Immunohistochemical distribution of regulatory peptides in the human fetal adenohypophysis

We have studied here the cellular distribution of several regulatory peptides in hormone-producing cells of the human pituitary during the fetal period. Immunohistochemistry was used to show the expression of several regulatory peptides, namely Angiotensin-II, Neurotensin and Galanin, at successive gestational stages and their co-localization with hormones in the human fetal adenohypophysis. Somatotrophs, gonadotrophs and thyrotrophs were differentiated earliest. At gestational week 9, Angiotensin-II immunoreactivity was co-localized only with growth hormone immunoreactivity in somatotrophs, one of the first hormone-producing cells to differentiate. This co-localization remained until week 37. Neurotensin immunoreactivity was present in gonadotrophs and thyrotrophs in week 23, after FSH and TSH hormone differentiation. Galanin immunoreactivity was present in all hormone-producing cell types except corticotrophs. The different pro-opiomelanocortin-derived peptides were detected at different stages of gestation and adrenocorticotrophic hormone immunoreaction was the last to be detected. Our results show an interesting relationship between regulatory peptides and hormones during human fetal development, which could imply that these peptides play a regulatory role in the development of pituitary function.     

Coexpression of galanin and adrenocorticotropic hormone in human pituitary and pituitary adenomas.

Galanin is a neuropeptide that regulates the secretion of several pituitary hormones, including prolactin (PRL) and growth hormone (GH). Galaninlike immunoreactivity (Gal-IR) and galanin mRNA in the rat anterior pituitary is cell lineage specific, with predominant expression in lactotrophs and somatotrophs. The authors examined the cellular distribution of human Gal-IR in seven normal postmortem pituitaries and 62 pituitary tumors by immunoperoxidase staining. In contrast to the rat, Gal-IR in human anterior pituitaries was present in corticotrophs scattered throughout the gland, but not in lactotrophs, somatotrophs, thyrotrophs, or gonadotrophs. Distinct Gal-IR also was present in hyperplastic and neoplastic corticotrophs in 19 of 22 patients with Cushing's disease. In noncorticotroph cell tumors, unequivocal Gal-IR was present in 5 of 11 GH-secreting tumors associated with clinical acromegaly, 9 of 18 nonfunctioning pituitary adenomas, and 2 of 14 prolactinomas. Of these galanin-positive tumors, four of the five GH-secreting adenomas, six of the nine nonfunctioning adenomas, and both of the prolactinomas also contained adrenocorticotropic hormone immunoreactivity (ACTH-IR). Immunostaining and in situ hybridization on adjacent sections using an 35S-labeled probe complementary to human galanin mRNA demonstrated predominant galanin expression in normal corticotrophs. Immunoelectron microscopy confirmed the presence of Gal-IR in pituitary cells characteristic of corticotrophs in both normal and neoplastic pituitaries. Thus, as in the rat, galanin gene expression in the human pituitary is cell-type specific. Unlike the rat, however, human galanin gene expression is restricted to the corticotroph lineage. Studies of tumors confirmed the observed coexpression of galanin and adrenocorticotropic hormone. The divergent cell type specificity of galanin production in human and rat pituitaries reflects different patterns of gene activation in these two species. In addition, these results suggest that galanin in the human pituitary may participate locally in the regulation of the hypothalamic-pituitary-adrenal axis.     

Glycoconjugate localization with lectin and PA-TCH-SP cytochemistry in rat hypophysis

Glycoconjugates were localized by light microscopy with lectin-peroxidase conjugates and by electron microscopy with the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) sequence in immunocyto-chemically or morphologically identified cell types in rat pituitary. Lectin histochemistry demonstrated sialic acid and glycoconjugates with N-glycosidically linked oligosaccharides in gonadotrophs, thyrotrophs, and corticotrophs. Galactose penultimate to sialic acid was observed mostly in gonadotrophs. The terminal galactose-N-acetylgalactosamine disaccharide was detected in a few gonadotrophs and in a moderate number of mammotrophs. Fucose was localized in only corticotrophs with two fucose-binding lectins and in thyrotrophs with another. Several different monosaccharides were seen in glycoconjugates in melanotrophs and in Herring bodies. Melanotrophs displayed heterogeneous staining with fucose-binding lectins. A small number of nonsecretory cells were also visualized in the pars distalis by virtue of their glycogen content. PA-TCH-SP staining revealed complex carbohydrates in secretory granules and some Golgi cisternae in all types of hormone-producing cells in the pars distalis except for the somatotrophs. Melanotrophs of pars intermedia exhibited stained secretory granules and irregular dense bodies containing a stained meshwork. Corticotrophs of the pars distalis lacked the latter bodies, although they from the same glycoprotein precursor hormone as melanotrophs. Lectin conjugates and the PA-TCH-SP sequence stained some groups of secretion granules in Herring bodies, possibly representing vasopressin-containing granules as well as other cell types in the pars nervosa.     

