vasoactive-intestinal-peptide and Pituitary-Neoplasms

Multiple Endocrine Neoplasia type 1 (MEN1) is a rare autosomal dominant hereditary cancer syndrome presented mostly by tumours of the parathyroids, endocrine pancreas and anterior pituitary, and characterised by a very high penetrance and an equal sex distribution. It occurs in approximately one in 30,000 individuals. Two different forms, sporadic and familial, have been described. The sporadic form presents with two of the three principal MEN1-related endocrine tumours (parathyroid adenomas, entero-pancreatic tumours and pituitary tumours) within a single patient, while the familial form consists of a MEN1 case with at least one first degree relative showing one of the endocrine characterising tumours. Other endocrine and non-endocrine lesions, such as adrenal cortical tumours, carcinoids of the bronchi, gastrointestinal tract and thymus, lipomas, angiofibromas, collagenomas have been described. The responsible gene, MEN1, maps on chromosome 11q13 and encodes a 610 aminoacid nuclear protein, menin, with no sequence homology to other known human proteins. MEN1 syndrome is caused by inactivating mutations of the MEN1 tumour suppressor gene. This gene is probably involved in the regulation of several cell functions such as DNA replication and repair and transcriptional machinery. The combination of clinical and genetic investigations, together with the improving of molecular genetics knowledge of the syndrome, helps in the clinical management of patients. Treatment consists of surgery and/or drug therapy, often in association with radiotherapy or chemotherapy. Currently, DNA testing allows the early identification of germline mutations in asymptomatic gene carriers, to whom routine surveillance (regular biochemical and/or radiological screenings to detect the development of MEN1-associated tumours and lesions) is recommended.

Topics: Adolescent; Adrenal Cortex Neoplasms; Adult; Aged; Aged, 80 and over; Angiofibroma; Carcinoid Tumor; Child; Facial Neoplasms; Female; Gastrinoma; Genetic Testing; Humans; Insulinoma; Lipoma; Male; Meningioma; Middle Aged; Multiple Endocrine Neoplasia Type 1; Pancreatic Neoplasms; Parathyroid Neoplasms; Pituitary Neoplasms; Prolactinoma; Proto-Oncogene Proteins; Thyroid Neoplasms; Vasoactive Intestinal Peptide; Young Adult

Topics: Animals; Growth Substances; Hormones; Humans; Kallikrein-Kinin System; Peptides; Pituitary Gland, Anterior; Pituitary Neoplasms; Renin-Angiotensin System; Vasoactive Intestinal Peptide

Somatostatin is a peptide synthesized in many tissues that can act as a neurotransmitter, a systemic hormone, or a local hormone, and inhibits the secretion of hormones or other cell products. A long-acting synthetic analogue of somatostatin (SMS 201-995) has been developed which when administered subcutaneously has a biologic half-life of 90 to 120 minutes and can be administered 2 or 3 times per day. SMS 201-995 can lower plasma concentrations of growth hormone and somatomedin-C in patients with pituitary acromegaly, but no controlled trials to assess symptomatic response or change in tumor size have been done. In patients with pituitary thyrotropin-producing pituitary tumors, SMS 201-995 has been remarkably effective in producing biochemical and clinical responses and is the drug of first choice in this syndrome when tumor resection is not possible. In patients with the carcinoid syndrome, SMS 201-995 effectively reduces diarrhea, is the best available drug for treatment of carcinoid flush (effective in approximately 90% of cases), and is useful in treating carcinoid crisis. Eighty-five percent of patients with pancreatic islet cell tumors that produce vasoactive intestinal peptide will respond to SMS 201-995 with a reduction in diarrhea that often has been resistant to all other therapy. SMS 201-995 may also be useful in treating the symptoms in some patients with glucagonomas, growth hormone releasing hormone-producing tumors and insulinomas. Whether SMS 201-995 has a significant effect on gut neuroendocrine tumor growth remains uncertain. Certain nonmalignant diseases of the gut respond to somatostatin, including secretory diarrhea and fistulas of unknown cause. In general, SMS 201-995 has proved safe with few significant side effects, but whether the long-term use of the drug will result in an iatrogenic form of the somatostatinoma syndrome is uncertain.

Topics: Acromegaly; Adenoma, Islet Cell; Diarrhea; Gastrointestinal Diseases; Gastrointestinal Hemorrhage; Gastrointestinal Neoplasms; Growth Hormone; Humans; Intestinal Fistula; Malignant Carcinoid Syndrome; Octreotide; Pancreatic Neoplasms; Pancreatitis; Pituitary Neoplasms; Thyrotropin; Vasoactive Intestinal Peptide

Topics: Adenoma; Adenylyl Cyclases; Adrenocorticotropic Hormone; Animals; Cyclic AMP; Growth Hormone; Haplorhini; Humans; Hypothalamus; In Vitro Techniques; Neurosecretion; Pituitary Gland, Anterior; Pituitary Neoplasms; Prolactin; Rats; Vasoactive Intestinal Peptide

As sensitive radioimmunoassays for the detection of polypeptide hormones are developed, the exciting discovery of a diffusely distributed system of interrelated endocrine cells has begun a new era of endocrinology. This system, although anatomically disassociated, is bound together by a number of common features such as its biosynthetic mechanism, histochemical and ultrastructural features, and embryologic origin (Table I). The most prominent feature, however, is their biosynthetic pathways for hormone production, from which the acronym APUD has been derived. These are the capacity for Amine Precursor Uptake such as DOPA and then subsequent Decarboxylation, resulting in the synthesis of bioactive amines or polypeptide hormones. Hyperplasias or neoplasms of these cells are defined as apudomas. In the last ten years a great deal of research has rapidly altered the original concepts of this system, especially in terms of its embryologic origin, physiologic interrelationships, classification, as well as the addition of many new APUD cell members. These will be reviewed, and the origin, diagnosis, and treatment of each recognized apudoma will be synthesized in light of its membership within the APUD system.

Topics: Adenoma, Islet Cell; APUD Cells; Apudoma; Carcinoid Tumor; Carcinoma; Endocrine Glands; Humans; Neural Crest; Neuroblastoma; Paraganglioma; Pheochromocytoma; Pituitary Neoplasms; Somatostatin; Thyroid Neoplasms; Vasoactive Intestinal Peptide

We examined a possible GH-releasing activity of vasoactive intestinal peptide (VIP) and its homologous peptide, peptide histidine methionine (PHM), in 22 patients with hyperprolactinemia (HPRL) who comprised 19 cases of prolactinoma (PRLoma) and 3 cases of hypothalamic HPRL. Each patient underwent iv bolus injections of VIP (100 micrograms) and PHM (100 micrograms) on separate days, and plasma levels of GH and PRL were measured. The plasma GH response to VIP and PHM were considered positive (a paradoxical increase) when an increase over baseline of at least 50% occurred. In agreement with previous reports, the PRL-releasing activity of VIP and PHM in our patients with HPRL were subnormal. Thirteen (59%) patients showed a paradoxical rise in GH after VIP, and 4 (18%) patients did so after PHM. It is to be noted that all the 3 patients with hypothalamic HPRL responded to VIP with a significant rise in GH. 3 of the 4 PHM-responders were also responsive to VIP, which suggests that PHM may have activated VIP receptors in the pituitary of the PHM-responders as a partial agonist of the VIP receptor. The responders and nonresponders to VIP or PHM, respectively, had similar results with respect to the mean age, and the mean basal PRL and GH levels in the plasma. Since these paradoxical GH responses were observed in not only the patients with PRLoma but also those with hypothalamic HPRL, it may be that these anomalous GH responses in HPRL were due to the HPRL itself rather than due to the neoplastic lactotrophs.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adolescent; Adult; Female; Growth Hormone; Humans; Hyperprolactinemia; Hypothalamic Diseases; Male; Middle Aged; Peptide PHI; Pituitary Neoplasms; Prolactinoma; Sequence Homology, Amino Acid; Vasoactive Intestinal Peptide

Vasoactive intestinal polypeptide (VIP) was infused into normal volunteers and patients with hyperprolactinaemia. Heart rate increased from 62 +/- 3 to 75 +/- 3 beats min-1 (P = 0.001) in controls and from 70 +/- 2 to 78 +/- 3 beats min-1 (P = 0.001) in hyperprolactinaemics. Similarly, haematocrit increased from 38 +/- 2 to 44 +/- 1% (P = 0.001) and from 40 +/- 1 to 43 +/- 2% (P = 0.002) and plasma renin activity from 910 +/- 59 to a peak of 3344 +/- 282 pg ml-1 h-1 (P = 0.001) and from 1577 +/- 671 to a peak of 4954 +/- 1364 pg ml-1 h-1 (P = 0.001) in the two groups, respectively. Prolactin concentrations rose in the control group only, from 134 +/- 11 to a peak of 377 +/- 35 mU 1(-1) (P = 0.001), whilst in the hyperprolactinaemics little change occurred from the pre-infusion concentration of 3873 +/- 2179 reaching a peak of 3998 +/- 2347 mU 1(-1) (P greater than 0.07). In separate studies, the normal subjects were pretreated with either bromocriptine or dexamethazone. Dexamethazone did not alter any parameter of the response to VIP. Bromocriptine did not affect the heart rate, haematocrit or renin response to VIP but clearly inhibited the rise in prolactin which remained at unmeasurable concentrations throughout the infusion.

Topics: Adenoma; Adult; Bromocriptine; Dexamethasone; Female; Heart Rate; Hematocrit; Humans; Male; Pituitary Neoplasms; Prolactin; Renin; Vasoactive Intestinal Peptide

Suprasellar tumors with compression of the optic chiasm are associated with an impaired sleep-wake rhythm. We hypothesized that this reflects a disorder of the biological clock of the human brain, the suprachiasmatic nucleus (SCN), which is located just above the optic chiasm. In order to test this hypothesis, we investigated the expression of two key neuropeptides of the SCN, that is, arginine vasopressin (AVP) and vasoactive intestinal peptide (VIP), as assessed by quantitative immunocytochemistry in post-mortem hypothalamic tissue of patients with a suprasellar tumor inducing permanent visual field defects. Post-mortem hypothalamic tissue of 5 patients with a suprasellar tumor inducing permanent visual field defects (acromegaly n = 2, nonfunctioning macro-adenoma n = 1, macroprolactinoma n = 1, infundibular metastasis of a colorectal adenocarcinoma n = 1) and 15 age- and gender-matched controls was obtained from the Netherlands Brain Bank. Total AVP immunoreactivity in the SCN was lower in patients with a suprasellar tumor than in controls (P = 0.03). By contrast, total VIP immunoreactivity was not different between patients and controls (P = 0.44). Suprasellar tumors leading to permanent visual field defects are associated with reduced AVP, but not VIP immunoreactivity, in the SCN. These findings raise the possibility that selective impairment of the SCN contributes to sleep-wake disturbances in these patients.

Topics: Aged; Aged, 80 and over; Arginine Vasopressin; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Perceptual Disorders; Pituitary Neoplasms; Postmortem Changes; Suprachiasmatic Nucleus; Vasoactive Intestinal Peptide; Visual Fields

Clinical studies have noted the common presentation of pituitary tumours with significant headache. This has been considered to be one, or a combination of, increased cranial pressure, tumour size with dural stretch, or cavernous sinus invasion. Newer hypotheses suggest an association between the presence of pituitary tumour-associated headaches and the expression and release of nociceptive substances. Vasoactive intestinal polypeptide (VIP), a marker of the cranial parasympathetic system, is increased during acute attacks of some primary headaches, and with its expression in the pituitary may link some pituitary tumours to their headache presentations.. Using immunohistochemical techniques, VIP expression in pituitary tumour specimens was examined to determine if there was a relationship between the presence or absence of pituitary-associated headache and the expression of VIP immunoreactivity (VIP-IR). A qualitative analysis of the VIP-IR in pituitary cells was performed by observers blinded to the headache status of each patient. The presence of VIP-IR and headache were treated as binary variables and associations tested with chi-square tests.. Forty-five per cent of specimens positive for VIP were from patients presenting with headache. There was no statistically significant association between the presence of VIP-IR and headache (chi(2) = 0.077, P = 0.781).. Although the significance of VIP positivity in pituitary tumour-associated headache is unknown it seems unrelated to headache. It remains possible that the mechanism of these headaches relates to the production of either an as yet unidentified peptide, or a combination of nociceptive peptides.

Topics: Biomarkers; Female; Headache; Humans; Immunohistochemistry; Male; Middle Aged; Neuroprotective Agents; Pituitary Neoplasms; Vasoactive Intestinal Peptide

We have previously described a sexual dimorphism in oestrogen-induced anterior pituitary tumorigenesis in Fischer 344 rats, with female tumours averaging twice the size of those of males. Neonatal androgenization of female Fischer 344 rats with 100 micro g of testosterone propionate reverted that effect, causing a 'male-like' phenotype. The peptides galanin and vasoactive intestinal peptide (VIP) are possible mediators of oestrogen effects on the anterior pituitary, including hyperprolactinemia and lactotroph proliferation. To further extend our previous findings, we investigated the expression of galanin and VIP in the anterior pituitary of control and oestrogenized male, female and neonatally androgenized female Fischer 344 rats. At 3 months of age, rats were deprived of their gonads and divided into control and diethylstilbestrol (DES)-treated groups. In the anterior pituitary of control rats, galanin and VIP immunoreactive cells were absent. However, in DES-treated rats, pituitaries from normal ovariectomized females showed higher number of galanin and VIP positive cells than pituitaries from neonatally androgenized ovariectomized females and gonadectomized males. This pattern correlated with changes in anterior pituitary weight and serum prolactin. Our study suggests that sexual differences in oestrogen-induced pituitary tumorigenesis could be due to the differential expression of galanin and VIP. Furthermore, our data support the fact that neonatal exposure to androgens, as in normal males and androgenized females, may condition the response of the pituitary gland to oestrogens in adult life.

Topics: Animals; Animals, Newborn; Diethylstilbestrol; Female; Galanin; Male; Pituitary Neoplasms; Prolactinoma; Rats; Rats, Inbred F344; Sex Characteristics; Sex Differentiation; Testosterone; Vasoactive Intestinal Peptide; Virilism

Neuropeptides act within the pituitary as autocrine or paracrine factors, modulating the synthesis and release of primary pituitary hormones, and possibly regulating cell proliferation and/or plasticity. Manipulation of the endocrine status of rats produces dramatic long-term changes in the pituitary expression of several peptides, including the neuropeptides galanin and vasoactive intestinal peptide (VIP). Whether or not these changes are caused indirectly by hypothalamic factors, or by hormone actions directly in the pituitary, has been only partially addressed. To determine if estrogen or thyroid hormone can act directly within the pituitary to regulate VIP and galanin gene expression, cultured female rat pituitary cells were treated with 10 nM 1,17 beta-estradiol (E2) or triiodothyronine (T(3)). E2 treatment for three days resulted in an approximate 5-fold and 7-fold increase in VIP and galanin mRNA, respectively. In contrast, T(3) treatment reduced the mRNA levels of these neuropeptides to approximately 40% and 30% of control values. A time course study indicated that the actions of estrogen on VIP and galanin mRNA, and of thyroid hormone on VIP mRNA were readily apparent after 24h. The rat pituitary tumor cell line RC-4B/C was found to express easily detectable levels of galanin but not VIP mRNA. Galanin gene expression in these cells was moderately increased by E2 and decreased by T(3). Transfection of a series of luciferase plasmids containing 5 kb to 131 bp of the bovine galanin promoter fused to luciferase revealed cell-type specific enhancer sequences located between -452 and -131 bp of the galanin gene transcription start site. However, transfected plasmids were minimally responsive to E2 and T(3) treatment. Overall the results suggest that E2 and T(3) exert significant local actions in the pituitary on VIP and galanin gene expression. The bovine galanin gene fragment used in these studies contains a potential pituitary cell-type specific enhancer, but appears to lack strong E2-and T(3)-responsive sequences.

Topics: Animals; Cattle; Cells, Cultured; Enhancer Elements, Genetic; Estradiol; Female; Galanin; Gene Expression Regulation; Genes, Reporter; Genes, Synthetic; Luciferases; Mice; Organ Specificity; Pituitary Gland, Anterior; Pituitary Neoplasms; Promoter Regions, Genetic; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; Recombinant Fusion Proteins; RNA, Messenger; Transfection; Triiodothyronine; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Six GH adenomas and three prolactinomas were investigated by light- and electron-microscopic morphological and immunocytochemical methods and the effect of vasoactive intestinal polypeptide (VIP) on growth hormone (GH) and prolactin (PRL) secretion was tested in vitro. The tumour cells of the acromegalic patients revealed both GH and PRL immunoreactivity while prolactinomas showed only PRL activity. All the adenomas stained immunocytochemically also for VIP. By electron microscopy, the tumours included two densely and two sparsely granulated GH, two mixed GH/PRL, and three sparsely granulated PRL adenomas. The dissociated cells were explanted, and cultured in vitro. The cultures in micro test plates were treated with VIP at different concentrations between 10(-5)-10(-12) M. GH and PRL contents in the culture media were measured by radioimmunoassay. GH release was significantly stimulated by VIP in a dose-dependent manner over the whole concentration range, while VIP was effective on the PRL release only at 10(-6)-10(-7) M concentration. The cells of a mixed adenoma were grown in Petri dishes and used for ultrastructural and immunocytochemical studies. The cytoplasmic structure of the cells treated with VIP corresponded to that of active hormone-secreting cells with large ergastoplasmic fields and Golgi zones containing secretory granules. Massive exocytotic events were encountered mainly in the GH-type cells. GH and PRL double immunocytochemistry showed the predominance of GH cells, many of them containing low amounts of PRL as well. Cells predominantly containing PRL were spread among them, they also might contain GH as well. Some of the cells contained only a single immunoreactive hormone. The intensity of gold labelling of the secretory granules appeared higher in the VIP-treated cells than in the untreated control ones which showed a cytoplasmic structure characteristic of glandular cells with low secretory activity. As all the adenoma cells both contained and reacted to VIP, our results are in agreement with an autocrine or paracrine effect of this peptide. The fine structure of the cells in the cultures treated with VIP supply an additional argument to the assumption that VIP may serve as a growth factor for these cell types.

