underwent the following experimental trials. In experiment 1, adult male and female rats had blood samples collected at -15 minutes, -7.5 minutes, and immediately preceding a subcutaneous (s.c.) injection of GAL at a dose of either 50 or 200 microg/kg. Blood samples were collected at 5, 10, 15, 30, and 60 minutes, and the GH concentration was measured using a radioimmunoassay. The tissues were collected and analyzed for mRNA levels of hypothalamic and pituitary GAL. In experiment 2, adult male and female rats were treated long-term with 200 microg/kg GAL for 7 days s.c., and the pituitary and hypothalamus were analyzed for GAL mRNA. Serum GH concentrations were significantly increased in acutely dosed male and female rats regardless of the dosage level. For the male and female animals acutely dosed with both 50 and 200 microg/kg GAL, hypothalamic GAL mRNA was decreased, whereas pituitary GAL mRNA was affected by 200 microg/kg GAL only in females (increased). For the animals treated long-term with GAL, hypothalamic GAL mRNA was decreased while mRNA for pituitary GAL was increased. We conclude that regardless of the dosage and duration of treatment, administration of GAL negatively regulates hypothalamic GAL mRNA in a non-gender-specific way. Pituitary GAL synthesis appears to be stimulated particularly during chronic SCGAL administration.