Characterization of pituitary cell populations in rats with induced polycystic ovaries

Several hypotheses have been proposed about the pathogenesis of polycystic ovarian syndrome (PCOS), however, the fundamental physiological interactions that initiate the development of follicular cysts have not yet been elucidated. Hence, in this study the proliferation, density and population of gonadotrophs, mammotrophs, somatotrophs and corticotrophs of the pituitary glands of rats with induced follicular cysts have been investigated by 2 experimental models (continuous light exposition and estradiol valerate-treated rats). Specific immunoreactivity associated with follicle-stimulating hormone, luteinizing hormone, prolactin, growth hormone and adrenocorticotropic hormone was evaluated by immunohistochemistry with specific hormone antibodies and proliferation of secretory cells by their colocalization (double-labeling) with proliferating cell nuclear antigen. The results indicate a reduction in the density and proliferation of gonadotrophs in both experimental groups. A reduction in the average density, proliferation and population of lactotrophs and corticotrophs was also observed in estradiol valerate-treated animals. However, no significant differences were found in somatotrophs. The present study contributes to the information about alterations of some cell populations that occur in the pituitary gland of rats with polycystic ovaries, and will enhance our understanding of the pathogenesis of this disease.     

Direct actions of adrenergic agents on rat anterior pituitary cells

We studied the effects of adrenergic agents on the five main cell types of the rat anterior pituitary by monitoring the changes of the cytosolic free [Ca2+] ([Ca2+]i) in single cells that were identified by multiple sequential primary immunocytochemistry at the end of the Ca2+ measurements. Adrenaline (100 nM) increased [Ca2+]i in 30% of the cells. Responses were most prominent in somatotrophs and corticotrophs (40-65% of the cells responded) whereas the other three cell types, lactotrophs, thyrotrophs and gonadotrophs, gave poorer responses. Selective agonists and antagonists revealed the presence of both alpha1- and beta-adrenergic receptors. Alpha1-receptors dominated in corticotrophs, beta-receptors in somatotrophs. The alpha1-adrenergic responses increased with culture of the cells. The beta-adrenergic responses were mediated by cAMP and consisted of stimulation of Ca2+ entry through L-type voltage-gated channels. Stimulation of alpha1-receptors released Ca2+ from intracellular stores in corticotrophs and induced cAMP-independent Ca2+ entry in somatotrophs. The effects of alpha1-agonists were additive with those of the releasing factors growth hormone-releasing hormone (GHRH) and corticotropin releasing factor (CRF) whereas those of the beta-agonists were not. Our results suggest that direct effects of plasma catecholamines on AP cells may contribute to the hormonal response to stress.     

An immunoperoxidase study of a human pituitary adenoma associated with Cushing's syndrome

A large endocrine active pituitary adenoma causing a Cushing's syndrome was investigated for the presence of subunits of the corticotropin-lipotropin precursor by immunohistology. A quantitative study revealed immunoreactivity (ir) for ACTH in 87.1% of the adenoma cells, beta-lipotropin-ir in 77.1%, beta-endorphin-ir in 75.3%, alpha-MSH-ir in 22.9% and methionine-enkephalin-ir in 7.8%. The adjacent distal pituitary gland showed a six-fold increase of prolactin-ir cells indicating the release of biologically active endogenous opiates. The strong alpha-MSH-ir within the adenoma cells in contrast to those of the distal pituitary may signify that alpha-MSH was being secreted by the adenoma. The proportions of other endocrine cell types within the anterior pituitary were normal (3.1% corticotrophs, 49.4% somatotrophs, 8.2% gonadotrophs and 7.1% thyrotrophs). 0.5% of the cells of the distal pituitary and 0.1% within the adenoma were VIP-ergic, which may be due to a vasoregularitory system and/or be involved in prolactin release.     