Topics: Acromegaly; Adenoma; Adult; Cytoplasm; Cytoplasmic Granules; Exocytosis; Female; Human Growth Hormone; Humans; Immunohistochemistry; Male; Microscopy, Electron; Middle Aged; Pituitary Neoplasms; Prolactin; Prolactinoma; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

To study the secretion from endocrine cells in culture, we have developed a cell column perfusion system with a time resolution of 4 s. The core of the system is a perfusion chamber with a cell-supporting matrix of monosized polystyrene beads. The particles are solid and can withstand a high pressure gradient without deformation. The minimum amount of cell material required to obtain detectable levels of secretory products is a function of the assay sensitivity, perfusion flow, fraction volume and time resolution. The volume of the perfusion chamber is therefore adjustable to satisfy varying demands of minimum cell number. The general flow characteristics of the system were characterized using radiolabeled substances of various molecular sizes. Using the system in secretory studies of rat pituitary tumor (GH4C1) cells, we have identified differences in secretion profiles that may be related to the kinetics of the different transmembrane and intracellular mechanisms involved.

Topics: Animals; Cells, Immobilized; Kinetics; Microscopy, Electron, Scanning; Microspheres; Peptides; Perfusion; Pituitary Neoplasms; Prolactin; Rats; Rheology; Thyrotropin-Releasing Hormone; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP), members of the glucagon-secretin family, have recently been suggested to be involved in the regulation of corticotropin (ACTH) secretion. In this study, we examined the effects of both peptides on POMC gene expression. Using AtT20PL, a clone of the AtT20 mouse corticotroph tumor cells stably transfected with 0.7 kb of the rat POMC 5' promoter-luciferase fusion gene, the effects of both peptides on the POMC promoter activity were estimated by a luciferase assay. PACAP stimulated POMC 5' promoter activity as well as cAMP generation and ACTH secretion in a dose- and time-dependent manner, with the maximal effect being observed 3 h after the start of incubation. A similar effect was observed with VIP. Although the combined effects of PACAP/CRH or VIP/CRH were greater than that of either hormone alone, no such effect was observed between PACAP and VIP. Furthermore, RT-PCR analysis showed the presence of only the PVR3 receptor subtype in this cell line, which is known to have a similar affinity to PACAP and VIP, indicating that both peptides exert their effects through the same receptor. In contrast to the effect of CRH, which was completely abolished by a protein kinase A inhibitor H89, the effects of PACAP/VIP on POMC expression persisted during H89 treatment, suggesting the involvement of alternative intracellular signaling pathway(s) distinct from the protein kinase A system. Our results suggest that PACAP and VIP have positive effects on POMC gene expression and that multiple signaling pathways are involved in the transcriptional event.

Topics: Adenylyl Cyclases; Adrenocorticotropic Hormone; Animals; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation; Isoquinolines; Mice; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Pituitary Neoplasms; Polymerase Chain Reaction; Pro-Opiomelanocortin; Rats; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Hormone; Receptors, Vasoactive Intestinal Peptide; Sulfonamides; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Mastoparan has been reported to induce a wide variety of cellular actions by activating GTP-binding proteins (G proteins) in various cells. Here, we demonstrate that mastoparan is able to stimulate the secretion of PRL from rat anterior pituitary tumor GH3 cells in dose- and time-dependent manners. Mastoparan had no effect on the accumulation of intracellular cAMP; however, it induced a rapid increase in the intracellular Ca2+ concentration in GH3 cells. Extracellular Ca2+ was required for mastoparan-induced PRL secretion, which was inhibited by nifedipine, an L-type Ca2+ channel blocker. Incubation of mastoparan with myo-[3H]inositol-labeled GH3 cells also resulted in the increased formation of inositol phosphates (InsPs) compared with control cells. Neomycin sulfate and U73122, both phospholipase C inhibitors, suppressed mastoparan-induced PRL secretion. Guanosine 5'-1beta-thioldiphosphate (GDPbetaS) encapsulated in GH3 cells by reversible electropermeabilization suppressed the response to mastoparan. However, pretreatment with pertussis toxin had no effect on the stimulation of PRL secretion by mastoparan, and both Mas7 (a highly active analogue of mastoparan) and Mas17 (an inactive analogue) enhanced the secretion of PRL to a similar level to that of mastoparan-induced GH3 cells. In contrast, the substance P-related peptide GPant-2A, a Gq antagonist, inhibited mastoparan-induced PRL release, whereas GPant-2, a G(i/o) antagonist, did not in electropermeabilized GH3 cells. Moreover, a specific G(q/11) antibody against the carboxyl terminus of the G(q/11) alpha-subunit blocked the stimulatory effect of mastoparan on secretion and mastoparan-stimulated InsPs production in digitonin-permeabilized GH3 cells. These results indicate that mastoparan induces the Ca2+-regulated secretion of PRL from GH3 cells by activating G(q/11) and the phospholipase C pathway.

Topics: Animals; Calcium; Calcium Channel Blockers; Cyclic AMP; Exocytosis; GTP-Binding Proteins; Inositol Phosphates; Intercellular Signaling Peptides and Proteins; Nifedipine; Peptides; Pituitary Gland, Anterior; Pituitary Neoplasms; Prolactin; Rats; Thyrotropin-Releasing Hormone; Tumor Cells, Cultured; Type C Phospholipases; Vasoactive Intestinal Peptide; Wasp Venoms

Topics: ACTH Syndrome, Ectopic; Adenoma; Adrenocorticotropic Hormone; Adult; Aged; Cushing Syndrome; Female; Humans; Male; Middle Aged; Petrosal Sinus Sampling; Pituitary Neoplasms; Prolactin; Vasoactive Intestinal Peptide

We have investigated the prevalence of MEN 1 in patients with recognized pituitary adenomas. Since hyperparathyroidism is present in nearly 95-100% of patients with MEN 1 and frequently is the first condition to be identified, the study was limited to the identification of patients with hyperparathyroidism while the screening for gastroenteropancreatic (GEP) lesions was carried out in patients with both pituitary and parathyroid lesions.. Serum total and ionized calcium, phosphate and intact PTH 1-84 (EASIA) were measured in 166 patients (68 with non-functioning pituitary adenoma, 42 with prolactinoma, 35 with GH-secreting adenoma, 17-with ACTH-screening adenoma, 1 with TSH-secreting adenoma, 1 with FSH-secreting adenoma and 2 with an only alpha-subunit secreting adenoma) referred to our clinic from 1990 to 1996. Plasma gastrin, somatostatin, pancreatic polypeptide and vasoactive intestinal peptide were measured by RIA in patients with hyperparathyroidism.. Eight of 166 patients (4.8%) were found to have primary hyperparathyroidism and among these 2 also had a gastrinoma while there was no evidence of other GEP tumours. Considering the tumour type, 6 had prolactinoma (14.3%), 1 GH-secreting adenoma (2.8%) and 1 non-functioning adenoma (1.5%). In most patients the diagnosis of pituitary tumour was made several years before that of hyperparathyroidism (from 1 to 15 years) although 6 patients had previously suffered from urolithiasis and one had undergone gastric resections for recurrent peptic ulcers. One patient was identified as a MEN 1 gene carrier and 2 had relatives with signs and symptoms referable to parathyroid or GEP lesions.. The study shows a prevalence of 4.8% of primary hyperparathyroidism in unselected patients with known pituitary tumours similar to that reported in a previous study. By contrast, the prevalence of MEN 1 in patients with prolactinoma was definitely high (14.3%). In most patients the diagnosis of pituitary tumours was made several years before that of hyperparathyroidism. Although the patients were believed to harbour a sporadic pituitary tumour, most of them had had signs and/or symptoms referable to one or both of the other organs involved in MEN 1, often concomitantly with those of pituitary tumours. These data indicate that the diagnosis of MEN 1 syndrome is missed in a substantial proportion of patients with prolactinomas and therefore the screening of these patients for the syndrome is strongly recommended.

Topics: Adenoma; Adolescent; Adult; Aged; Biomarkers; Calcium; Female; Gastrinoma; Gastrins; Humans; Hyperparathyroidism; Male; Middle Aged; Multiple Endocrine Neoplasia Type 1; Pancreatic Polypeptide; Parathyroid Hormone; Phosphates; Pituitary Neoplasms; Prevalence; Prolactinoma; Somatostatin; Vasoactive Intestinal Peptide

The effects of gastrin-releasing peptide (GRP) and vasoactive intestinal polypeptide (VIP) on the prolactin gene transcription of cultured pituitary of male Sprague-Dawley (SD) rats PRL releasing tumor (PPRT) (induced by estradiol) cells were studied. The PRL mRNA levels were determined by in situ hybridization of cytoplasmic RNA with a DIG-labeled PRL cDNA probe. PRL mRNA levels didn't change when the PPRT cells were incubated with 10(-8) mol/L or 10(-7) mol/L GRP for 24 h, but decreased by 20% when GRP was increased to 10(-6) mol/L (P < 0.05). The PRL mRNA level increased to 1.60, 2.10, 2.21 times of the control group when the PPRT cells were respectively incubated with 10(-8), 10(-7), 10(-6) mol/L VIP for 24 h (P < 0.05). The PRL mRNA level didn't change when the PPRT tumor cells were incubated with 10(-8) mol/L E2 for 48 h, but did increase to 2.80 and 2.92 times of the control group respectively when 10(-7) mol/L and 10(-6) mol/L E2 were used. The results above indicated that GRP and VIP exert an inhibitory and a stimulatory effect on RPL gene transcription respectively, while the stimulatory action of E2 on PRL secretion is a direct one.

Topics: Animals; Estradiol; Gastrin-Releasing Peptide; Male; Pituitary Neoplasms; Prolactin; Prolactinoma; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Signals responsible for expression of the vasoactive intestinal peptide (VIP)-stimulated prolactin gene in GH3 pituitary tumor cells were examined. Transfection with a deoxyribonucleic acid (DNA) construct containing the chloramphenicol acetyltransferase (CAT) gene fused to the 2.5-kb prolactin 5'-upstream regulatory sequence indicated that VIP stimulated CAT expression. However, this effect could not be mimicked by 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), and was inhibited by the L-type Ca(2+)-channel blocker verapamil. While KCl had little effect on CAT activity, combined treatment with KCl and 8-Br-cAMP synergistically activated CAT expression. Potentiation between KCl and 8-Br-c-AMP was also seen with c-fos messenger ribonucleic acid (mRNA) expression. In addition, KCl and 8-Br-cAMP synergistically activated cAMP response element (CRE)-mediated CAT expression, and the synergism was abolished by verapamil. In the presence of okadaic acid, cAMP had no significant activation on CRE-driven CAT expression, whereas KCl-stimulated CAT expression was greatly potentiated. These results indicate that cAMP and Ca2+ synergistically activated CRE-driven gene expression through non-overlapping phosphorylation events in GH3 cells.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Calcium; Calcium Channel Blockers; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Drug Synergism; Gene Expression Regulation, Neoplastic; Genes, fos; Phosphorylation; Pituitary Neoplasms; Potassium Chloride; Prolactin; Promoter Regions, Genetic; Rats; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The presence of the pertussis toxin (PTX) insensitive GTP-binding proteins (C-proteins) G(q) alpha and/or G(11) alpha has been demonstrated in three different prolactin (PRL) and growth hormone (GH) producing pituitary adenoma cell lines. Immunoblocking of their coupling to hormone receptors indicates that G(q) and/or G(11) confer throliberin (TRH) responsive phospholipase C (PL-C) activity in these cells. The contention was substantiated by immunoprecipitation analyses showing that anti G(q)/11 alpha-sera coprecipitated PL-C activity. In essence, only G(q)/11 (but neither G(12) G(13) nor G(o)) seems to mediate the TRH-sensitive PL-C activity, while G(o) may be coupled to a basal or constitutive PL-C activity. Immunoblocking studies imply that the B gamma-complex also, to some extent, may stimulate GH(3) pituitary cell line PL-C activity. Finally, the steady state levels of G(q)/(11) alpha mRNA and protein were down regulated upon long term exposure of the GH(3) cells to TRH (but not to vasoactive intestinal peptide = VIP).

Topics: Adenoma; Animals; Enzyme Activation; Female; GTP-Binding Proteins; Liver; Pertussis Toxin; Pituitary Gland; Pituitary Neoplasms; Rats; Rats, Sprague-Dawley; RNA, Messenger; Thyrotropin-Releasing Hormone; Tumor Cells, Cultured; Type C Phospholipases; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

Gonadotropes, like other cells, receive informational input from multiple receptor types, acting through multiple intracellular signaling pathways, and are therefore faced with the task of integrating this input in order to respond appropriately to their environment. In recent years an increasing number of examples of functional interactions occurring between the PIC and adenylyl cyclase signaling pathways in gonadotropes have been described, and the discovery that these cells are targets for PACAP has provided a physiological context for earlier work on gonadotrope regulation by cyclic AMP. The development of the alpha T3-1 cell line has greatly facilitated investigation of the interaction between these signaling systems. In these cells we have obtained no evidence for interaction between the GnRH and PACAP receptor-effector systems at the level of receptor occupancy or expression, but these systems clearly do have reciprocal modulatory effects on second messenger generation and/or mobilization. We are now faced with the challenge of determining the physiological and/or pathophysiological relevance of such interactions.

Topics: 1-Methyl-3-isobutylxanthine; Adenylyl Cyclases; Animals; Cell Line; Cyclic AMP; Follicle Stimulating Hormone; Gonadotropin-Releasing Hormone; Inositol; Kinetics; Luteinizing Hormone; Neuropeptides; Neurotransmitter Agents; Pituitary Adenylate Cyclase-Activating Polypeptide; Pituitary Gland; Pituitary Neoplasms; Receptors, LHRH; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Hormone; RNA, Messenger; Second Messenger Systems; Transcription, Genetic; Vasoactive Intestinal Peptide

Experiments were performed to determine whether PRL secretion in the rat diethylstilbestrol (DES)-induced prolactinoma model is affected by the addition of thyrotropin-releasing hormone (TRH) and/or immunoneutralization of intrapituitary vasoactive intestinal polypeptide (VIP) in vitro. Male Fischer 344 rats were implanted with either a 10 mg DES or placebo pellet 30 days prior to obtaining the anterior pituitary glands for perifusion. The anterior pituitaries were quartered and used in three different perifusion experiments. In Experiment I, placebo-treated tissue channels were perifused for 2 baseline hr followed consecutively by a 30-min exposure to 1:100 nonimmune rabbit serum (NRS), a 30-min wash, and a final 30-min exposure to 10(-5) M TRH. Additional placebo channels were run as above except 1:100 VIP antiserum (AVIP) was substituted for NRS and AVIP was added to the TRH. In Experiment II, the same perifusion protocol was used as in Experiment I, except DES-induced tumor tissue was used instead of placebo tissue. Results from Experiment I and II reveal that AVIP significantly decreased PRL secretory rate in both DES and placebo groups. In the tumor group, both TRH alone and in the presence of AVIP significantly increased the PRL secretory rate. In Experiment III DES-induced tumor tissue channels were perifused with a similar protocol, except the concentrations of NRS and AVIP were increased to 1:10. Both NRS and AVIP significantly decreased PRL secretory rate; however, AVIP had a significantly greater effect than NRS. In this experiment, 1:10 AVIP overcame the stimulatory effect of TRH. In conclusion, AVIP decreases and TRH increases, even in the presence of AVIP, PRL release in DES-induced prolactinoma tissue in vitro. Increasing the AVIP concentration 10-fold diminished the PRL-releasing action of TRH in the tumor tissue. These data suggested that PRL secretion is not autonomous in these prolactinomas and can be affected by exogenous TRH and partial immunoneutralization of endogenous VIP.

Topics: Animals; Diethylstilbestrol; Immune Sera; In Vitro Techniques; Male; Pituitary Gland, Anterior; Pituitary Neoplasms; Placebos; Prolactin; Prolactinoma; Rabbits; Radioimmunoassay; Rats; Rats, Inbred F344; Thyrotropin-Releasing Hormone; Vasoactive Intestinal Peptide

Ergovaline inhibition of radioligand binding to the D2 dopamine receptor and ergot alkaloid inhibition of vasoactive intestinal peptide (VIP)-stimulated cyclic AMP production in GH4ZR7 cells, stably transfected with a rat D2 dopamine receptor, were evaluated. Ergovaline inhibition of the binding of the D2-specific radioligand, [3H]YM-09151-2, exhibited a KI (inhibition constant) of 6.9 +/- 2.6 nM, whereas dopamine was much less potent (370 +/- 160 nM). Ergot alkaloids were also effective in inhibiting VIP-stimulated cyclic AMP production, with EC50 values for ergovaline, ergonovine, alpha-ergocryptine, ergotamine, and dopamine of 8 +/- 2, 47 +/- 2, 28 +/- 2, 2 +/- 1, and 8 +/- 1 nM, respectively. Inhibition of cyclic AMP production by ergovaline was blocked by the dopamine receptor antagonist, (-)-sulpiride (IC50, 300 +/- 150 nM). Our results indicate that ergot compounds, especially ergovaline, bind to D2 dopamine receptors and elicit second messenger responses similar to that of dopamine. These findings suggest that some of the deleterious effects of consumption of endophyte-infected tall fescue, which contains several ergot alkaloids including ergovaline, may be due to D2 dopamine receptor activation.

