Glial cell line-derived neurotrophic factor gene therapy ameliorates chronic hyperprolactinemia in senile rats.

Progressive dysfunction of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons during normal aging is associated in the female rat with chronic hyperprolactinemia. We assessed the effectiveness of glial cell line-derived neurotrophic factor (GDNF) gene therapy to restore TIDA neuron function in senile female rats and reverse their chronic hyperprolactinemia. Young (2.5 months) and senile (29 months) rats received a bilateral intrahypothalamic injection (10(10) pfu) of either an adenoviral vector expressing the gene for beta-galactosidase; (Y-betagal and S-betagal, respectively) or a vector expressing rat GDNF (Y-GDNF and S-GDNF, respectively). Transgenic GDNF levels in supernatants of GDNF adenovector-transduced N2a neuronal cell cultures were 25+/-4 ng/ml, as determined by bioassay. In the rats, serum prolactin (PRL) was measured at regular intervals. On day 17 animals were sacrificed and neuronal nuclear antigen (NeuN) and tyrosine hydroxylase (TH) immunoreactive cells counted in the arcuate-periventricular hypothalamic region. The S-GDNF but not the S-betagal rats, showed a significant reduction in body weight. The chronic hyperprolactinemia of the senile females was significantly ameliorated in the S-GDNF rats (P<0.05) but not in the S-betagal rats. Neither age nor GDNF induced significant changes in the number of NeuN and TH neurons. We conclude that transgenic GDNF ameliorates chronic hyperprolactinemia in aging female rats, probably by restoring TIDA neuron function.

Gene therapy in the neuroendocrine system.

The implementation of experimental gene therapy in animal models of neuroendocrine diseases is an area of growing interest. In the hypothalamus, restorative gene therapy has been successfully implemented in Brattleboro rats, an arginine vasopressin (AVP) mutant which suffers from diabetes insipidus, and in Koletsky (fa(k)/fa(k)) and in Zucker (fa/fa) rats which have leptin receptor mutations that render them obese, hyperphagic and hyperinsulinemic. In the above models, viral vectors expressing AVP, leptin receptor b and proopiomelanocortin, respectively, were stereotaxically injected in the relevant hypothalamic regions. In rats, aging brings about a progressive degeneration and loss of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons, which are involved in the tonic inhibitory control of prolactin secretion and lactotropic cell proliferation. Stereotaxic injection of an adenoviral vector expressing insulin-like growth factor I corrected their chronic hyperprolactinemia and restored TIDA neuron numbers. Spontaneous intermediate lobe pituitary tumors in a retinoblastoma (Rb) gene mutant mouse were corrected by injection of an adenoviral vector expressing the human Rb cDNA and experimental prolactinomas in rats were partially reduced by intrapituitary injection of an adenoviral vector expressing the HSV1-thymidine kinase suicide gene. These results suggest that further implementation of gene therapy strategies in neuroendocrine models may be highly rewarding.

Altered functional responses with preserved morphology of gonadotrophic cells in congenitally athymic mice.

Neonatal thymectomy or congenital absence of the thymus induces severe reproductive deficiencies in female mice, which are associated with reduced levels of circulating and pituitary gonadotropins. In contrast, the reproductive function is well preserved in nude males. It was therefore of interest to assess gonadotrophic cell morphology and function in congenitally athymic male mice. Circulating gonadotropins were measured under basal and stressful conditions, taking as a reference their haired counterparts. Adult normal (+/+), heterozygous nude (nu/+), and homozygous (nu/nu) CD-1 mice were subjected to 1-h immobilization stress. Serum levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were assessed by RIA at 0, 30, and 60 min poststress. Athymic animals showed significantly lower basal levels of serum LH and FSH than their heterozygous littermates. Immunohistochemical assessment of LH and FSH cell populations revealed a normal morphology and cell number in the athymic animals compared to their normal littermates. Immobilization stress induced a significant reduction in gonadotrophin levels, particularly LH, in normal mice but had only a weak effect in athymic animals. It is concluded that congenital athymia in the adult male mouse is associated with decreased basal levels of serum LH and FSH, in the presence of a normal gonadotroph number and morphology. The anomalous responses of athymic mice to stress do not appear to be due to primary hypopituitarism but, rather, to an altered modulation of pituitary hormone secretion. .