Fine structural features of secretion in adenomas of human pituitary gland

Ultrastructural investigation of 274 human pituitary adenomas and 39 nontumorous adenohypophyses revealed two distinct types of secretion. Exocytosis was characteristic of prolactin cell adenomas, mixed growth hormone-prolactin cell adenomas, acidophil stem cell adenomas, and nontumorous prolactin cells. The second type, termed "transmembrane effusion," was noted in corticotroph, thyrotroph, gonadotroph, undifferentiated cell adenomas, and oncocytomas, as well as nontumorous corticotrophs, thyrotrophs, and gonadotrophs. It differed from that of prolactin cells and resembled diacrine secretion of gastrointestinal gastrin cells and membrane release in the neurohypophysis. In adenomatous and nontumorous growth hormone cells, neither exocytosis nor transmembrane effusion were apparent, hence a third type of release is suggested. Electron microscopic study of release mechanisms is helpful in the differential diagnosis of pituitary adenomas, since discharge of secretory products is not identical in the various tumor types.     

Hypothalamic input is required for development of normal numbers of thyrotrophs and gonadotrophs, but not other anterior pituitary cells in late gestation sheep

To evaluate the hypothalamic contribution to the development of anterior pituitary (AP) cells we surgically disconnected the hypothalamus from the pituitary (hypothalamo-pituitary disconnection, HPD) in fetal sheep and collected pituitaries 31 days later. Pituitaries ( n = 6 per group) were obtained from fetal sheep (term = 147 ± 3 days) at 110 days (unoperated group) of gestation and at 141 days from animals that had undergone HPD or sham surgery at 110 days. Cells were identified by labelling pituitary sections with antisera against the six AP hormones. Additionally, we investigated the colocalization of glycoprotein hormones. The proportions of somatotrophs and corticotrophs were unchanged by age or HPD. Lactotrophs increased 80% over time, but the proportion was unaffected by HPD. Thyrotrophs, which were unaffected by age, increased 70% following HPD. Gonadotrophs increased with gestational age (LH+ cells 55%; FSH+ cells 19-fold), but this was severely attenuated by HPD. We investigated the possible existence of a reciprocal effect of HPD on multipotential glycoprotein-expressing cells. Co-expression of LH and TSH was extremely rare (< 1%) and unchanged over the last month of gestation or HPD. The increase of gonadotrophs expressing FSH only or LH and FSH was attenuated by HPD. Therefore, the proportions of somatotrophs, lactotrophs and corticotrophs are regulated independently of hypothalamic input in the late gestation fetal pituitary. In marked contrast, the determination of the thyrotroph and gonadotroph lineages over the same time period is subject to complex mechanisms involving hypothalamic factors, which inhibit differentiation and/or proliferation of thyrotrophs, but stimulate gonadotrophs down the FSH lineage. Development of a distinct population of gonadotrophs, expressing only LH, appears to be subject to alternative mechanisms.     

Remodeling of Hyperplastic Pituitaries in Hypothyroid us-Subunit Knockout Mice After Thyroxine and 1713-Estradiol Treatment: Role of Apoptosis

Hyperplasia of pituitary thyrotrophs is often associated with hypothyroidism. In this study, the effects of thyroxine and 17β-estradiol on thyrotroph hyperplasia was analyzed using a hypothyroid mouse model resulting from targeted disruption of the glycoprotein hormone α-subunit (αSU) gene, which leads to lack of functional thyroid-stimulating hormone (TSH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) and underdevelopment of the thyroid and gonads. Thyroxine replacement for 2 mo resulted in a decrease in the relative percent of thyrotrophs and an increase of lactotrophs and somatotrophs numbers to normal values. A twofold increase in the relative percent of gonadotrophs was observed compared to wild-type mouse pitutary. Treatment for 2 mo with 17β-estradiol led to an increase in lactotroph numbers to normal levels, but had no influence on thyrotroph hyperplasia. Rearrangement of the hyperplastic pituitary phenotype after hormonal replacement proceeded without any evidence of pituitary cell necrosis. A slight increase in apoptotic cell death was observed in hormone-treated pituitaries, and this was localized to TSH cells by double-labeling experiments. Chronic thyroxine treatment resulted in increased expression of Bcl-2 protein in hypertrophied pituitary cells, whereas 17β-estradiol increased expression of Bad protein in prolactin cells. These results suggest that apoptotic cell death is involved in reversal of thyrotroph hyperplasia in the presence of thyroid hormone. Thyroxine and 17β-estradiol may influence cell death in this model by regulating expression of the Bcl-2 protein family in a cell-type specific manner.     