Topics: Adenosine Monophosphate; Animals; Cell Membrane; Ergotamines; Pituitary Neoplasms; Radioligand Assay; Rats; Receptors, Dopamine D2; Second Messenger Systems; Sulpiride; Transfection; Tumor Cells, Cultured; Vasoactive Intestinal Peptide; Vasoconstrictor Agents

Experiments were designed to examine whether vasoactive intestinal polypeptide (VIP), a known stimulator of basal prolactin (PRL) secretion, regulates PRL gene expression in the rat diethylstilbestrol (DES)-induced prolactinoma model. The VIP-induced increase in PRL release could result from increased PRL synthesis and/or decreased PRL degradation. Male Fischer 344 rats were implanted with 10 mg DES pellets 40 or 110 days prior to obtaining the anterior pituitary glands for cell dispersal. Cells were incubated in 1:10 normal rabbit serum or VIP antiserum (AVIP). After incubation, cells were pelleted, washed, and pooled for total nucleic acid extraction. The rat PRL (rPRL) mRNA abundance was quantitated using a solution hybridization/ribonuclease protection assay. Supernatant was collected and analyzed for PRL content using radioimmunoassay. Results from this experiment reveal partial immunoneutralization of intrapituitary VIP significantly decreased PRL secretory rate by rapid reduction in rRPL mRNA in the 40-day tumors. However, in the 110-day tumors the rPRL mRNA steady-state levels were unchanged but the basal release of PRL continued to be decreased by AVIP. These results indicate VIP exerts its effects on PRL secretion through at least two mechanisms.

Topics: Animals; Diethylstilbestrol; Immune Sera; Male; Pituitary Neoplasms; Prolactin; Prolactinoma; Rats; Rats, Inbred F344; Recombinant Proteins; RNA, Messenger; Time Factors; Vasoactive Intestinal Peptide

Detection of subjects from a multiple endocrine neoplasia type 1 family must rest on clinical, biochemical and radiological data, since study of the genome is unable to detect these subjects. In the new family described here, 6 out of the 14 subjects explored were affected. One had a confirmed pancreatic endocrine tumour and in 3 others a pancreatic endocrine tumour was highly probable, since insulin and glucagon levels, as well as ultrasonic exploration of the pancreas were pathological. Measurements of gastrointestinal hormones gave normal results in all cases. We conclude that to detect this endocrine neoplasia in subjects at risk it seems necessary to measure plasma insulin levels and perform an abdominal ultrasonography.

Topics: Adolescent; Adult; Aged; Blood Glucose; C-Peptide; Child; Female; Gastrins; Glucagon; Humans; Insulin; Male; Middle Aged; Multiple Endocrine Neoplasia; Pancreatic Neoplasms; Pancreatic Polypeptide; Pituitary Neoplasms; Risk Factors; Substance P; Ultrasonography; Vasoactive Intestinal Peptide

This study, carried out on 9 nonfunctioning pituitary adenomas, was undertaken in order to evaluate the ability of these tumors to synthesize and release gonadotropins and/or free alpha-subunit (alpha-SU) of glycoproteins. The morphological study included electron microscopy and immunofluorescence analysis while hormone release was evaluated by the reverse hemolytic plaque assay (RHPA) and measurements in culture media. By electron microscopy in all tumors (6 null cell adenomas and 3 oncocytomas), it was possible to identify rough endoplasmic reticulum, Golgi apparatus and secretory granules. By immunofluorescence, 5 of 6 tumors were immunoreactive for one or more gonadotropin subunits; in particular, 5 adenomas were positive for alpha-SU and LH-beta, and 3 for FSH-beta. By the RHPA, about 1% of cells obtained from one single tumor formed plaques for LH-beta and alpha-SU while the remaining tumors were negative. Similarly, the study of media concentrations of LH, FSH and alpha-SU in 2 h culture revealed very low amounts of released hormones. In these experimental conditions no modification was observed after the addition of stimulatory agents such as TRH, GnRH and VIP. The present study clearly indicates that although the large majority of nonfunctioning tumors are positive for gonadotropins their secretory capacity is very low in both basal and stimulated conditions.

Topics: Adenoma; Cytoplasmic Granules; Endoplasmic Reticulum; Fluorescent Antibody Technique; Follicle Stimulating Hormone; Glycoprotein Hormones, alpha Subunit; Golgi Apparatus; Gonadotropin-Releasing Hormone; Gonadotropins, Pituitary; Growth Hormone; Hemolytic Plaque Technique; Humans; Luteinizing Hormone; Microscopy, Electron; Pituitary Neoplasms; Prolactin; Thyrotropin-Releasing Hormone; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

It is well known that dopamine (DA) inhibits while vasoactive intestinal peptide (VIP) and angiotensin II (ANG II) stimulate prolactin (PRL) release from normal anterior pituitary lactotrophs; however, elucidation of the intracellular mechanisms involved in these effects has been hindered by the cellular heterogeneity of the anterior pituitary. MMQ cells, isolated from the PRL-secreting rat pituitary tumor 7315a is an interesting model since they only secrete PRL. In order to determine whether and which GTP-binding (G) proteins are involved in the modulation of cyclic 3',5'-adenosine monophosphate (cAMP) accumulation and phospholipids turnover and eventually PRL release, we have performed studies with MMQ cells. For this purpose, the levels of various G proteins (alpha o, alpha s, alpha i, alpha q and beta) and their mRNAs, measured by Western and Northern blots respectively, were correlated with intracellular cAMP accumulation in response to DA, VIP or DA plus VIP, and with inositol phosphates (IPx) formation in response to ANG II, DA or DA plus ANG II. This study shows that, when compared to normal pituitary tissue, the levels of alpha o, alpha o2 and alpha i3 were significantly decreased in MMQ cells; those of alpha o1, alpha i (alpha i1 + alpha i2), alpha s42 and alpha q were very low or undetectable while those of alpha s47 and beta were normal. DA was unable to inhibit basal PRL release and cAMP accumulation. VIP increased both cAMP accumulation and PRL release, while cAMP accumulation elicited by VIP could be suppressed by DA. BAY K 8644-induced PRL release also could be suppressed by DA.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Angiotensin II; Animals; Cyclic AMP; Dopamine; Drug Interactions; Exocytosis; Gene Expression Regulation, Neoplastic; GTP-Binding Proteins; Inositol Phosphates; Neoplasm Proteins; Phospholipids; Pituitary Neoplasms; Prolactin; Rats; RNA, Messenger; Signal Transduction; Thyrotropin-Releasing Hormone; Tumor Cells, Cultured; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

The presence of pituitary adenylate cyclase activating polypeptide (PACAP) receptors coupled to adenylate cyclase was investigated in four types of human pituitary adenomas: three null adenomas and five gonadotropin-, three ACTH-, four GH-, and four PRL-producing adenomas. In all samples, except in prolactinomas, PACAP(1-27) and PACAP(1-38) stimulated adenylate cyclase activity equally well and potently (K(act) around 3 nmol). Vasoactive intestinal polypeptide (VIP) was systematically 100- to 300-fold less potent than both PACAPs. In prolactinomas, PACAP(1-27), PACAP(1-38), and VIP were inactive despite a response of the enzyme to guanosine 5'-triphosphate, Gpp(NH)p, forskolin, and fluoride. [125I-AcHis1]PACAP(1-27) binding was detected in all samples except in prolactinomas. In addition, a detailed analysis of receptors was feasible in all five gonadotropin- and in two ACTH-producing adenomas, confirming the existence of selective PACAP receptors that recognized PACAP(1-27) and PACAP(1-38) with similar high affinity (IC50 0.8-1.5 nmol) and VIP with a low affinity (IC50 100 nmol/L).

Topics: Adenoma; Adenylyl Cyclases; Binding Sites; Humans; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Pituitary Neoplasms; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Hormone; Vasoactive Intestinal Peptide

The prolactin secreting rat pituitary tumor cell line, GH3, expresses high affinity receptors for both vasoactive intestinal peptide (VIP) and somatostatin (SS14). VIP induces prolactin secretion by GH3 cells, an action which is antagonized by SS14. This in vitro model was used to examine the mechanism of action of two synthetic somatostatin analogs, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-OH (octreotide; SMS 201-995) and cyclo(aminoheptanoyl-Phe-D-Trp-Lys-Thr (benzyl)) (cyclic pentapeptide; CPP). Octreotide and CPP bind to the pituitary somatostatin receptor with lower affinity than does SS14 (KD = 1.3 +/- 1.1; 80 +/- 29; 211 +/- 107 nM for SS14, octreotide and CPP, respectively). SS14 and octreotide were equally effective as inhibitors of VIP-mediated accumulation of cAMP (40% and 45% inhibition, respectively, P < 0.01). SS14 and octreotide also inhibited forskolin-mediated accumulation of cAMP (42% and 40% inhibition of cAMP production, respectively; P < 0.01). The inhibitory action of somatostatin and octreotide on both VIP- and forskolin-mediated cAMP accumulation was blocked by pre-treatment of GH3 cells with pertussis toxin (P < 0.001). Neither SS14 nor octreotide affects the apparent affinity of VIP for its specific receptors on GH3 cells; thus, the inhibitory action of SS14 and octreotide appears to be mediated at the locus of the G-protein-adenylate cyclase complex. In contrast, CPP inhibited VIP-mediated cAMP accumulation slightly, but had no effect on forskolin-mediated cAMP production. Pertussis toxin did not attenuate CPP affects on VIP-mediated cAMP accumulation. However, pre-incubation of GH3 cells with CPP decreased the apparent affinity of receptors for VIP, suggesting that effects of CPP are attributable to interference with VIP binding rather than inhibition at the G-protein-adenylate cyclase complex.

Topics: Amino Acid Sequence; Animals; Molecular Sequence Data; Octreotide; Pituitary Neoplasms; Protein Binding; Rats; Receptors, Somatostatin; Second Messenger Systems; Somatostatin; Structure-Activity Relationship; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Epidermal growth factor (EGF) stimulated the prolactin (PRL) synthesis and release from the GH4C1 cells in a dose-dependent manner. The ED50 was between 10(-11) and 10(-10) mol/l. The maximal effect was obtained at 10(-9) mol/l EGF for the release, and 10(-8) mol/l EGF for the synthesis. EGF stimulated the release of PRL from cell perfusion columns after a lag period of about 30 s. The maximal secretion of PRL occurred about 60 s after the start of stimulation. The PRL secretion declined to basal levels within 2 min. The EGF-stimulated PRL release was additive to the secretion evoked by thyrotropin-releasing hormone (TRH) and vasoactive intestinal peptide (VIP). An instantaneous increase in the intracellular concentration of free calcium, [Ca2+]i, of the GH4C1 cells was observed after the administration of EGF. EGF modified neither the basal nor the TRH-stimulated inositoltrisphosphate production in the GH4C1 cells, and EGF did not show any effect on the cyclic adenosine monophosphate production of these cells.

Topics: Animals; Calcium; Cyclic AMP; Dose-Response Relationship, Drug; Drug Synergism; Epidermal Growth Factor; Inositol 1,4,5-Trisphosphate; Pituitary Gland; Pituitary Neoplasms; Prolactin; Rats; Thyrotropin-Releasing Hormone; Time Factors; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

In contrast to lactotrophs, tumoral pituitary cells like GH3 and GH4C1 lack expression of dopamine D2short and D2long receptors. In GH4C1 cells, we observed that the expression of only the short isoform of D2 receptor can be induced after transfection with a plasmid which confers resistance to neomycin (pRSVNeo). High levels of fully functional D2short receptor were obtained in GH4C1 following transfection (528fmol/mg protein). Sequence, pharmacology and coupling of the induced-D2 receptor do not show any difference with the cloned rat D2 short receptor.

Topics: Animals; Apomorphine; Base Sequence; Colforsin; Cyclic AMP; Dopamine; Dose-Response Relationship, Drug; Ergolines; Gene Expression; Kanamycin Kinase; Kinetics; Molecular Sequence Data; Oligodeoxyribonucleotides; Phosphotransferases; Pituitary Neoplasms; Polymerase Chain Reaction; Potassium Chloride; Prolactin; Quinpirole; Rats; Receptors, Dopamine D2; Recombinant Fusion Proteins; Spiperone; Sulpiride; Transfection; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Several studies have shown that folliculo-stellate cells (FS cells) in the anterior pituitary gland exhibit paracrine functions. Recently, we established a pituitary FS-like cell line, TtT/GF, which was derived from an isologously transplantable pituitary thyrotropic tumor line induced by radiothyroidectomy. In studies to examine the function of FS cells, we found that two forms of a novel hypophysiotropic peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), were potent activators of TtT/GF cells. Both the 27- and 38-amino acid forms of PACAP (PACAP-27 and PACAP-38) and vasoactive intestinal peptide (VIP) increased the levels of cAMP in TtT/GF cells in a similar dose-dependent manner. PACAP-27 and PACAP-38 specifically stimulated the proliferation of TtT/GF cells dose dependently, whereas VIP was ineffective. The minimal effective concentration of the PACAPs inducing cell proliferation was between 10(-8)-10(-7) M. However, PACAP-27 was much less potent than PACAP-38 in stimulating cell proliferation and DNA synthesis. PACAP-38, PACAP-27, and VIP all stimulated the release of interleukin-6 (IL-6) from TtT/GF cells. PACAP 38 (10(-8) M) stimulated IL-6 production effectively within 1 h of incubation, and the level attained at 8 h of cultivation (620 pg/ml) was nearly 10-fold that in the absence of PACAP-38 (60 pg/ml). PACAP-38 and VIP stimulated IL-6 secretion significantly at 10(-10)-10(-9) M in a bell-shaped manner; the maximum values were 10(-7) and 10(-8) M, respectively. On the other hand, IL-6 secretion stimulated by PACAP-27 became saturated at 10(-8) M, and the maximum value (320 pg/ml) was about 25% of that stimulated by PACAP-38 (1280 pg/ml). These findings obtained using TtT/GF cells as a model of FS cells suggest that PACAP acts as a hypophysiotropic factor, which targets FS cells and stimulates their proliferation, adenylate cyclase activation, and IL-6 secretion.

Topics: Animals; Cell Division; Cyclic AMP; DNA; Interleukin-6; Mice; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Pituitary Gland, Anterior; Pituitary Neoplasms; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The thyroliberin receptor in GH3 pituitary tumour cells is known to couple to phospholipase C via a guanine-nucleotide-binding protein (G protein). Thyroliberin is postulated also to activate adenylyl cyclase, via the stimulatory G protein (Gs). In order to study this coupling, we constructed an antisense RNA expression vector that contained part of the Gs alpha-subunit cDNA clone (Gs alpha) in an inverted orientation relative to the mouse metallothionein promoter. The cDNA fragment included part of the coding region and all of the 3' non-translated region. Transient expression of Gs alpha antisense RNA in GH3 cells resulted in the specific decrease of Gs alpha mRNA levels, followed by decreased Gs alpha protein levels. Thyroliberin-elicited adenylyl cyclase activation in membrane preparations showed a reduction of up to 85%, whereas phospholipase C stimulation remained unaffected. Activation of adenylyl cyclase by vasoactive intestinal peptide was reduced by 30-40%. Investigation of the effects of thyroliberin and vasoactive intestinal peptide on adenylyl cyclase in GH3 cell membranes pretreated with antisera against Gs alpha and Gi-1 alpha/Gi-2 alpha support the results obtained by the use of the antisense technique. We conclude that thyroliberin has a bifunctional effect on GH3 cells, in activating adenylyl cyclase via Gs or a Gs-like protein in addition to the coupling to phospholipase C.

Topics: Adenylyl Cyclases; Animals; Blotting, Western; Enzyme Activation; Gene Expression; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Immune Sera; Pituitary Neoplasms; Plasmids; Rats; Receptors, Neurotransmitter; Receptors, Thyrotropin-Releasing Hormone; RNA, Antisense; RNA, Messenger; Thyrotropin-Releasing Hormone; Transfection; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Vasoactive intestinal polypeptide (VIP) is now considered to be a prolactin-releasing factor (PRF). The aim of this study was to determine the VIP concentration in peripheral blood in patients with prolactin-secreting adenoma compared to healthy subjects. We also examined the effect of bromocriptine administration on the plasma VIP concentration in patients with prolactinoma. Nine patients with prolactinoma (6 women and 3 men, aged 27-50) and 7 healthy control subjects (4 women and 3 men, aged 26-40) were examined. Blood samples for prolactin and VIP were collected at 06:00, 12:00, 18:00, 24:00. In prolactinoma blood was taken before and after bromocriptine administration. Serum prolactin concentration was determined by the radioimmunoassay. VIP concentration was measured by a specific radioimmunoassay Kit-INCSTAR Corp. (Minnesota, USA). Statistical significance was calculated using the analysis of variance. A single 5 mg oral dose of bromocriptine decreased the mean prolactin concentration during the first 24 hours of treatment. Plasma VIP concentration was higher in prolactinoma patients compared to healthy subjects. There was no change in plasma VIP level after bromocriptine administration.. in patients with prolactin secreting adenoma the plasma VIP concentration is increased.

Topics: Adult; Analysis of Variance; Bromocriptine; Female; Humans; Male; Middle Aged; Pituitary Neoplasms; Prolactinoma; Vasoactive Intestinal Peptide

We have investigated the modulation of different G protein alpha- and beta-subunit levels in prolactin (PRL) and growth hormone producing rat pituitary adenoma cells (GH3 cells) in culture after prolonged exposure (6-48 h) to the steroid hormones 17 beta-oestradiol and dexamethasone. Gi-3 alpha- and G beta-subunits were the only G protein subunits which increased in response to 10(-6) M oestradiol (to approximately 150 and 200% of controls, respectively), while the other alpha-subunits investigated (Gs alpha, Gi-2 alpha and G(o) alpha) remained relatively unchanged. Thyroliberin (TRH)--and guanosine 5'-[beta gamma-imido]trisphosphate (Gpp(NH)p)-elicited adenylyl cyclase (AC) activities were reduced during 6-12 h of oestradiol treatment (by 60 and 20%, respectively), while the inhibitory effect of somatostatin (SRIF) increased by approximately 100%. Dexamethasone (10(-6) M) increased levels of the stimulatory G protein Gs alpha (to approximately 340%) and decreased levels of Gi-3 alpha (to 25%). After 48 h, the AC response to TRH was reduced by approximately 70%, whereas the effect of the other modulators remained close to controls. We conclude that G protein subunits in GH3 cells are subject to specific regulation by steroid hormones and that this may be important in the tuning of the responsiveness of PRL secretion to hormones in the in vivo situation.

Topics: Adenylyl Cyclases; Animals; Dexamethasone; Estradiol; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Pituitary Neoplasms; Prolactin; Rats; Second Messenger Systems; Somatostatin; Thyrotropin-Releasing Hormone; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The 4-chlorophenylthio analogue of cyclic AMP evoked profound and long-lasting changes in cytosolic [Ca2+] ([Ca2+]i) in pituitary-derived GH3 cells. However, vasoactive intestinal peptide (VIP), a hormone considered to act via cyclic AMP, was ineffective in modulating [Ca2+]i. The ability of VIP to modulate [Ca2+]i was enhanced by treatments that increased intracellular cyclic AMP. Much greater concentrations of intracellular cyclic nucleotides were achieved by the analogue than with VIP, under any condition. Thus cyclic AMP may play a prominent role in regulating [Ca2+]i in these cells, but the ability of hormones to stimulate its synthesis is limited, leading to a weak action on [Ca2+]i.

Topics: 1-Methyl-3-isobutylxanthine; Adenosine Triphosphate; Animals; Calcium; Cell Line; Colforsin; Cyclic AMP; Cytosol; Kinetics; Pituitary Neoplasms; Rats; Thionucleotides; Time Factors; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Recent studies using both normal and tumoral pituitary cell cultures have demonstrated that growth hormone (GH) and prolactin (PRL) secreting populations contain cells which release either one or both of these hormones. In order to determine whether these two cell types can be differentially regulated by hypothalamic factors we performed the following study employing plaque assays for GH and PRL. Using cultures of GH3 cells, a rat tumor cell line which contains both of these cell types, we found that the hypothalamic factors vasoactive intestinal peptide (VIP) and thyrotropin releasing hormone (TRH) when used together had a greater influence on plaque formation than when each was used individually. This suggested that cells were present in culture that responded to one peptide but not the other. Estradiol-treated cultures (which contain only dual-secreting cells) were then evaluated for VIP and TRH responsiveness and found to respond to TRH but not VIP. Finally, we assessed the peptide sensitivity of cultures that were exposed to a conjugate of VIP and the A-chain of ricin (a potent cytotoxin). In addition to eliminating VIP-responsive cells, this treatment markedly reduced the proportions of cells secreting GH-only while having no appreciable influence on dual-hormone secretors. When taken together, our findings indicate that single and dual secretors respond differently to at least two hypothalamic secretagogues and suggest that regulatory differences between these cell types may be important in the control of GH and PRL secretion.

Topics: Animals; Estradiol; Growth Hormone; Neoplasm Proteins; Pituitary Neoplasms; Prolactin; Rats; Ricin; Secretory Rate; Thyrotropin-Releasing Hormone; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Secretion of beta-endorphin from mouse pituitary AtT20 cells is stimulated by a variety of compounds that raise intracellular cAMP and Ca2+. To investigate the role of cAMP-dependent protein kinases in secretion, AtT20 cells were transfected with an expression vector coding for a regulatory (R) subunit of cAMP-dependent protein kinase containing mutations in both cAMP-binding sites. Expression of the mutant regulatory subunit in stable transformants (RAB cells) results in a dominant inhibition of cAMP-dependent protein kinase activity. Isoproterenol (1 microM) or analogs of cAMP stimulated beta-endorphin secretion from AtT20 cells, but failed to stimulate secretion in RAB cells expressing the mutant R subunit. Secretion in response to CRF (100 nM) was inhibited by 80% in these mutant clones, whereas the secretory response to vasoactive intestinal peptide (VIP; 100 nM) or phorbol ester (100 nM phorbol myristate acetate) was not inhibited by the R subunit mutation. Intracellular cAMP was elevated in response to CRF (11- to 15-fold), isoproterenol (5- to 10-fold), and VIP (4- to 8-fold) in RAB cells. Similar concentrations of VIP were required to evoke beta-endorphin secretion in either RAB cells or AtT20 cells. As with most secretagogues, VIP-induced secretion was inhibited in the presence of either EGTA or a voltage-sensitive Ca2+ channel antagonist, PN200-110. The secretory response to VIP was unaffected by down-regulation of protein kinase-C. These results suggest that CRF and isoproterenol work via cAMP-dependent protein kinase to activate beta-endorphin secretion, whereas VIP can act by a different mechanism that does not involve cAMP-dependent protein kinase or protein kinase-C.

Topics: Animals; beta-Endorphin; Calcium Channels; Cell Line, Transformed; Cholera Toxin; Cyclic AMP; Gene Expression; Isoproterenol; Mice; Mutagenesis; Pituitary Neoplasms; Protein Kinase C; Protein Kinases; Tetradecanoylphorbol Acetate; Thionucleotides; Transfection; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The effects of hypothalamic peptides (TRH, GnRH, arginine vasopressin, vasoactive intestinal peptide, GHRH, CRH, and SRIH) on cytosolic free calcium concentrations ([Ca2+]i) and adenylyl cyclase (AC) activity were evaluated in 12 nonfunctioning pituitary adenomas. TRH, GnRH, and arginine vasopressin induced a marked [Ca2+]i rise in 10/12, 4/12, and 2/5 tumors, respectively. The transients induced by these peptides were due to both Ca2+ mobilization from the intracellular stores and Ca2+ influx from the extracellular medium. AC activity was evaluated in 10 adenomas; 1 microM vasoactive intestinal peptide induced a 2- to 6-fold stimulation of the enzyme activity in all tumors, while neither GHRH nor CRH were effective. Moreover, in 5/10 tumors 1 microM SRIH reduced both AC activity and [Ca2+]i, while in 2/10 the peptide caused a significant rise in [Ca2+]i despite the AC inhibition and in 3/10 SRIH did not modify either AC activity or [Ca2+]i. This study indicates that in nonfunctioning pituitary adenomas a wide spectrum of hypothalamic peptides modulate [Ca2+]i and AC activity. Moreover, the presence of biologically active receptors may offer a possible target for therapeutic intervention.

Topics: Adenoma; Adenylyl Cyclases; Adult; Aged; Arginine Vasopressin; Calcium; Corticotropin-Releasing Hormone; Cytosol; Female; Gonadotropin-Releasing Hormone; Growth Hormone-Releasing Hormone; Humans; Hypothalamic Hormones; Male; Middle Aged; Pituitary Neoplasms; Second Messenger Systems; Somatostatin; Thyrotropin-Releasing Hormone; Vasoactive Intestinal Peptide

The rat pituitary cell line GH3 contains a high molecular weight microtubule-associated protein with properties characteristic of microtubule-associated protein-2 (MAP-2). The 280-kDa protein is selectively immunoprecipitated by antibodies to authentic bovine brain MAP-2 and is phosphorylated at appropriate sites by cAMP-dependent protein kinase (cAMP kinase) and multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Although MAP-2 is a minor cellular constituent, it can be immunoprecipitated from [32P]Pi-labeled GH3 cells and shown to contain a high level of basal phosphorylation. Vasoactive intestinal peptide, forskolin, 3-isobutyl-1-methylxanthene, or cholera toxin, treatments which increase cellular cAMP levels, or dibutyryl cAMP stimulate phosphorylation of specific sites on MAP-2 without significantly increasing its high state of basal phosphorylation. Phosphopeptide mapping reveals that the sites phosphorylated by cAMP kinase in vitro are the same sites whose phosphorylation in situ increases following stimulation of GH3 with agents that activate cAMP kinase. Increasing intracellular Ca2+ levels in GH3 cells also stimulates phosphorylation of MAP-2 but at sites distinct from those phosphorylated following treatment with cAMP inducing agonists. Phosphopeptide mapping indicates that the sites phosphorylated by CaM kinase in vitro are the same sites whose phosphorylation in situ increases following Ca2(+)-mediated stimulation. We conclude that activation of cAMP- and Ca2(+)-based signaling pathways leads to phosphorylation of MAP-2 in GH3 cells and that cAMP kinase and CaM kinase mediate phosphorylation by these pathways, respectively.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Bucladesine; Calcium; Cell Line; Cholera Toxin; Colforsin; Cyclic AMP; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Homeostasis; Kinetics; Microtubule-Associated Proteins; Peptide Mapping; Phosphates; Phosphopeptides; Phosphorylation; Pituitary Neoplasms; Protein Kinases; Rats; Signal Transduction; Vasoactive Intestinal Peptide

Rolipram (4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone) represents a new class of specific low Km cAMP phosphodiesterase (PDE) inhibitors. This compound enhances basal, hormone- and forskolin-elicited cAMP accumulation in prolactin (PRL) producing rat pituitary adenoma (GH4C1) cells in culture (ED50 = 5.10(-8) M). This effect is due to a selective inhibition of the low Km cAMP PDE (type III), since neither basal nor hormone-stimulated adenylate cyclase (AC) nor the Ca2+/calmodulin-dependent PDE were affected by rolipram. The drug enhanced vasoactive intestinal polypeptide (VIP)-stimulated PRL-secretion, while thyroliberin (TRH)- and 12-0-tetradecanoyl phorbol-13-acetate (TPA)-elicited PRL egress were slightly reduced indicating a cAMP-mediated reduction of protein kinase C (PK-C) mediated PRL release. Interestingly, inhibition of PRL secretion by somatostatin (SRIH) was completely suppressed suggesting cAMP-mediated inactivation of some GTP-binding protein(s) of the alpha i family (G alpha i2 or Gk). Rolipram did not affect phosphoinositide metabolism (i.e. IP3 accumulation), neither acutely nor after long term administration. Rolipram, like the cAMP PDE inhibitor Ro 20-1724, did not influence AC and PDE I, but dose-dependently inhibited PDE III activity. Long term incubation of GH4C1 cells with rolipram in the presence of noradrenaline (NA) exerted a marginal decrease of beta-receptor number, AC activation and cAMP accumulation, while Ro 20-1724 brought about a marked down-regulation and desensitization of the AC complex. In summary, rolipram selectively interacts with PDE III in rat pituitary adenoma cells in culture and does not result in beta-adrenoceptor AC downregulation. These features are not shared by the other drugs tested.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenoma; Adenylyl Cyclases; Animals; Colforsin; Cyclic AMP; Enzyme Activation; Norepinephrine; Phosphatidylinositols; Phosphodiesterase Inhibitors; Pituitary Neoplasms; Prolactin; Pyrrolidinones; Rats; Receptors, Adrenergic, beta; Rolipram; Thyrotropin-Releasing Hormone; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The influence of protein kinase C (PKC) activation on cyclic AMP production in GH3 cells has been studied. The stimulation of cyclic AMP accumulation induced by forskolin and cholera toxin was potentiated by 4 beta-phorbol 12,13-dibutyrate (PDBu). Moreover, PDBu, which causes attenuation of the maximal response to vasoactive intestinal polypeptide (VIP), also induced a small right shift in the dose-response curve for VIP-induced cyclic AMP accumulation. PDBu-stimulated cyclic AMP accumulation was unaffected by pretreatment of cells with pertussis toxin or the inhibitory muscarinic agonist, oxotremorine. PDBu stimulation of adenylate cyclase activity required the presence of a cytosolic factor which appeared to translocate to the plasma membrane in response to the phorbol ester. The diacylglycerol-generating agents thyroliberin, bombesin and bacterial phospholipase C each stimulated cyclic AMP accumulation, but, unlike PDBu, did not attenuate the stimulation induced by VIP. These results suggest that PKC affects at least two components of the adenylate cyclase complex. Stimulation of cyclic AMP accumulation is probably due to modification of the catalytic subunit, whereas attenuation of VIP-stimulated cyclic AMP accumulation appears to be due to the phosphorylation of a different site, which may be the VIP receptor.

Topics: 1-Methyl-3-isobutylxanthine; Adenylyl Cyclases; Bombesin; Cholera Toxin; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Oxotremorine; Phorbol 12,13-Dibutyrate; Pituitary Neoplasms; Protein Kinase C; Tumor Cells, Cultured; Type C Phospholipases; Vasoactive Intestinal Peptide

Previous work has shown that corticotropin releasing factor, vasoactive intestinal peptide, phorbol ester, and forskolin cause the secretion of adrenocorticotropic hormone and beta-endorphin from the AtT-20 mouse pituitary cell line. Human recombinant interleukin 1 alpha and 1 beta also stimulated adrenocorticotropic hormone and beta-endorphin secretion from AtT-20 cells in a time- and dose-related manner. The effect appeared only after pretreatment with interleukin 1 (IL-1) for at least 18 hr and was maximum at 24 hr. After pretreatment of the cells over a period of time with IL-1, the secretion induced by corticotropin releasing factor and vasoactive intestinal peptide was increased in more than an additive manner. The enhancement of corticotropin releasing factor-induced beta-endorphin release produced by IL-1 was apparent after 12 hr and reached a maximum at 24 hr. IL-1 did not affect forskolin-induced cAMP generation but enhanced the effect of forskolin on beta-endorphin secretion. This suggests that IL-1 does not induce adenylate cyclase and that forskolin causes the secretion of beta-endorphin by a mechanism independent of cAMP. IL-1 enhanced phorbol ester-induced beta-endorphin secretion. After prolonged treatment with phorbol ester (an activator of protein kinase C), the secretion induced by phorbol ester was abolished as well as the enhancement induced by IL-1. However, prolonged treatment with phorbol ester had no effect on IL-1-induced beta-endorphin secretion. These observations suggest that IL-1 enhances peptide-generated secretion of beta-endorphin by inducing protein kinase C.

Topics: Animals; beta-Endorphin; Colforsin; Corticotropin-Releasing Hormone; Cyclic AMP; Drug Synergism; Enzyme Activation; Interleukin-1; Kinetics; Mice; Pituitary Neoplasms; Protein Kinase C; Recombinant Proteins; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Different peptide hormones influence hormone secretion in pituitary cells by diverse second messenger systems. Recent data indicate that luteinizing-hormone-releasing hormone (LHRH) stimulates and somatostatin inhibits voltage-dependent Ca2+ channels of GH3 cells via pertussis-toxin-sensitive mechanisms [Rosenthal et al. (1988) EMBO J. 7, 1627-1633]. In other pituitary cell lines, somatostatin has been shown to cause a pertussis-toxin-sensitive decrease in adenylate cyclase activity, and LHRH and thyrotropin-releasing hormone (TRH) stimulate phosphoinositol lipid hydrolysis in a pertussis-toxin-independent manner. Whether stimulation of Ca2+ influx by TRH is affected by pertussis toxin is not known. In order to elucidate which of the hormone receptors interact with pertussis-toxin-sensitive and -insensitive G-proteins, we measured the effects of LHRH, somatostatin and TRH on high-affinity GTPases in membranes of GH3 cells. In control membranes, both LHRH and TRH stimulated the high-affinity GTPase by 20%, somatostatin by 25%. Maximal hormone effects were observed at a concentration of about 1 microM. Pretreatment of cells with pertussis toxin abolished pertussis-toxin-catalyzed [32P]ADP-ribosylation of 39-40-kDa proteins in subsequently prepared membranes and reduced basal GTPase activity. The toxin also reduced by more than half the increases in GTPase activity induced by LHRH and TRH; stimulation of GTPase by somatostatin was completely suppressed. Stimulation of adenylate cyclase by vasoactive intestinal peptide (VIP) was not impaired by pretreatment of cells with pertussis toxin. Somatostatin but not LHRH and TRH decreased forskolin-stimulated adenylate cyclase activity. The results suggest that the activated receptors for LHRH and TRH act via pertussis-toxin-sensitive and -insensitive G-proteins, whereas effects of somatostatin are exclusively mediated by pertussis-toxin-sensitive G-proteins.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclases; Animals; Cell Line; Cell Membrane; Gonadotropin-Releasing Hormone; GTP Phosphohydrolases; GTP-Binding Proteins; Kinetics; Pertussis Toxin; Phosphoric Monoester Hydrolases; Pituitary Neoplasms; Somatostatin; Thyrotropin-Releasing Hormone; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

Recent evidence indicates that vasoactive intestinal peptide (VIP) may be involved in normal pituitary function. Immunocytochemistry was used to localize VIP in human biopsied pituitary adenomas and postmortem anterior pituitary glands. Paraffin sections were immunostained for VIP with the avidin-biotin-peroxidase technique. Strong VIP-like immunoreactivity (VIP-LI) was observed in 16 of 17 prolactinomas, 12 of 14 growth hormone-secreting tumors associated with acromegaly, four of 12 ACTH-secreting tumors, and 14 of 18 nonfunctioning pituitary adenomas. In most cases, VIP was colocalized with the classical pituitary hormones. Six of the 18 nonfunctioning tumors had no demonstrable hormone immunoreactivity; five of these stained strongly for VIP, whereas one was negative. Of 18 normal anterior pituitaries, 12 showed strong diffuse staining for VIP throughout the gland. One pituitary with VIP-LI came from an individual who had undergone pituitary stalk transection. Double-immunoenzyme labeling and immunoelectron microscopy demonstrated VIP-LI in many lactotrophs, scattered thyrotrophs, corticotrophs, and in an occasional gonadotroph. These results suggest the following: 1) VIP is present in more than one cell type in normal and adenomatous human pituitaries; and 2) VIP may be involved in the function and development of pituitary tumors.