Ultrastructure of the fetal rat anterior pituitary gland as term and during prolonged gestation.

The ultrastructure of the fetal rat anterior pituitary gland is described at term (day 22) and during experimentally prolonged gestation (days 23, 24, 25). The latter was achieved by daily subcutaneous injections of five mg progesterone to gravid females from the twentieth through the twenty-fourth day. Using morphological criteria for classifying pituitary cells (Moriarty, '73), six different types were observed: thyrotrophs, gonadotrophs, corticotrophs, mammotrophs, somatotrophs and non-granulated cells. During prolonged gestation (days 24 and 25 only), cells designated as corticotrophs revealed changes suggesting increased activity, i.e.,proliferation and dilation of the endoplasmic reticulum, dilated Golgi membranes and a redction of secretory granules. In addition, meconium staining , which is indicative of fetal distress, was also observed on days 24 and 25. The ultrastructural changes noted suggest an increased in corticotroph activity due to fetal hypoglycemia and hypoxia which are known to occur as a result of placental insufficiency during prolonged gestation (Roux et al., '64; Vorherr, '75; Thliveris, '76.     

Morphology of the pituitary gland in ferrets (Mustela putorius furo) with hyperadrenocorticism

Pituitary tumours are the cause of hyperadrenocorticism in a variety of species, but the role of the pituitary gland in hyperadrenocorticism in ferrets is not known. In this species, the disease is mediated by the action of excess gonadotrophins on the adrenal cortex and is characterized by an excessive secretion of sex steroids. In this study, the pituitary gland of four healthy control ferrets, intact or neutered, and 10 neutered ferrets with hyperadrenocorticism was examined histologically following immunohistochemical labelling for adrenocorticotrophic hormone, alpha-melanocyte-stimulating hormone, growth hormone, thyroid-stimulating hormone, luteinizing hormone, follicle-stimulating hormone, and prolactin. Immunohistochemistry revealed that somatotrophs, thyrotrophs and lactotrophs were the most abundant cell types of the pars distalis of the pituitary gland in the healthy ferrets. The distribution of corticotrophs was similar to that in the dog and man. In ferrets, as in dogs, the melanotrophic cell was almost the only cell type of the pars intermedia. Gonadotrophs were found in the pars distalis of neutered, but not intact ferrets. All the ferrets with hyperadrenocorticism had unilateral or bilateral alterations of the adrenal gland. In addition, in the pituitary gland of two of these ferrets a tumour was detected. These tumours were not immunolabelled by antibodies against any of the pituitary hormones, and had characteristics of the clinically non-functional gonadotroph tumours seen in man. In some of the other ferrets low pituitary immunoreactivity for gonadotrophic hormones was detected, which may have been due to the feedback of autonomous steroid secretion by the neoplastic transformation of the adrenal cortex. It is concluded that initially high concentrations of gonadotrophins resulting from castration may initiate hyperactivity of the adrenal cortex. The low incidence of pituitary tumours and the low density of gonadotrophin-positive cells in non-affected pituitary tissue in this study suggest that persistent hyperadrenocorticism is not dependent on persistent gonadotrophic stimulation.     

Retention of peptide hormones during partial secretion in pituitary somatotrophs and corticotrophs

The secretion of peptide hormones during exocytosis of an individual vesicle can result in either complete discharge of vesicle content or can occur in a partial manner in which some hormone is retained during transient fusion. In anterior pituitary lactotrophs, the retained hormone prolactin was internalized and recycled into a pool of vesicles that underwent preferential use during subsequent exocytic stimulations [Bauer et al., (2004) J Cell Sci. 117:2193–2202]. The aim of the present study was to determine whether retention and preferential recycling of retained hormones occurred in other anterior pituitary cells. Stimulation of somatotrophs with high K⁺ resulted in 50 discrete puncta per cell that were positive for growth hormone immunoreactivity. Identical stimulation of corticotrophs resulted in 150 puncta per cell that were anti-adrenocorticotrophic hormone (ACTH) positive. However, unlike what was observed for lactotrophs, the number of structures containing retained growth hormone and ACTH decreased to less than 10% of the initial value in 80 min in somatotrophs and in less than 10 min in corticotrophs. Our results indicate that functional recycling of retained hormones is not shared by all anterior pituitary cell types.     