Topics: Adenoma; Adult; Aged; Female; Humans; Immunohistochemistry; Male; Microscopy, Electron; Middle Aged; Pituitary Gland; Pituitary Neoplasms; Vasoactive Intestinal Peptide

The growth of two human prolactin-secreting cell lines developed in our laboratory has been investigated in response to a number of factors. Oestrogen stimulated the synthesis of DNA and protein and increased prolactin secretion. Dexamethasone had the opposite effect to oestrogen. In the presence of serum, epidermal growth factor (EGF) inhibited cell growth at concentrations of 5 ng/ml. Known secretagogues of prolactin (vasoactive intestinal peptide (VIP), TRH, bombesin and neurotensin) were investigated for their action on cell growth but only VIP had a stimulatory effect. Two preparations of fibroblast growth factor (FGF) were studied. One form, derived from bovine pituitary glands, stimulated human pituitary cell growth. In contrast, another FGF, of the basic type (rFGF), was inhibitory to cell growth, increasing the time for cell doubling from 30 to 72 h. This inhibitory effect of rFGF was modified but not abolished by serum, oestradiol, platelet-derived growth factor or EGF. We conclude that bovine pituitary contains at least two fibroblast growth factors, both of which stimulate fibroblast cell growth, but one stimulates and the other inhibits human pituitary tumour cell growth.

Topics: Cell Line; DNA; Estradiol; Fibroblast Growth Factors; Humans; Neoplasm Proteins; Pituitary Neoplasms; Prolactin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Phorbol esters alter cyclic AMP levels in a number of tissues, including the anterior pituitary. We report that membrane preparations from GH3 cells exposed to phorbol esters exhibit decreased vasoactive intestinal peptide (VIP)-stimulated and enhanced forskolin-stimulated adenylate cyclase activity. The responsiveness of adenylate cyclase activity to NaF, guanylyl-imidodiphosphate, and Mn2+ was also reduced by phorbol ester treatment. The ability of somatostatin to inhibit forskolin-stimulated adenylate cyclase activity was reduced while phorbol ester exposure had no apparent effect on somatostatin inhibition of VIP-stimulated adenylate cyclase activity. We suggest that protein kinase C alters at least two distinct components of the adenylate cyclase system. One modification disrupts hormone receptor-Gs interaction (lowering VIP efficacy) and the second perturbation augments the activity of the adenylate cyclase catalytic subunit.

Topics: Adenylyl Cyclases; Animals; Colforsin; Cyclic AMP; Enzyme Activation; Guanylyl Imidodiphosphate; Manganese; Pituitary Neoplasms; Protein Kinase C; Rats; Sodium Fluoride; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Although dopamine inhibits PRL release from the normal anterior pituitary lactotroph, a conclusive demonstration of the mechanisms involved in this response has been impeded by the presence of other cell types in the anterior pituitary. To circumvent this problem, we have isolated a clonal cell line, designated MMQ, from the 7315a rat pituitary tumor. The MMQ cell is an exemplary model for our use because it only secretes PRL. Our studies show that dopamine inhibits secretagogue-induced PRL release from these cells. In addition, dopamine decreases the intracellular cAMP concentration in MMQ cells that have been exposed to forskolin, cholera toxin, or vasoactive intestinal polypeptide, each a stimulator of cAMP generation. This inhibition is, in turn, reversed by the dopamine antagonist haloperidol and by pertussis toxin, an inactivator of the GTP-binding coupling protein. Dopamine also decreases the uptake and fractional efflux of 45Ca2+ by MMQ cells that have been exposed to the calcium channel activator maitotoxin. It seems, therefore, that dopamine decreases PRL release from MMQ cells at least in part by decreasing intracellular cAMP levels and calcium uptake. In additional experiments, we have found that MMQ cells are responsive to somatostatin, estrogen, progesterone, and acetylcholine, but not to TRH, angiotensin II, neurotensin, or bombesin. Furthermore, these cells possess a functional protein kinase-C system, as evidenced by the increase in PRL release and decrease in stimulated intracellular cAMP levels that occur in response to treatment with phorbol diesters. We suggest that the MMQ cell line will prove a useful model system for study of the biochemical effects of dopamine and other factors that modify PRL release.

Topics: Calcium; Cholera Toxin; Colforsin; Cyclic AMP; Dopamine; Enzyme Activation; Estradiol; Haloperidol; Immunohistochemistry; Marine Toxins; Oxocins; Pertussis Toxin; Pituitary Neoplasms; Prolactin; Protein Kinase C; Tumor Cells, Cultured; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

The GH4C1 pituitary cell line contains specific plasma membrane receptors for the inhibitory neuropeptide somatostatin (SRIF). Unlike other peptides which bind to cell surface receptors on these cells, SRIF is not rapidly internalized via receptor-mediated endocytosis. Here we examined the effects of chronic SRIF pretreatment on the subsequent ability of GH4C1 cells to bind and respond to this hormone. Treatment of cells with 100 nM SRIF increased [125I-Tyr1]SRIF binding to a maximum value of 220% of control after 20 h. Scatchard analysis demonstrated that the number, but not the affinity, of the receptors was altered. The effect of SRIF was dose-dependent (ED50 = 2.3 +/- 0.4 nM), was not mimicked by an inactive analog, and was specific for the SRIF receptor. Furthermore, pretreatment of cells with other agents, which mimic SRIF's action to decrease intracellular cAMP and free Ca2+ concentrations, did not mimic the SRIF-induced increase in receptor number. Thus, occupancy of the SRIF receptor was required for SRIF receptor up-regulation. Inhibition of protein synthesis with cycloheximide did not prevent the SRIF-induced increase in receptors, consistent with an effect of SRIF to either reduce receptor degradation or cause slow redistribution of preexisting receptors to the plasma membrane. In contrast to the effects on receptor binding, pretreating cells with SRIF did not alter either basal cAMP levels or the potency of SRIF to inhibit cAMP accumulation (ED50 = 0.5 +/- 0.2 nM). However, the maximum cAMP produced by stimulators of adenylyl cyclase was increased. The observation that chronic SRIF exposure did not cause homologous desensitization in GH4C1 cells and increased rather than decreased SRIF receptor number is consistent with the fact that this neuropeptide is not rapidly internalized by receptor-mediated endocytosis.

Topics: Animals; Colforsin; Cycloheximide; Dose-Response Relationship, Drug; Pituitary Neoplasms; Rats; Receptors, Neurotransmitter; Receptors, Somatostatin; Somatostatin; Time Factors; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Hormonal activation and inhibition of the GH4Cl1 cell adenylate cyclase complex is delineated. In the presence of the guanyl nucleotide GTP, enzyme activity was enhanced twofold by thyroliberin, sixfold by vasoactive intestinal peptide (VIP), twofold by prostaglandin E2 and twofold by isoproterenol. The diterpene, forskolin, increased, the activity 14-fold. In the presence of high GTP (400 microM) and NaCl (150 mM) concentrations, somatostatin inhibited (ED50 = 0.5 microM) the cyclase activity by 40%. In the presence of 10 microM somatostatin, the ED50 values (5 nM) for thyroliberin- and VIP-stimulated adenylate cyclase activities were shifted to 20 nM. Forskolin-elicited activation was, however, not affected by somatostatin. Cholera-toxin and pertussis-toxin pretreatment of the enzyme brought about some 20-fold and twofold activation, respectively. Inhibition by somatostatin was abolished upon pre-exposure to pertussis toxin. Mild alkylation by N-ethylmaleimide increased basal and hormone-activated adenylate cyclase while somatostatin again failed to express its inhibitory potential. Further alkylation caused a gradual decline and convergence of hormone-modulated cyclase activities towards zero. The N-ethylmaleimide-induced attenuation of thyroliberin-elicited activity was paralleled by a decrease in [3H]thyroliberin binding. Trifluoperazine and an anti-calmodulin serum reduced basal and net thyroliberin-, VIP- and forskolin-enhanced cyclase activities by some 30%, 100%, 70% and 80%, respectively. The Vmax of basal and thyroliberin-stimulated adenylate cyclase was diminished by 65%, leaving the apparent Km values (7.2 mM and 2.6 mM, respectively) for Mg2+ unaltered. Finally, the phorbol ester 12-O-tetra-decanoyl-phorbol 13-acetate (TPA) doubled the activity. This effect was counteracted by the protein kinase C inhibitor, polymyxin B, while thyroliberin-enhanced adenylate cyclase remained unaffected. In summary, we have described an adenylate cyclase with stimulatory (Rs) and inhibitory (Ri) receptors coupled to a calmodulin-sensitive holoenzyme through the Gs and Gi type of GTP-binding proteins. The ratio of the Gs to Gi is high. It appears that the GH4C1 cell adenylate cyclase is also activated by protein kinase C by interference with Gi. Apparently, thyroliberin activates the cyclase both directly through Gs and indirectly via protein kinase C stimulation.

Topics: Adenylyl Cyclases; Animals; Cell Line; Cell Membrane; Cholera Toxin; Colforsin; Dinoprostone; Hormones; Isoproterenol; Kinetics; Pituitary Neoplasms; Prolactinoma; Somatostatin; Thyrotropin-Releasing Hormone; Vasoactive Intestinal Peptide

We have previously identified a group of cytoplasmic phosphoproteins (proteins 1-11) whose phosphorylation could be related, on a pharmacological basis, to the multihormonal regulation of PRL synthesis and release in the anterior pituitary tumor-derived GH cell lines. Phosphoproteins with identical migration properties on two-dimensional electrophoresis gels were also detectable in normal rat anterior pituitary cells in culture. We designed appropriate culture and [32P] phosphate-labeling conditions allowing to analyze the regulation of the phosphorylation of these proteins in normal pituitary cells. TRH, 12-O-tetradecanoylphorbol-13-acetate, and vasoactive intestinal peptide induced the same qualitative changes in phosphorylation of proteins 1-11 in normal as in GH cells. Quantitative differences observed are most likely due to the heterogeneity of primary pituitary cultures. Phosphorylation changes affecting proteins 14-16, not previously detected in GH cells, were also observed with normal anterior pituitary cells. GH cell lines have lost the sensitivity of pituitary lactotrophs for dopamine, an important physiological inhibitor of PRL synthesis and release. In normal anterior pituitary cells in culture, dopamine inhibited also the TRH-stimulated phosphorylation of proteins 1-10, thus strengthening the correlation between phosphorylation of these proteins and multihormonal regulation of pituitary cell functions. Our results indicate: 1) that the same phosphoproteins as in GH cells are related to the multihormonal regulation of nontumoral, normal anterior pituitary cells in culture; 2) that dopamine acts by interfering with the phosphorylation of these proteins. The phosphoproteins described here are therefore likely to be part, in normal anterior pituitary cells and possibly in other cell types, of the intracellular machinery involved in the cascade of transducing events leading from the binding of extracellular signals to the regulation of their target biological functions.

Topics: Animals; Cell Line; Cells, Cultured; Dopamine; Female; Phosphates; Phosphorus Radioisotopes; Phosphorylation; Pituitary Gland, Anterior; Pituitary Neoplasms; Prolactin; Rats; Rats, Inbred Strains; Reference Values; Tetradecanoylphorbol Acetate; Thyrotropin-Releasing Hormone; Vasoactive Intestinal Peptide

The clonal rat pituitary tumor cell line GH4C1 secretes PRL but does not respond to dopamine, a physiological inhibitor of PRL. In an attempt to generate a dopamine-responsive cell line, GH4C1 cells, which lack the enzyme hypoxanthine-guanine phosphoribosyltransferase, were fused with cells from the pituitary glands of lactating rats to generate cell hybrids. The GH4C1 cells were fused with dispersed normal pituitary cells by either chemical fusion in 40% polyethylene glycol or electrofusion. The fused cells were grown in medium with hypoxanthine, aminopterin, and thymidine (HAT) for 4 weeks to select for hybrid cells. Control fusions between GH4C1 cells only or normal cells only did not produce viable colonies. Of 36 HAT-selected colonies, 3 responded to 10 nM bromocryptine (BCR) with inhibition of TRH-stimulated PRL release. These hybrid colonies had an inhibitory response similar to that of normal pituitary cells in culture. Both TRH- and vasoactive intestinal peptide-stimulated PRL release were inhibited to basal levels by 10 nM BCR, with an IC50 for BCR of approximately 0.25 nM. Basal hormone release was not inhibited by BCR. The BCR-sensitive hybrid cells grew more slowly than the parental GH4C1 line both in culture and when passaged in female Wistar-Furth rats. The response of the hybrid cells to the dopamine agonist and the characteristic of slow growth were lost after 9 months of continuous culture and after freezing cells. The parental GH4C1 cells were grown in female Wistar-Furth rats, the resulting tumors were dissociated, and the cells were grown in culture. This resulted in a brief establishment of the dopamine response. Stimulated PRL and GH release from freshly dispersed GH4C1 tumor cells was inhibited by BCR at concentrations from 0.1-10 nM, and spiroperidol reversed the inhibition. The inhibitory response to the dopaminergic agonist was lost quickly as the cells were carried in culture. These results demonstrate that GH4C1 cells may have the genetic information necessary for dopaminergic inhibition of PRL synthesis, but that the dopamine response is not observed under standard tissue culture conditions.

Topics: Animals; Bromocriptine; Cell Fusion; Cell Line; Cells, Cultured; Dopamine; Female; Growth Hormone; Hybrid Cells; Pituitary Gland; Pituitary Neoplasms; Prolactin; Rats; Rats, Inbred WF; Spiperone; Thyrotropin-Releasing Hormone; Vasoactive Intestinal Peptide

The functions of vasoactive intestinal polypeptide (VIP) and the many other neuropeptides that are localized in the anterior pituitary gland remain unknown although VIP of hypothalamic origin is established to act as a PRL-releasing factor. Evidence is presented here that locally-produced VIP acts in an autocrine fashion to stimulate PRL release. VIP antibodies or a VIP antagonist profoundly but reversibly suppressed PRL secretion in primary cultures of rat pituitary cells or the GH3 cell line. This evidence was obtained with the use of a reverse hemolytic plaque assay for microscopic demonstration of PRL release from individual cells under conditions precluding cell-cell interaction. We suggest that most of the high rate of "spontaneous" PRL secretion attributed to lactotropes deprived of hypothalamic influence is due in fact to the stimulatory effects of VIP acting in an autocrine fashion.

Topics: Animals; Cell Line; Cells, Cultured; Female; Homeostasis; Immune Sera; Kinetics; Pituitary Gland, Anterior; Pituitary Neoplasms; Prolactin; Rats; Vasoactive Intestinal Peptide

1H- and 31P-NMR spectroscopy has been applied to rats carrying implanted tumours in vivo, and used to observe simultaneous changes in intracellular pH (pHi) and lactate concentration during the stimulatory action of vasoactive intestinal polypeptide (VIP). A maximal decrease in pHi to a mean of 0.29 units below basal values was recorded. At the same time, 11 min after VIP, a maximal increase in tumour lactate was found, with a mean value of 150% of the basal concentration. The magnitude of these changes was compatible with in vitro measurements of basal lactate concentration and buffering capacity made on the same tumour line. It is concluded that VIP stimulates glycolysis by the tumour cells, resulting in an accumulation of lactate and a consequent fall in pHi.

Topics: Adenoma; Animals; Glycolysis; Hydrogen; Kinetics; Lactates; Magnetic Resonance Spectroscopy; Phosphorus; Pituitary Neoplasms; Rats; Rats, Inbred WF; Vasoactive Intestinal Peptide

The effects of the prolactin secretagogue vasoactive intestinal peptide (VIP) on the membrane excitability of clonal prolactin-secreting (GH3/B6) cells were studied using the whole-cell configuration of the patch recording technique. Submicromolar concentrations of VIP affected membrane excitability in more than half the cells tested, increasing the frequency of spontaneous Ca2+-dependent action potentials and prolonging individual action potentials, as well as changing their rheobase. Under voltage clamp, VIP induced changes in several voltage-sensitive conductances at both instantaneous and steady-state times. Some of these changes in membrane excitability may be related to VIP's stimulatory effects on prolactin secretion.

Topics: Action Potentials; Animals; Calcium; Cell Line; Cell Membrane; Electric Conductivity; Membrane Potentials; Pituitary Neoplasms; Rats; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) stimulates prolactin (PRL) secretion from cultured rat pituitary cells (GH cells) (Bjøro et al. 1984). This study demonstrates the presence of specific receptors for 125I-VIP on the GH4C1 cells. Specific binding was rapid and biphasic giving a transient plateau lasting from 7 to 30 min. Thereafter specific binding declined to about one-third after 90 min. This coincided with enhanced degradation of 125I-VIP. The degradation was mainly cell-mediated and only partly receptor dependent. Trichloroacetic acid precipitation and absorption chromatography indicated that the degradation products were either 125I- and/or small labelled peptide fragments. Bioassay, RIA and rebinding studies also demonstrated degradation of VIP. Pretreatment of GH4C1 cells with trypsin decreased the rate of degradation of 125I-VIP, but also reduced the amount of specific binding. Scatchard analysis of binding data indicated the existence of two independent classes of receptors, one with Kd = 2.2 nM and Bmax = 15 fmol per 10(6) cells and another with Kd = 180 nM and Bmax = 550 fmol per 10(6) cells. The IC50 for VIP, PHI and secretin were 4, 5 and 500 nM, respectively. We conclude that the high affinity receptor is the most probable mediator of VIP on PRL secretion. The effect of VIP and PHI on PRL secretion in GH4C1 cells is mediated through one common receptor.

Topics: Animals; Cell Line; Pituitary Neoplasms; Prolactin; Rats; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

Vasoactive intestinal polypeptide (VIP) was administered (75 micrograms iv over 12 min) to 14 patients with Cushing's disease, 1 patient with Nelson's syndrome, and 8 normal subjects. VIP induced a significant rise of plasma ACTH levels in 6 patients with Cushing's disease, from a baseline of 13.2 pmol/l (9.9-18.5 pmol/l) to a peak of 24.5 pmol/l (7.7-18.9 pmol/l), median and range (P less than 0.05), and in the patient with Nelson's syndrome, from a baseline of 260.9 to 461.3 pmol/l. A significant elevation of cortisol levels was also observed, from a baseline of 567 nmol/l (185-842 nmol/l) to a peak of 727 nmol/l (364-1029 nmol/l); P less than 0.05. No modifications in plasma ACTH and cortisol levels were noticed in the other 8 patients with Cushing's disease, or in the normal subjects. In the responsive patients, the median plasma ACTH level reached after VIP was found to be less than that induced by CRH administration. In 2 of the responsive patients, VIP was injected again after successful microadenomectomy and did not then cause changes in ACTH and cortisol concentration. These data demonstrate that VIP specifically stimulates ACTH release in some patients with corticotropinomas but not in normal subjects; the disappearance of such abnormal ACTH responses after successful adenomectomy suggests the presence of specific VIP receptors only on the adenomatous corticotropes.