Proliferation and differentiation of pituitary somatotrophs and mammotrophs during late fetal and postnatal periods

Proliferation of somatotrophs and mammotrophs in the rat pituitary during late fetal and postnatal periods up to 4 weeks after birth was quantitatively studied with the double immunostaining of bromodeoxyuridine and the hormones produced by them. Somatotrophs were first detected in 18.5-day fetuses and rapidly increased in number throughout the periods studied. The cells labeled with both anti-BrdU and anti-GH were few in number until shortly before birth and then increased conspicuously during the first 10 days after birth. Mammotrophs were detected at gestational day 19.5 but they were few until the second week after birth, when their number began to increase rapidly. The percentage of the number of the cells double-labeled with both anti-BrdU and anti-GH to all somatotrophs was 8.3% at the most. This was about the same as that of corticotrophs during the late fetal period and that of thyrotrophs in the early postnatal period. In contrast, the percentage of double-labeled cells to all mammotrophs was 3.8% as a maximum, which is lower than the values for somatotrophs, corticotrophs, or thyrotrophs, indicating a smaller contribution of mitosis to mammotroph proliferation. It is possible that this smaller contribution is compensated for by transdifferentiation of cells committed to become the somatotroph lineage. However, coexistence of GH and PRL was not observed in the present material.     

The pituitary in cirrhosis: ultrastructure, growth hormone, and prolactin concentrations.

Micronodular cirrhosis was induced in male SUAH substrain Wistar rats by combined phenobarbitone and carbon tetrachloride treatment. Both pituitary and serum concentrations of growth hormone were significantly reduced in cirrhotic rats compared with age-related untreated rats or those treated only with phenobarbitone. Ultrastructurally growth hormone-secreting cells (somatotrophs) of pituitaries of cirrhotic rats appeared relatively inactive, having few hormone-containing granules, sparse rough endoplasmic reticulum, and small nuclei with areas of condensed chromatin. The cells themselves were smaller than similar cells of untreated rats with a reduced cytoplasmic area. In addition immunocytochemistry of pituitaries at light microscope level, using sheep anti-rat growth hormone antibody, showed that somatotrophs of cirrhotic rats were more heteromorphic and disorganized than those in controls. There was marked development of the folliculo-stellate cell system in pituitaries of cirrhotic rats, the cells were enlarged with distinct golgi, and numerous microvilli were projecting into dilated follicular lumena.     

Ghrelin increases intracellular Ca(2+) concentration in the various hormone-producing cell types of the rat pituitary gland

Ghrelin, isolated from the stomach as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), has potent growth hormone release ability in vivo and in vitro. Although GHS-R is abundantly expressed in the pituitary gland, there is no direct evidence of a relationship between hormone-producing cells and functional GHS-R in the pituitary gland. The aim of this study was to determine which anterior pituitary cells respond to ghrelin stimulation in male rats. We performed Fura-2 Ca(2+) imaging analysis using isolated pituitary cells, and performed immunocytochemistry to identify the type of pituitary hormone-producing cells. In Fura-2 Ca(2+) imaging analysis, ghrelin administration increased the intracellular Ca(2+) concentration in approximately 50% of total isolated anterior pituitary cells, and 20% of these cells strongly responded to ghrelin. Immunocytochemical analysis revealed that 82.9±1.3% of cells that responded to ghrelin stimulation were GH-immunopositive. On the other hand, PRL-, LH-, and ACTH-immunopositive cells constituted 2.0±0.3%, 12.6±0.3%, and 2.5±0.8% of ghrelin-responding pituitary cells, respectively. TSH-immunopositive cells did not respond to ghrelin treatment. These results suggest that ghrelin directly acts not only on somatotrophs, but also on mammotrophs, gonadotrophs, and corticotrophs in the rat pituitary gland.     

Hypophysial portal vascular infusion of TRH in the rat: An ultrastructural and radioimmunoassay study

The hypophysial portal vessels and anterior pituitary gland of adult male Wistar rats were exposed surgically. A hypophysial portal vessel was cannulated and infused for one minute with saline or thyrotrophin (TRH). Anterior pituitary glands were collected at 1, 5, 15, 30, or 60 minutes after cessation of infusion, for light and electron microscopic examination. Before and immediately after cannulation of a portal vessel, a 1-ml sample of blood was collected at 1, 5, 15, 30, or 60 minutes, from the femoral vein for radioimmunoassay (RIA) of growth hormone. Thyrotrophs from anterior pituitary glands of rats infused with TRH displayed emiocytic activity at all time-periods studied. Rough endoplasmic reticular (RER) cisternae were dilated at 15 minutes following infusion and remained dilated at 30 and 60 minutes. TRH was observed to stimulate emiocytic activity in most pituitary cell-types. Extensive dilations of RER cisternae were also observed in mammotrophs and gonadotrophs, but were not observed in somatotrophs or adrenocorticotrophs. The demonstration that thyrotrophs, mammotrophs, somatotrophs, and gonadotrophs respond to TRH suggests that some common features may be shared by these cells. Preliminary analysis of the RIA data show that TRH was potent in elevating radioimmunoassayable growth hormone levels. Significant increases (p < 0.02) in plasma GH levels were present at the earlier time periods studied (1, 5, and 15 minutes) following the infusion of TRH, but not at 30 or 60 minutes. These findings provide additional support for the non-specific action of TRH upon the various adenohypophysial cell types, and demonstrate that TRH stimulates these cells by a direct action on the adenohypophysis.     

Evidence for growth hormone (GH) autoregulation in pituitary somatotrophs in GH antagonist-transgenic mice and GH receptor-deficient mice

Growth hormone (GH) modulates the hypothalamic release of somatostatin and GH-releasing hormone; however, there has been no evidence of GH autoregulation on the pituitary somatotroph. To determine the effects of GH on its own regulation, we examined the pituitaries of giant transgenic mice expressing a GH agonist (E117L), dwarf transgenic mice expressing a GH antagonist (G119K), and dwarf mice devoid of the GH receptor/binding protein (GHR/BP). In the E117L transgenic mice, the number and distribution of pituitary GH-immunoreactive cells were unchanged from nontransgenic littermate controls; an ultrastructural examination revealed typical, densely granulated somatotrophs. In contrast, the pituitaries of the G119K mice contained both moderately granulated somatotrophs and a sparsely granulated (SG) population with well-developed synthetic organelles and a distinct juxtanuclear globular GH-staining pattern. GHR/BP-deficient mice exhibited a marked reduction in the intensity of cytoplasmic GH immunoreactivity; however, prominent GH staining in the juxtanuclear Golgi was seen. GH-immunoreactive cells were increased in number, and the reticulin network pattern was distorted; stains for proliferating cell nuclear antigen confirmed mild hyperplasia. Electron microscopy showed that the somatotrophs were hyperactive SG cells with prominent endoplasmic reticulum membranes, large Golgi complexes, and numerous mitochondria. These findings are consistent with synthetic and secretory hyperactivity in pituitary somatotrophs due to the reduced GH feedback regulation. The changes are most striking in animals that are devoid of GHR/BP and less marked in animals expressing a GH antagonist; both models had reduced insulin-like growth factor-I levels, but the more dramatic change in the GHR/BP animals can be explained by abrogated GH signaling. This represents the first evidence of direct GH feedback inhibition on pituitary somatotrophs, which may have implications for the use of GH analogs in different clinical settings.     

Differential calcium responses to the pituitary adenylate cyclase-activating polypeptide (PACAP) in the five main cell types of rat anterior pituitary

We have compared the effects of pituitary adenylate cyclase-activating polypeptide (PACAP-27) on the five main cell types of rat anterior pituitary in primary culture by monitoring changes in cytosolic Ca2+ concentration ([Ca2+]i) in single fura-2-loaded cells. Cells were typed by multiple sequential primary immunocytochemistry at the end of the Ca2+ measurements. PACAP-27 increased [Ca2+]i by three different mechanisms, each one dominant in a given cell type. These involved Ca2+ entry or release from the stores and mediation through different second messenger pathways: (1) stimulation of Ca2+ entry mediated by cAMP was the main mechanism in somatotrophs; (2) Ca2+ release from the intracellular Ca2+ stores mediated by phospholipase C (PLC) was the dominant modality in gonadotrophs; (3) stimulation of Ca2+ entry not mediated by cAMP was the main mechanism in lactotrophs. A minor fraction of somatotrophs (11%) may also use mechanism 3. Corticotrophs and thyrotrophs exhibited weak responses to PACAP (<10% of the cells responded), which in all cases were mediated by mechanism 1. Mechanism 3 represents a novel effect of PACAP which cannot be explained by interaction with the conventional PACAP receptor families.