Topics: Adenoma; Adolescent; Adrenocorticotropic Hormone; Adult; Cushing Syndrome; Female; Humans; Hydrocortisone; Male; Middle Aged; Nelson Syndrome; Pituitary Neoplasms; Radioimmunoassay; Vasoactive Intestinal Peptide

A culture of clonal tumour cells from rat pituitary gland that secrete both prolactin and growth hormone were used to investigate whether the pineal hormone melatonin can act directly on the pituitary gland to control prolactin production. Melatonin inhibition of prolactin and growth hormone production was significant but mild. Concentrations of between 10(-8) M and 10(-6) M reduced both prolactin and growth hormone production and prolactin secretion by 10-50%. 17 beta-oestradiol (OE) and thyrotrophin-releasing hormone (TRH) stimulated prolactin production but had no significant effect on growth hormone production. Melatonin reduced the effects of both of these compounds. Both TRH and vasoactive intestinal peptide (VIP) stimulated secretion of prolactin, and TRH also of growth hormone. Melatonin reduced these effects significantly. TRH and VIP increased cAMP production two- and 12-fold, respectively. Melatonin had no effect on basal or stimulated cAMP production. The melatonin-induced changes in prolactin and growth hormone production and secretion seen here do not approach the magnitude of the fluctuations seen in plasma in vivo. It is concluded that while melatonin does have a direct effect on the lactotroph in the regulation of prolactin production, its main physiological target must be elsewhere.

Topics: Animals; Cyclic AMP; Estradiol; Growth Hormone; Melatonin; Pituitary Gland; Pituitary Neoplasms; Prolactin; Rats; Thyrotropin-Releasing Hormone; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Long-acting somatostatin analogues such as SMS 201-995 (Sandoz) are being evaluated in a wide range of clinical indications, including gut neuroendocrine tumours and acrogemaly. Long-term continuous SMS 201-995 treatment has achieved useful symptomatic improvement in diarrhoea in 4 patients with metastatic VIPomas who had relapsed following previous treatment. Clinical improvement has outlasted suppression of VIP secretion (suggesting an additional direct antisecretory action of SMS 201-995) and has occurred despite expansion of hepatic metastases. In 6 patients with tumours secreting gastrin and/or glucagon, secretion of these peptides was acutely inhibited by SMS 201-995. However, endocrine and clinical responses to chronic treatment have been less consistent. SMS 201-995 is active orally at doses of 4-8 mg and when given thrice-daily to 6 patients with active acromegaly, suppressed mean 24-h growth hormone levels by 51-88%. Despite significantly reduced plasma insulin concentrations, glucose tolerance did not deteriorate. SMS 201-995 was also effective in suppressing thyroid-stimulating hormone (TSH) and thyroid hormone secretion in a patient with mild thyrotoxicosis due to non-tumoural inappropriate TSH hypersecretion. In all cases SMS 201-995 treatment has been well tolerated and has few side-effects.

Topics: Acromegaly; Adenoma; Adult; Aged; Diarrhea; Female; Gastrointestinal Neoplasms; Glucagonoma; Growth Hormone; Humans; Hyperthyroidism; Liver Neoplasms; Male; Middle Aged; Neoplasms, Glandular and Epithelial; Octreotide; Pancreatic Neoplasms; Pituitary Neoplasms; Somatostatin; Streptozocin; Thyrotropin; Vasoactive Intestinal Peptide; Vipoma; Zollinger-Ellison Syndrome

Prolactin secretion by a human pituitary tumour cell line produced in our laboratory was stimulated by TRH, vasoactive intestinal peptide (VIP) and epithelial growth factor (EGF). All raised the intracellular concentration of free calcium (Ca2+ i) of cells loaded with a fluorescent quinoline Ca2+ indicator in suspension, but the effect of TRH was much more rapid and less prolonged than that of VIP and EGF. Both TRH and VIP also increased Ca2+ i in GH3 rat pituitary tumour cells, but in this cell line the effect of VIP was only found in attached cells grown on cover-slips. In both human and rat cell lines, the increase in Ca2+ i produced by TRH was independent of extracellular calcium, whereas this was a requirement for the action of VIP and EGF. It is concluded that the prolactin secretagogues, VIP and probably EGF, increase Ca2+ i through an effect on plasma membrane calcium channels and that this effect differs from that of TRH.

Topics: Animals; Calcium; Cell Line; Growth Substances; Humans; Pituitary Neoplasms; Prolactin; Thyrotropin-Releasing Hormone; Time Factors; Vasoactive Intestinal Peptide

In order to investigate the influence of dopamine (DA) in modulating prolactin (PRL) response to vasoactive intestinal polypeptide (VIP), VIP (75 micrograms i.v. over 12 min) was administered to normal women and patients with microprolactinomas during saline or DA (0.06 micrograms/kg BW/min) infusion. In 8 normal women VIP caused PRL to rise from 7.9 +/- 0.8 (mean +/- SE) to 33.4 +/- 17.1 ng/ml (p less than 0.01), the peak occurring at 15 min, while it did not elicit any significant modification in serum PRL levels in 18 patients with microprolactinomas. DA infusion lowered serum PRL by 67 and 58.2% at 120 min in normal women and in patients with microprolactinomas, respectively, and abolished VIP-induced PRL response in normal women without influencing PRL response in patients with microprolactinomas. PRL responsiveness to VIP was not restored by dopaminergic disinhibition (domperidone 10 mg i.v.) in 4 patients tested. Two previously unresponsive patients showed a VIP-induced PRL increase, superimposable on that recorded in normal women, after successful selective adenomectomy. These data suggest that DA, although able to suppress VIP-induced PRL response in normals, does not play any major role in causing unresponsiveness to VIP in prolactinomas. The lack of PRL responsiveness to VIP might be intrinsic to the adenoma or due to alterations of PRL secretion regulatory mechanisms other than DA secondary to the presence of the tumor, or to a depletion of the readily releasable pool of PRL.

Topics: Adolescent; Adult; Domperidone; Dopamine; Female; Humans; Pituitary Neoplasms; Prolactin; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP), an dopamine have been measured in the hypothalamus and anterior pituitaries of female rats at three different ages. Rats aged 19 months had a high incidence (35%) of prolactin-secreting tumours of the pituitary and these animals had increased hypothalamic and pituitary VIP compared with three-month-old rats or rats without tumours. VIP in old animals without tumours was similar to that in young animals. The third group, with an even more advanced age of 22-28 months, had a lower incidence of PRL-secreting tumours, and pituitary and hypothalamic VIP concentrations similar to those in young animals. Dopamine was significantly increased in the pituitaries of 22-28-month-old nontumorous rats and slightly raised in 19-month-old rats. We conclude that the increased pituitary and hypothalamic VIP content of 19-month-old rats could be a factor in the development of tumours seen at this age, and the differences seen in the two groups of old rats may be related to changes in the steroid environment.

Topics: Age Factors; Animals; Dopamine; Hypothalamus; Norepinephrine; Pituitary Gland, Anterior; Pituitary Neoplasms; Prolactin; Rats; Rats, Inbred Strains; Vasoactive Intestinal Peptide

It was shown that somatostatin (SRIF) inhibited cAMP-dependent vasoactive intestinal peptide (VIP)-stimulated prolactin (PRL) release by a GH3 clonal strain of rat pituitary tumor cells and decreased basal PRL secretion and inhibited PRL release in response to thyrotropin releasing hormone (TRH) whose action was independent of prior synthesis of cAMP. Pretreatment of these cells with pertussis toxin prevented SRIF's inhibitory effects on basal and TRH-stimulated hormone secretion as well as its VIP-stimulated responses. The blockade of SRIF's inhibitory effect on the actions of TRH or VIP was dependent on both the duration of preincubation and concentration of the toxin and was correlated with the ability of the toxin to catalyze the ADP-ribosylation of the 39,000-Da membrane protein. It is likely that this pertussis toxin substrate is involved in signal transduction of SRIF on cAMP-dependent actions of VIP and cAMP-independent action of TRH. However, the mechanism of SRIF's action on TRH is not clear, since SRIF did not affect the intracellular responses by TRH, neither intracellular Ca2+ mobilization nor the increase of 1,2-diacylglycerol formation following the breakdown of polyphosphoinositides.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Cell Line; Cyclic AMP; Diglycerides; Dose-Response Relationship, Drug; Pertussis Toxin; Phosphatidic Acids; Pituitary Neoplasms; Prolactin; Rats; Somatostatin; Thyrotropin-Releasing Hormone; Time Factors; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

It is now well known that dopamine (DA) plays a major role in the inhibitory control of prolactin (PRL); however, the mechanisms that are physiologically involved in the stimulation of PRL release are still under investigation. Indeed, although suppression of DA inhibitory tonus, administration of thyrotropin-releasing hormone (TRH) or vasoactive intestinal peptide (VIP) are all known PRL releasers, it is not clear whether they interact during physiological periods of PRL release such as suckling and estrus. No clear indications exist, furthermore, on whether they all act upon a same pituitary pool that may become depleted following repeated exposure to stimuli. Refractoriness to a single or a repeated stimulus has been reported to occur in prolactinoma-bearing or normal humans, respectively, the mechanism of which is still matter for discussion. Our present studies performed by perifusing normal or adenomatous rat lactotrophs attached to Cytodex I microcarrier beads was undertaken to try and answer some of these questions. The experimental period consisted in perifusing the cells for 1 h with Dulbecco's modified Eagle's medium (DMEM) containing DA 10(-5) M, then for 2 h with either DMEM, DMEM and TRH 10(-8) M, DMEM and VIP 10(-7) M, then again with DA in DMEM for 1 h, and finally with DMEM, DMEM and TRH, or DMEM and VIP. Three experiments of various combinations were performed. Lower PRL levels were observed under DA, while two periods (first and second) of PRL release followed the suppression of DA infusion with or without the addition of either one of the two peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenoma; Animals; Dopamine; Female; Pituitary Gland; Pituitary Neoplasms; Prolactin; Rats; Rats, Inbred F344; Rats, Inbred Strains; Thyrotropin-Releasing Hormone; Vasoactive Intestinal Peptide

4 beta phorbol-12, 13-dibutyrate (PDBu) stimulated cyclic AMP accumulation in GH3 pituitary tumour cells in the presence of isobutylmethylxanthine. This effect persisted after preincubation of cells with cholera or pertussis toxins. In contrast, vasoactive intestinal polypeptide (VIP)-stimulated cyclic AMP accumulation was inhibited by PDBu in a dose dependent fashion (IC50 = 5.1 nM). Thyroliberin (TRH) had a similar, but non-additive, stimulatory effect on cyclic AMP accumulation with PDBu, however it did not inhibit VIP stimulation. These results suggest that TRH may stimulate cyclic AMP accumulation through protein kinase C and that stimulation of adenylate cyclase by PDBu and TRH may occur distal to the guanine nucleotide binding regulatory proteins, Ns and Ni.

Topics: 1-Methyl-3-isobutylxanthine; Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Animals; Bacterial Toxins; Cell Line; Cholera Toxin; Cyclic AMP; GTP-Binding Proteins; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Pituitary Gland; Pituitary Neoplasms; Rats; Thyrotropin-Releasing Hormone; Time Factors; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

The influence of vasoactive intestinal peptide (VIP), TRH, and dexamethasone (Dex) on PRL mRNA was investigated in PRL-producing GH3 cells using cytoplasmic dot hybridization and RNA blot analysis. Total cytoplasmic RNA was transferred to nitrocellulose filter paper and quantitated by hybridization to PRL recombinant DNA probes labeled with 32P. Incubation of GH3 cells with VIP for 25 h increased the content of cytoplasmic PRL mRNA. This increase was dose dependent, being significant at 2 X 10(-8) M and reaching a maximum at 2 X 10(-7) M. VIP at 2 X 10(-9) M had no effect on cytoplasmic PRL mRNA content. TRH (2 X 10(-7) M) also increased whereas Dex (2 X 10(-7) M) decreased the content of PRL mRNA. The inhibitory effect of Dex (2 X 10(-7) M) on cytoplasmic PRL mRNA was reversed by VIP (2 X 10(-7) M). Changes in medium PRL levels after these various hormone treatments paralleled those changes observed in PRL mRNA content. Examination of total poly(A)+ RNA demonstrated that incubation with VIP (2 X 10(-7) M) for 6 h increased the content of the mature PRL mRNA and its processing intermediates. Dex (2 X 10(-7) M) decreased the content of all species of PRL mRNA. These data suggest that VIP-stimulated PRL release is the result of an increase in the content of PRL mRNA and its precursors.

Topics: Animals; Cell Line; Cell Nucleus; Dexamethasone; DNA, Recombinant; Nucleic Acid Hybridization; Pituitary Neoplasms; Poly A; Prolactin; Rats; RNA, Messenger; Thyrotropin-Releasing Hormone; Vasoactive Intestinal Peptide

In an attempt to characterize GH and PRL secretion in acromegaly, the effects of various stimuli on GH and PRL release by cultured pituitary adenoma cells derived from acromegalic patients were studied. In addition, the PRL responses of somatotroph adenoma cells were compared to those of prolactinoma cells. GH-releasing hormone-(1-44) (GHRH) consistently stimulated GH secretion in all 14 somatotroph adenomas studied in a dose-dependent manner. The sensitivity as well as the magnitude of the GH responses to GHRH were highly variable in individual tissues. Somatotroph adenomas that did not respond to dopamine were more sensitive and had greater GH responses to GHRH. In 8 of 9 somatotroph adenomas that concomitantly secreted PRL, the addition of GHRH likewise increased PRL release. Omission of extracellular Ca2+ blocked the stimulatory effect of GHRH on GH and PRL secretion. When cells were coincubated with 0.1 nM somatostatin, GH and PRL secretion induced by 10 nM GHRH were completely blocked in most adenomas. Similarly, coincubation of dopamine resulted in inhibition of GHRH-induced hormone secretion in some adenomas. Addition of TRH to the incubation medium, on the other hand, significantly stimulated GH secretion in 8 of 14 adenomas, while TRH stimulated PRL release in all of the adenomas. Vasoactive intestinal peptide (VIP) and corticotropin-releasing hormone (CRH) produced an increase in GH and PRL secretion in other adenomas. In prolactinoma cells, somatostatin and dopamine unequivocally suppressed PRL secretion; however, other stimuli including GHRH, VIP, and CRF were ineffective. TRH induced a significant increase in PRL secretion in only one prolactinoma. These results suggest that responsiveness to GHRH and somatostatin is preserved in somatotroph adenomas; the responsiveness to GHRH is inversely correlated to that to dopamine; and PRL cells associated with somatotroph adenomas possess characteristics similar to those of GH cells. Further, the GH stimulatory actions of TRH and VIP are different.

Topics: Acromegaly; Adenoma; Adult; Cells, Cultured; Corticotropin-Releasing Hormone; Dopamine; Female; Growth Hormone; Growth Hormone-Releasing Hormone; Humans; Male; Middle Aged; Pituitary Neoplasms; Prolactin; Radioimmunoassay; Somatostatin; Thyrotropin-Releasing Hormone; Vasoactive Intestinal Peptide

GH3 cells were used to study the effect of rat growth hormone-releasing factor on adenylate cyclase activity and its interaction with somatostatin. Rat GRF stimulates adenylate cyclase activity (ED5 0 = 6 X 10(-8) M) and somatostatin-14 inhibits this GRF-stimulated activity in a non-competitive manner without affecting the basal enzyme levels. Inhibition by somatostatin-14 is observed at concentrations as low as 10(-11) M and the half-maximal effect is obtained with 10(-8) M. Somatostatin-28 is more potent than SS-14 and has an ED5 0 of 3 X 10(-11) M. VIP is more active than rat GRF in stimulating adenylate cyclase activation. We conclude that in GH3 cells rat GRF behaves as a partial VIP agonist by interacting with VIP-preferring receptors and its effects are inhibited by somatostatin.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Cell Line; Enzyme Activation; Growth Hormone-Releasing Hormone; Pituitary Neoplasms; Rats; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Somatostatin; Somatostatin-28; Vasoactive Intestinal Peptide

The effect of GH-releasing factor(1-44)(GRF) alone, or together with somatostatin (SRIF), dopamine (DA), vasoactive intestinal peptide (VIP) or cycloheximide was studied in a total of ten human somatotrophinomas using a static cell culture system. Growth hormone-releasing factor (2.0 X 10(-8) mol/l) significantly (P less than 0.05) stimulated GH release from nine out of ten tumours over 4-h incubations, and a dose-related effect (2.0 X 10(-10) -2.0 X 10(-8) mol/l) was observed in five tumours thus studied. Repeated GRF (2.0 X 10(-8) mol/l)-mediated GH release was seen during 96% (n = 25) of experiments performed on six tumours over 4 h and up to 27 days in culture. Growth hormone-releasing factor (2.0 X 10(-8) mol/l) also stimulated GH release from five out of seven somatotrophinomas during 60-min incubations. Somatostatin (6.1 X 10(-9) mol/l) completely inhibited GRF-induced GH secretion from four tumours studied over 4 h, but in each case there was significant (P less than 0.05) 'rebound' of GH release from cultures exposed to both GRF and SRIF during a subsequent recovery period. Dopamine suppressed basal GH release from two out of four tumours, but in each case had a greater inhibitory effect on GRF-mediated GH release. Vasoactive intestinal peptide directly stimulated GH release from two out of three tumours, and the effects were additive to maximal stimulatory doses of GRF. Cycloheximide significantly (P less than 0.01) enhanced GRF-stimulated release of GH during a 60-min incubation, but inhibited both basal and GRF-stimulated release over 4 and 8 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cells, Cultured; Cycloheximide; Dopamine; Drug Interactions; Growth Hormone; Growth Hormone-Releasing Hormone; Humans; In Vitro Techniques; Peptide Fragments; Pituitary Gland; Pituitary Neoplasms; Somatostatin; Vasoactive Intestinal Peptide

To characterize the functional aspect of prolactin (Prl) cells coexisting with corticotroph adenomas, pituitary adenoma cells obtained from a patient with Cushing's disease and a patient with Nelson's syndrome, who were associated with hyperprolactinaemia, were cultured in monolayer and their Prl responses to various secretagogues were compared with those of prolactinoma cells in culture. Immunohistochemistry performed in one of these two adenomas demonstrated the presence of Prl-containing cells in addition to ACTH cells. When ACTH-Prl adenoma cells were exposed to ovine corticotrophin-releasing factor (CRF), a dose-dependent increase in both ACTH and Prl secretion was observed, which was blocked by coincubation with hydrocortisone. In contrast, no stimulatory effect of CRF on Prl release was observed in all of the experiments using prolactinoma cells. Thyrotrophin-releasing hormone, which consistently stimulated Prl secretion in ACTH-Prl adenomas, was effective in triggering Prl release in only 25% of the prolactinomas. Exposure of the cultured cells to lysine vasopressin, growth hormone-releasing factor and vasoactive intestinal peptide resulted in an increase in ACTH and Prl secretion in one ACTH-Prl adenoma, however, none of the prolactinomas responded to these stimuli to secrete Prl. Dopamine and somatostatin, on the other hand, uniformly suppressed Prl secretion from ACTH-Prl adenomas as well as from prolactinoma cells. These results suggest that the mode of Prl secretion by mixed ACTH-Prl pituitary adenomas is not identical to that by pure prolactinomas and is, at least in part, common to that of ACTh secretion.

Topics: Adenoma; Adrenocorticotropic Hormone; Adult; Cells, Cultured; Corticotropin-Releasing Hormone; Cushing Syndrome; Dopamine; Female; Haloperidol; Humans; Lypressin; Nelson Syndrome; Pituitary Neoplasms; Prolactin; Radioimmunoassay; Somatostatin; Thyrotropin-Releasing Hormone; Vasoactive Intestinal Peptide

The hypothalamic peptide vasoactive intestinal peptide (VIP) stimulates ACTH and endorphin secretion by the AtT20/D16 clonal strain of mouse pituitary tumor cells. The dose dependence for VIP stimulation of hormone release is biphasic, indicating that VIP is able to activate at least two classes of receptors in D16 cells (ED50 = 1.6 and 160 nM). We show that at high concentrations (ED50 greater than or equal to 150 nM), other natural peptides with primary structures homologous to that of VIP also increased ACTH secretion by D16 cells, whereas structurally unrelated peptides did not. The stimulatory actions of GH-releasing factor (GRF) and porcine heptacosapeptide with amino-terminal histidine and carboxy-terminal isoleucine amide (PHI) were mediated by high affinity VIP receptors because their effects were not additive with that of 10 nM VIP. In addition, GRF and PHI behaved as antagonists at low affinity VIP receptors; both peptides inhibited stimulation by 1 microM VIP. In contrast, glucagon and gastric inhibitory polypeptide appeared to stimulate ACTH release via low affinity VIP receptors because their effects were additive with that of 10 nM, but not 1 microM, VIP. Since all of the VIP-like peptides increased ACTH secretion only at high concentrations, they were unlikely to represent a physiological ligand for the receptor activated by high concentrations of VIP. Therefore, we determined whether cross-reactivity occurred between VIP-like peptides and corticotropin-releasing factor (CRF), a potent stimulator of ACTH secretion both in vitro and in vivo. The dose-response curve for CRF stimulation of ACTH secretion by D16 cells extended over more than a 1000-fold range of concentrations and was biphasic (ED50 = 2.6 and greater than 300 nM), indicating that CRF interacted with multiple receptor types in D16 cells. However, since the effect of 10 nM CRF was additive with that of 1 microM VIP, the CRF receptor was not the site at which high concentrations of VIP stimulated ACTH release. In contrast, the effect of 1 microM CRF was not additive with that of 1 microM VIP or other VIP-like peptides. Therefore, high concentrations of CRF and the previously recognized VIP-like peptides stimulated ACTH secretion by overlapping pathways. Comparison of the amino acid sequence of CRF with those of the VIP-like peptides showed that 18 of the 41 amino acids in CRF match a corresponding amino acid in at least 1 member of the VIP peptide family.(ABSTRACT TRUNCATED AT 40

Topics: Adrenocorticotropic Hormone; Amino Acid Sequence; Animals; Cells, Cultured; Corticotropin-Releasing Hormone; Cyclic AMP; Dose-Response Relationship, Drug; Mice; Pituitary Neoplasms; Receptors, Cell Surface; Receptors, Somatotropin; Receptors, Vasoactive Intestinal Peptide; Somatostatin; Vasoactive Intestinal Peptide

Pertussis toxin, PT, abolishes inhibitory regulation of adenylate cyclase by cell surface receptors. Inhibitors of adenylate cyclase in GH3 cells, namely somatostatin and the muscarinic cholinergic agonist carbachol, lower the cytosolic free Ca2+ concentration. [Ca2+]i and cause hyperpolarization. These responses are selectively abolished by PT. It is concluded that the effects of somatostatin and carbachol to lower [Ca2+]i and to hyperpolarize are secondary to their inhibitory action on adenylate cyclase. In contrast, PT does not impair the TRH induced rise in [Ca2+]i in GH3 cells demonstrating that the coupling of TRH receptors to Ca2+ mobilization is not mediated by a PT substrate.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclases; Animals; Bacterial Toxins; Calcium; Carbachol; Cell Line; Cytosol; Pertussis Toxin; Pituitary Neoplasms; Rats; Somatostatin; Thyrotropin-Releasing Hormone; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

GH3 cells can be used effectively to study the in vitro mechanism of action of GRF. In these cells, there is a time and concentration-dependent release of cAMP into the medium. Rat hypothalamic GRF, (rGRF) is 7 to 10 fold more active than human hypothalamic GRF (hGRF). VIP, a peptide which is structurally homologous to GRF, stimulates cAMP efflux in GH3 cells, with a higher affinity than hGRF or rGRF. We propose that in contradistinction to the normal rat pituitary, the stimulation of cAMP release by GRF in GH3 cells occurs via activation of VIP-preferring receptors and that GRF (rGRF in particular) behaves as a partial VIP agonist.

Topics: Animals; Cell Line; Cyclic AMP; Growth Hormone; Growth Hormone-Releasing Hormone; Humans; Hypothalamus; Kinetics; Pituitary Gland, Anterior; Pituitary Neoplasms; Rats; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

Although purine nucleosides have been shown to regulate the secretion of several peptide and steroid hormones, effects on pituitary hormone release have not been reported. We show here that in the clonal GH4C1 pituitary cell line maximal concentrations of adenosine (greater than or equal to 50 microM) inhibited PRL and GH secretion by 40%. Adenosine deaminase abolished the inhibitory effect of adenosine but not that of SRIF or (-)N6(R-2-phenylisopropyl)adenosine (PIA), a nonhydrolyzable adenosine analog. Furthermore, this enzyme increased basal secretion by 50%, and analysis of the incubation medium by HPLC demonstrated that the cells secreted biologically effective concentrations of adenosine. These results indicate that adenosine produced in culture tonically inhibits hormone release. In other target cells, adenosine inhibition is mediated by two types of binding sites: an extracellular Ri-site requiring an intact ribose moiety or an intracellular P-site requiring an intact purine ring. Four lines of evidence indicate that in GH4C1 cells, adenosine acts at an Ri-site. PIA, an Ri-site-specific agonist, was a potent inhibitor of hormone release (ED50 = 30 nM). Theophylline, an Ri-site antagonist, competitively inhibited the action of PIA (Ki = 2.4 microM). 3) 2'5'-Dideoxyadenosine, a P-site-specific agonist, did not inhibit PRL release even at a concentration of 1 mM. 4) Dipyridamole, an adenosine uptake inhibitor, did not reduce adenosine inhibition. In addition to its effect on basal secretion, PIA inhibited stimulation of hormone release by vasoactive intestinal peptide and TRH. PIA also reduced vasoactive intestinal peptide-stimulated cAMP accumulation by 75%, consistent with its action to inhibit adenylate cyclase via Ri receptors in other targets. Since PIA inhibition of PRL release and cAMP accumulation was not additive with the effects of SRIF and carbamyl choline, these inhibitors may act via a common rate-limiting step. Our results demonstrate that adenosine activates an Ri-type of adenosine receptor in GH4C1 cells and that the production of adenosine under normal culture conditions causes autocrine inhibition of secretion.

Topics: Adenine; Adenosine; Adenosine Deaminase; Animals; Carbachol; Cell Line; Chromatography, High Pressure Liquid; Cyclic AMP; Deoxyadenosines; Dideoxyadenosine; Dipyridamole; Growth Hormone; Phenylisopropyladenosine; Pituitary Gland; Pituitary Neoplasms; Prolactin; Rats; Thyrotropin-Releasing Hormone; Vasoactive Intestinal Peptide

The effect of an iv bolus injection of 1 microgram/kg body weight of vasoactive intestinal polypeptide (VIP) on plasma prolactin (Prl) levels was tested in 13 normal volunteers and 15 patients with hyperprolactinaemia of various aetiology: 9 with Prl-producing pituitary tumours (6 prolactinoma, 3 mixed pituitary adenoma, secreting Prl and growth hormone (GH)), 6 with hyperprolactinaemia secondary to a hypothalamic lesion (4 craniopharyngioma, 1 hypothalamic germinoma, 1 meningoencephalitis). In the normal subjects, an iv injection of VIP caused a prompt increase in plasma Prl with peaks 2- to 3-fold greater than the basal values. On the other hand, none of the 9 patients with a Prl producing pituitary tumour showed any obvious Prl rise after VIP irrespective of a marked difference in their basal Prl levels. Lack of a Prl response to VIP was also found in the 2 patients with hypothalamic lesions (1 craniopharyngioma, 1 hypothalamic germinoma) whose basal Prl concentration was higher than 100 ng/ml. However, in the remaining 4 patients with hypothalamic lesions whose basal Prl concentration was less than 100 ng/ml, VIP injection resulted in a stimulation of the Prl secretion with a maximal net increment of 11.3 +/- 3.8 ng/ml, which is not different statistically form that (16.3 +/- 3.3 ng/ml) in the normal subjects, but significantly higher than that (-2.3 +/- 2.7 ng/ml) in the 4 patients with Prl-secreting adenoma and a basal Prl concentration of less than 100 ng/ml. These results indicate that the VIP test may be a useful diagnostic tool for discriminating a Prl-producing tumour from a hypothalamic lesion in patients with mild hyperprolactinaemia.

Topics: Adenoma; Adolescent; Adult; Child; Diagnosis, Differential; Female; Humans; Hyperprolactinemia; Hypothalamic Diseases; Injections, Intravenous; Male; Middle Aged; Pituitary Neoplasms; Prolactin; Vasoactive Intestinal Peptide

We studied the in vitro responsiveness of prolactin-secreting MtTW15 and 7315a pituitary tumor cells to stimulation by selected secretagogues using a perifusion technique. Prolactin release by these cells was refractory to thyrotropin-releasing hormone (TRH) and vasoactive intestinal peptide (VIP). In contrast, 50 mM K+, dibutyryl cAMP, theophylline, phospholipase A2 and phorbol myristate acetate all increased prolactin release from both tumor cell types. Phospholipase C increased prolactin release from 7315a but not from MtTW15 cells. TRH increased 32P incorporation into phosphatidylinositol in the 7315a but not in the MtTW15 tumor cells. Therefore, the refractoriness of these tumors to TRH and VIP may be at least partially due to a defect in the receptor or in the process that couples receptor binding and intracellular biochemical processes. In the MtTW15 tumor at least part of the defect may be related to phospholipid hydrolysis.

Topics: Animals; Bucladesine; Cell Line; Female; Phosphates; Phosphatidylinositols; Phospholipases A; Phospholipases A2; Pituitary Neoplasms; Prolactin; Rats; Rats, Inbred BUF; Rats, Inbred Strains; Tetradecanoylphorbol Acetate; Theophylline; Thyrotropin-Releasing Hormone; Type C Phospholipases; Vasoactive Intestinal Peptide

The interaction between forskolin and vasoactive intestinal polypeptide (VIP) in the regulation of cyclic AMP production in GH3 pituitary tumour cells was investigated. Both forskolin (10nM-10 microns) and VIP (10pM-10nM) increased the cyclic AMP content of GH3 cells. Forskolin (50-100nM) was additive with VIP in stimulating cyclic AMP accumulation when low concentrations (less than 1 nM) of the peptide were used, but exhibited a synergistic interaction with higher VIP concentrations (10-100 nM). These effects on cyclic AMP accumulation were reflected in a leftward shift in the concentration-response curve for VIP-stimulated prolactin release from GH3 cells, a process known to be regulated by intracellular cyclic AMP concentrations. The synergy observed did not appear to be related to changes in cyclic nucleotide phosphodiesterase activity, since it was even more marked in the presence of isobutylmethylxanthine, a phosphodiesterase inhibitor. Studies of the time-course of VIP-induced changes in GH3-cell cyclic AMP content revealed that, with high concentrations of VIP, production ceased within 2 min of addition. This attenuation of cyclic AMP synthesis was still observed in the presence of isobutylmethylxanthine, but was markedly inhibited by low concentrations of forskolin (50-100nM). The results suggest that VIP-induced cyclic AMP production rapidly becomes desensitized. This process, which is prevented by forskolin, may be related to changes in the ability of the guanine nucleotide regulatory protein to couple receptor occupancy to activation of adenylate cyclase.

Topics: Animals; Clone Cells; Colforsin; Cyclic AMP; Diterpenes; Drug Synergism; Pituitary Neoplasms; Prolactin; Radioimmunoassay; Rats; Stimulation, Chemical; Time Factors; Vasoactive Intestinal Peptide

The studies reported here were directed toward establishing the mechanism by which TRH acutely stimulates PRL secretion in GH3 pituitary cells. Studies of TRH stimulation of PRL secretion were conducted on a time scale which enables comparison with other reported rapid effects of TRH on GH3 cells. TRH stimulation of secretion was found to be extremely rapid in onset (less than or equal to 10 sec) and biphasic (phase I, 0-2 min; phase II, 5-60 min). The earliest (phase I) secretory response was observed to be independent of medium Ca+2 concentration or Ca+2 influx, but to be dependent on an intracellular Ca+2 pool. The phase II response to TRH was found to depend, in part, on medium Ca+2. The phase I response to TRH could be mimicked only by agents known to influence Ca+2 translocation in GH3 cells (60 mM K+, A23187, ionomycin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and carbonyl cyanide m-chlorophenylhydrazone). These agents failed to promote sustained PRL release characteristic of phase II. It is concluded that the ability of TRH to rapidly stimulate PRL secretion (phase I) is correlated with its ability to rapidly promote a transient cytoplasmic Ca+2 concentration rise from an intracellular Ca+2 pool.

Topics: Animals; Calcimycin; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Line; Diglycerides; Gallopamil; Glycerides; Pituitary Neoplasms; Potassium; Prolactin; Rats; Receptors, Cell Surface; Receptors, Thyrotropin-Releasing Hormone; Thyrotropin-Releasing Hormone; Vasoactive Intestinal Peptide

The inhibition of prolactin secretion and cyclic AMP accumulation in GH3 cells by muscarinic agonists was blocked by preincubation of the cells with pertussis toxin (islet-activating protein). There was a lag of approx. 80 min in the onset of the effect on secretion. These results suggest that muscarinic agonists decrease prolactin secretion by inhibiting adenylate cyclase activity.

Topics: Adenylate Cyclase Toxin; Animals; Bacterial Toxins; Cell Line; Cyclic AMP; Oxotremorine; Parasympathomimetics; Pertussis Toxin; Pituitary Gland, Anterior; Pituitary Neoplasms; Prolactin; Rats; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

Somatostatin (SRIF) inhibits vasoactive intestinal peptide (VIP)-stimulated cAMP accumulation in the GH4C1 strain of rat pituitary tumor cells, and this effect is responsible for SRIF inhibition of VIP-stimulated hormone release. In this study we examined the interaction between the SRIF receptor and adenylate cyclase in GH4C1 cell membranes. Maximal concentrations of VIP (50 nM) increased membrane adenylate cyclase activity 4.2-fold; half-maximal stimulation was observed with 0.75 nM VIP. SRIF noncompetitively inhibited the stimulatory effect of VIP, but it did not alter basal adenylate cyclase activity. The relative potencies of SRIF and two SRIF analogs as inhibitors of VIP-stimulated adenylate cyclase activity in membranes and of VIP-stimulated cAMP accumulation in intact cells were similar. Furthermore, the concentration of SRIF that caused half-maximal inhibition of adenylate cyclase activity (ED50 = 2.3 nM) was close to the equilibrium dissociation constant for SRIF (Kd = 0.40 nM) measured in membrane preparations in the presence of GTP. Therefore, SRIF inhibition of adenylate cyclase appears to be receptor mediated. As with receptors known to regulate adenylate cyclase by interaction with a guanine nucleotide regulatory subunit, SRIF receptor binding was decreased in the presence of guanine nucleotides. Addition of GTP (150 microM) or the nonhydrolyzable GTP analog guanyl-5'-yl-imidodiphosphate (100 microM) decreased the specific binding of [125I-Tyr1]SRIF to 31% and 13% of the control value, respectively. This decrease in specific binding was due entirely to decreased receptor affinity for SRIF. GTP (150 microM) increased the equilibrium dissociation constant for SRIF from 0.11 to 0.40 nM, whereas the number of binding sites was unaffected by the nucleotide (Bmax = 0.2 pmol/mg protein). Analysis of dissociation kinetics demonstrated that in the absence of guanyl nucleotides, the rate of [125I-Tyr1]SRIF dissociation was first order (t 1/2 = 180 min). However, in the presence of a half-maximal concentration of guanyl-5'-yl-imidodiphosphate (0.3 microM), [125I-Tyr1]SRIF dissociation occurred with biphasic kinetics. Fifty percent of the specifically bound peptide dissociated at the same rate as that observed in the absence of nucleotide, whereas the remainder dissociated 15 times more rapidly (t 1/2 = 9.6 min).(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Adenylyl Cyclases; Animals; Cell Line; Cell Membrane; Cyclic AMP; Guanine Nucleotides; Pituitary Neoplasms; Rats; Receptors, Cell Surface; Receptors, Somatostatin; Somatostatin; Time Factors; Vasoactive Intestinal Peptide

Acetylcholine, oxotremorine and carbachol, compounds that exhibit muscarinic agonist activity, maximally inhibited basal prolactin secretion from GH3 cells by approx. 50% and intracellular cyclic AMP levels by approx. 20%. Both parameters were inhibited with similar potencies by each agonist. These inhibitory effects were blocked by a muscarinic but not by a nicotinic receptor antagonist. In the presence of VIP or IBMX, which raise intracellular cyclic AMP levels and stimulate hormone release, the degree of muscarinic inhibition was increased, but the potency remained unchanged. Similar changes in the secretory rate of prolactin and growth hormone occurred in these and in cell perifusion experiments. These results suggest that the inhibition of hormone secretion from GH3 cells by muscarinic agonists is mediated by a decrease in intracellular cyclic AMP levels.

Topics: 1-Methyl-3-isobutylxanthine; Acetylcholine; Animals; Carbachol; Cell Line; Cyclic AMP; Growth Hormone; Kinetics; Oxotremorine; Pituitary Gland, Anterior; Pituitary Neoplasms; Prolactin; Rats; Receptors, Muscarinic; Vasoactive Intestinal Peptide

We have studied the effects of vasoactive intestinal peptide (VIP) on PRL secretion in a Bio-Gel column parafusion system containing rat pituitary tumour cells (GH4C1). A dose-dependent increase in PRL release was observed with half-maximal and maximal effect (2.1-fold) at 8 X 10(-8) and 5 X 10(-6) M, respectively. The PRL-stimulatory effect of VIP was instantaneous and maintained during the parafusion experiments (up to 60 min). On a molar basis VIP was always less effective than thyroliberin (THR), and the maximum stimulation of PRL release obtained with TRH was 1.2-3.0-fold higher (n = 12) than the maximum effect seen after VIP administration. The PRL-releasing effects of VIP, THR and high extracellular K+ were almost completely abolished in the presence of two inhibitors of voltage-sensitive Ca2+ channels, CoCl2 (10(-3) M) and verapamil (10(-4) M). In Ca2+-free buffer VIP, TRH and high extracellular K+ had only negligible effects, but the responses were fully restored in the presence of normal concentrations of extracellular Ca2+. In contrast to TRH, VIP had no demonstrable effect on the Ca2+-dependent action potentials of the GH4 cells.

Topics: Animals; Calcium; Cell Line; Dose-Response Relationship, Drug; Electrophysiology; Pituitary Neoplasms; Potassium; Prolactin; Rats; Thyrotropin-Releasing Hormone; Vasoactive Intestinal Peptide

Intravenous thyrotrophin releasing hormone (TRH) caused a 6.5-fold increase in plasma prolactin (PRL) in rats carrying implanted pituitary tumours. Vasoactive intestinal polypeptide (VIP) had no effect, but TRH given after VIP raised TRH stimulated secretion 13-fold above basal. 31P NMR spectroscopy showed that VIP caused a decrease in high energy metabolites (depleted phosphocreatine, elevated inorganic phosphate and lowered intracellular pH). TRH alone caused a similar but smaller effect; given after VIP, it caused no detectable depletion. We suggest that the changes in high energy metabolite concentrations reflect increased cellular energy consumption consistent with a priming process (stage 1) in PRL secretion, followed by hormone release (stage 2). VIP induces stage 1 whereas RTH induced both stages.

Topics: Animals; Drug Interactions; Female; Magnetic Resonance Spectroscopy; Neoplasm Transplantation; Pituitary Gland; Pituitary Neoplasms; Prolactin; Rats; Rats, Inbred Strains; Thyrotropin-Releasing Hormone; Vasoactive Intestinal Peptide; Vasodilation

Topics: Adrenocorticotropic Hormone; Animals; Cell Line; Dexamethasone; Dose-Response Relationship, Drug; Endorphins; Gastrointestinal Hormones; Mice; Pituitary Gland; Pituitary Neoplasms; Somatostatin; Vasoactive Intestinal Peptide

The involvement of cyclic AMP in mediating regulatory peptide-controlled prolactin release from GH3 pituitary tumour cells was investigated. Cholera toxin and forskolin elicited concentration-dependent increases in both GH3 cell cyclic AMP content and prolactin release. The maximum rise in prolactin release with these agents was 2-fold over basal. 8-Bromo-cyclic AMP produced a similar stimulation of prolactin release. The phosphodiesterase inhibitor isobutylmethylxanthine also produced an increase in prolactin release and GH3 cell cyclic AMP content. However, the magnitude of the stimulated prolactin release exceeded that obtained with any other agent. Thyrotropin-releasing hormone (thyroliberin) and vasoactive intestinal polypeptide produced a concentration-dependent rise in both cell cyclic AMP content and prolactin release. However, only vasoactive intestinal polypeptide elicited an increase in cell cyclic AMP content at concentrations relevant to the stimulation of prolactin release. Vasoactive intestinal polypeptide and thyrotropin-releasing hormone, when used in combination, were additive with respect to prolactin release. Vasoactive intestinal polypeptide and forskolin, at concentrations that were maximal upon prolactin release, were, when used in combination, synergistic upon GH3 cell cyclic AMP content but were not additive upon prolactin release. In conclusion the evidence supports a role for cyclic AMP in the mediation of vasoactive intestinal polypeptide- but not thyrotropin-releasing hormone-stimulated prolactin release from GH3 cells. A quantitative analysis indicates that a 50-100% rise in cyclic AMP suffices to stimulate cyclic AMP-dependent prolactin release fully.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Cells, Cultured; Cholera Toxin; Colforsin; Cyclic AMP; Diterpenes; Dose-Response Relationship, Drug; Pituitary Neoplasms; Prolactin; Rats; Thyrotropin-Releasing Hormone; Vasoactive Intestinal Peptide

Topics: Adenoma; Adenylyl Cyclases; Bucladesine; Culture Techniques; Dopamine; Dose-Response Relationship, Drug; Female; Gastrointestinal Hormones; Humans; Male; Pituitary Neoplasms; Prolactin; Sodium Fluoride; Vasoactive Intestinal Peptide

PRL responsitivity after Vaso-active intestinal Peptide (VIP) infusion was studied during a Dopamine (DA) infusion, in normal women and women with a prolactinoma. VIP alone (6.2 micrograms/min., 12 min.) increases PRL in normal females, but does not remove DA-induced PRL-decrease (1 microgram X kg X X min.-1). It has no effect in women with prolactinoma. In these women, PRL increases when VIP is administered at the 200th minute of DA infusion (1 microgram X kg/min.). A PRL release by VIP in women with a prolactinoma, could imply the restitution of a DA inhibitory control. The same dose of VIP does not modify PRL, when administered during a 4 microgram X kg/min. infusion of DA, in women with a prolactinoma.

Topics: Dopamine; Female; Humans; Pituitary Neoplasms; Prolactin; Vasoactive Intestinal Peptide

The effects of vasoactive intestinal polypeptide (VIP), dopamine, and somatostatin (SRIF) on GH secretion were examined in vitro in perifused pituitary adenoma tissues obtained at surgery from seven patients with acromegaly. The perifusion of VIP at 5 x 10(-8) M resulted in a significant increase in effluent GH levels in five of the seven adenomas. A dose-related GH response was observed from 5 x 10(-9) to 5 x 10(-7) M VIP in two adenomas examined. SRIF at 5 x 10(-8) to 10(-7) M suppressed not only baseline secretion of GH but also inhibited GH rises elicited by VIP in six of the seven adenomas. Dopamine at 5 x 10(-7) to 5 x 10(-6) M decreased the baseline secretion of GH in six of the seven adenomas. In four of the six adenomas responsive to dopamine, dopamine suppressed VIP-induced GH release when perifused simultaneously. In the remaining two dopamine-sensitive adenomas in which VIP alone failed to affect GH release, the inhibition by dopamine of GH release was blocked by VIP perifused concomitantly with dopamine. Synthetic TRH or theophylline perifused at the end of the experiment stimulated GH release in all of the adenomas, indicating the viability of tumor cells throughout the study. These results suggest that VIP stimulates GH release by its direct action on pituitary adenoma cells of acromegalic patients and that VIP, SRIF, and dopamine interact at the pituitary level in modulating GH secretion from these adenomas.

Topics: Acromegaly; Adenoma; Adult; Dopamine; Female; Gastrointestinal Hormones; Growth Hormone; Humans; Male; Middle Aged; Pituitary Neoplasms; Somatostatin; Theophylline; Thyrotropin-Releasing Hormone; Vasoactive Intestinal Peptide

The effect of vasoactive intestinal peptide (VIP), cholecystokinin octapeptide (CCK), bombesin, arginine vasopressin (AVP), and hydrocortisone (HC) on ACTH release from human corticotropinoma cells in culture has been studied. Tumor tissue was obtained from 6 patients with pituitary corticotropinomas. Eleven to 21 cultures yielding 0.7-2.0 X 10(6) cells/culture, were obtained from each tumor and maintained for periods of 4 weeks to longer than 6 months. VIP (500 ng/ml) significantly (P less than 0.005) stimulated ACTH release from all tumors studied, and a dose (5-500 ng/ml)-response effect was observed in 3 of 5 tumors. Stimulation by VIP was seen at 2,4, and 24 h and was maximal at 4 h. CCK and bombesin were without effect on ACTH release from 4 tumors studies at 4 h. AVP (1-10 mU/ml) stimulated ACTH from 4 tumors studied at 60 min or 4 h. Coincubation of cultures with VIP (50-500 ng/ml) and AVP (1-10 mU/ml) resulted in at least an additive effect. HC (100 ng/ml) significantly (P less than 0.025) inhibited basal ACTH secretion from 2 of 4 tumors at 4 h and from 3 of 4 (P less than 0.005) at 24 h. Simultaneous coincubation of cultures with VIP (50 ng/ml) and HC (100 ng/ml) resulted in an attenuation or blockade of the VIP-stimulated ACTH release, whereas prior incubation of cultures with HC for 28 h before exposure to VIP did not. The results demonstrate that VIP is a potent ACTH secretagogue from human corticotropinoma cells in culture; its effects are additive to those of AVP and modulated by HC.

Topics: Adenoma; Adrenocorticotropic Hormone; Arginine Vasopressin; Cells, Cultured; Dose-Response Relationship, Drug; Drug Interactions; Female; Gastrointestinal Hormones; Humans; Hydrocortisone; Pituitary Neoplasms; Vasoactive Intestinal Peptide

The effect of vasoactive intestinal polypeptide (VIP) on in vitro ACTH release and adenylate cyclase activity was investigated in human ACTH-secreting pituitary adenomas from 4 patients with Cushing's disease and 2 patients with Nelson's syndrome. In all the tumors tested, VIP elicited a dose-dependent stimulation of hormone release from adenoma fragments (90-247% at 10-7 M VIP) and of cAMP formation in membrane preparations (75-140% at 3 X 10-6 M VIP). Therefore a role of VIP in the control of ACTH secretion in human ACTH-secreting adenomas is suggested; a cAMP-dependent mechanism of action can also be hypothesized.

Topics: Adenoma; Adenylyl Cyclases; Adrenocorticotropic Hormone; Animals; Gastrointestinal Hormones; Humans; Pituitary Neoplasms; Stimulation, Chemical; Swine; Vasoactive Intestinal Peptide

Topics: Adrenocorticotropic Hormone; Animals; Cell Line; Corticotropin-Releasing Hormone; Cyclic AMP; Drug Synergism; Isoproterenol; Mice; Pituitary Gland, Anterior; Pituitary Neoplasms; Vasoactive Intestinal Peptide

Effects of Ca2+ and calmodulin on the adenylate cyclase activity of a prolactin and growth hormone-producing pituitary tumor cell strain (GH3) were examined. The adenylate cyclase activity of homogenates was stimulated approx. 60% by submicromolar free Ca2+ concentrations and inhibited by higher (microM range) concentrations of the cation. A 2-3-fold stimulation of the activity in response to Ca2+ was observed at physiologic concentrations of KCl, with both the stimulatory and inhibitory responses occurring at respectively higher free Ca2+ concentrations. Calmodulin in incubations at low KCl concentrations increased the enzyme activity at all Ca2+ concentrations tested. In incubations conducted at physiologic KCl concentrations, both the inhibitory and stimulatory responses to Ca2+ were shifted by calmodulin to lower respective concentrations of the cation, without significant change occurring in the maximal rate of enzymic activity at optimal free Ca2+ X Mg2+ concentrations in the incubation also influenced the Ca2+ concentration dependence of adenylate cyclase; at high Mg2+ more Ca2+ was required to obtain maximal activity. Trifluoperazine inhibited adenylate cyclase of GH3 cells only in the presence of Ca2+; as Ca2+ concentrations in the assay were increased, higher drug concentrations were required to inhibit the enzyme. Ca2+ was also observed to reduce the extent of enzyme destabilization which occurred during pretreatments at warm temperatures. Vasoactive intestinal polypeptide and phorbol myristate acetate, which stimulate prolactin secretion in intact GH3 cells, enhanced enzyme activity 4- and 2.5-fold, respectively, without added Ca2+. Increasing free Ca2+ concentrations reduced the enhancement by VIP and eliminated the stimulation by PMA.

Topics: Adenylyl Cyclases; Animals; Calcium; Calmodulin; Cell Line; Egtazic Acid; Kinetics; Magnesium; Pituitary Neoplasms; Potassium; Rats; Tetradecanoylphorbol Acetate; Vasoactive Intestinal Peptide

Topics: Adult; Female; Gastrointestinal Hormones; Humans; Pituitary Neoplasms; Prolactin; Vasoactive Intestinal Peptide

The effects of vasoactive intestinal polypeptide (VIP), TRH, dopamine, and rat median eminence extract on GH release from GH-secreting pituitary adenomas were studied in vitro using a sensitive superfusion method. Dispersed pituitary tumor cells obtained from three patients with acromegaly were placed in a superfusion column, and the amounts of GH in the superfusate were determined. The addition of VIP (10(-6) M) to the perfusion system resulted in a marked increase in GH release in all three cases, and a dose-response relationship in VIP (10(-8) 10(-6) M) induced GH secretion was observed in one case studied. TRH (10(-7) M) and median eminence extract (1 equivalent/ml) also caused an abrupt and marked increase in GH release in all of the experiments. The infusion of either dopamine (10(-7) M) or bromocriptine (10(-7) M) inhibited GH secretion. These results suggest that VIP as well as TRH stimulate GH secretion by a direct action on GH-secreting pituitary tumor cells in at least some acromegalic patients.

Topics: Adenoma; Adult; Dopamine; Dose-Response Relationship, Drug; Female; Gastrointestinal Hormones; Growth Hormone; Humans; In Vitro Techniques; Male; Median Eminence; Perfusion; Pituitary Neoplasms; Thyrotropin-Releasing Hormone; Tissue Extracts; Vasoactive Intestinal Peptide

Topics: Animals; Cells, Cultured; Cholecystokinin; Dopamine; Gastrointestinal Hormones; Humans; Pituitary Gland; Pituitary Neoplasms; Prolactin; Rats; Vasoactive Intestinal Peptide

Topics: Adenoma; Adenylyl Cyclases; Egtazic Acid; Gastrointestinal Hormones; Guanosine Triphosphate; Humans; In Vitro Techniques; Pituitary Neoplasms; Prolactin; Vasoactive Intestinal Peptide

The authors present a brief report on a familial case of Wermer's syndrome, and review the principal characteristics of this "multiple endocrine neoplasm" which usually affects the parathyroids, pancreas, and anterior pituitary.

Topics: Adult; Female; Gastrins; Glucagon; Humans; Insulin; Multiple Endocrine Neoplasia; Pancreatic Neoplasms; Parathyroid Neoplasms; Pituitary Neoplasms; Syndrome; Vasoactive Intestinal Peptide

Topics: 1-Methyl-3-isobutylxanthine; Animals; Cell Line; Cyclic AMP; Dose-Response Relationship, Drug; Gastrointestinal Hormones; Kinetics; Pituitary Neoplasms; Prolactin; Rats; Thyrotropin-Releasing Hormone; Vasoactive Intestinal Peptide

Receptors for the Vasoactive Intestinal Peptide (VIP) were characterized in particles enriched in plasma membranes obtained from a human prolactin-secreting pituiatry tumor. Native VIP inhibited competitively the binding of 125I-VIP to the particles and stimulated cyclic AMP production; both these effects were observed at concentrations of VIP as low as 10(-11)-10(-10) M, which are compatible with VIP concentrations in the hypothalamopituitary portal blood.

Topics: Adenylyl Cyclases; Adult; Cell Membrane; Enzyme Activation; Gastrointestinal Hormones; Humans; Kinetics; Male; Pituitary Neoplasms; Prolactin; Receptors, Cell Surface; Vasoactive Intestinal Peptide