Studies on the prolactin-releasing mechanism of histones H2A and H2B.

In previous studies we demonstrated that histone preparations possess multiple effects in vivo on pituitary hormone secretion. We have now studied the specificity and signal transduction pathways involved in the prolactin (PRL)-releasing activity of histones H2A and H2B on perifused and incubated rat pituitary cells. In the perifusion experiments, freshly dispersed pituitary cells were packed into short columns and were continuously perifused with serum-free medium. The substances to be tested (stimuli) were pumped through the perifusion circuit, at the end of which perifusate fractions were collected and PRL measured by specific RIA. In the incubation studies, freshly dispersed pituitary cells were incubated in a metabolic incubator with different stimuli at different doses and for varying times. Perifusion of cells with median eminence extract (1/30), histone H2A (30 microM) or histone H2B (30 microM), generated clear PRL release responses. Cells incubated with histone H2A and H2B showed a dose- and time-dependent stimulatory effect on PRL release which, for H2A, was blocked by peptide MB-35, an 86-120 amino acid synthetic fragment of histone H2A. The polycation, poly-lys was unable to mimic the action of histones. To detect the possible signal transduction pathways involved in the response of lactotrophs to histones, cells were incubated with the calcium ionophore A23187, the calcium chelator EGTA, the intracellular phosphoinositide enhancer LiCl, the intracellular cAMP enhancers caffeine, NaF and forskolin, and the protein kinase C inhibitor, trifluoperazine (TFP). Both EGTA (or EGTA plus A23187 ionophore) and TFP were able to reduce significantly the response of lactotrophs to histones. Our results confirm previous evidence that histones may act as hypophysotropic signals. The data also suggest that calcium- and diacylglycerol-associated pathways participate in these effects.

Studies on the gonadotropin-releasing activity of thymulin: changes with age.

We assessed the ability of thymulin, a zinc-dependent nonapeptide produced by the thymic epithelial cells, to influence the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from dispersed anterior pituitary (AP) cells from young, adult, and senescent female rats. Perifusion of young and senescent AP cells with thymulin doses of 10(-6) to 10(-5) M gave a significant stimulatory response for LH but not FSH. Gonadotropin release was always lower in the senescent cells. AP cells from both age groups incubated with 10(-8) to 10(-3) M thymulin showed a time- and dose-dependent response for both gonadotropins, with a maximal stimulation at 10(-7) M. Preincubation of thymulin with an antithymulin serum completely quenched the secretagogue activity of the hormone. Coincubation of thymulin with the secretagogue gonadotropin-releasing hormone (GnRH) revealed a synergistic effect on LH release and an additive effect on the release of FSH. The calcium chelator EGTA blocked the gonadotropin-releasing activity of thymulin in AP cells. The cAMP enhancers, caffeine, NaF, and forskolin significantly increased the thymulin-stimulated release of gonadotropins. The inositol phosphate enhancer LiCl potentiated the action of thymulin on gonadotropins. It is concluded that the gonadotropin-releasing activity documented here for thymulin is an age- and receptor-dependent effect mediated in part by calcium, cAMP, and inositol phosphates.

Growth hormone-releasing activity of thymulin on pituitary somatotropes is age dependent.

Thymulin is a Zn-bound nonapeptide produced by the thymic epithelial cells (TEC) whose secretion is modulated by growth hormone (GH), among others. We assessed the ability of thymulin to influence the release of GH from dispersed anterior pituitary (AP) cells from young, middle-aged and senescent Sprague-Dawley female rats. Perifused and incubated AP cells were used in different sets of experiments and GH release was measured by RIA. Perifusion of young and senescent AP cells with thymulin doses, ranging from 10(-8) to 10(-5) M, gave a logarithmic dose-response pattern of GH. Supernatants from TEC lines also showed GH secretagogue activity. The GH release was always lower in the senescent cells. AP cells incubated with 10(-8)-10(-3) M thymulin showed a time- and dose-dependent response, the latter being bell-shaped with a maximum at 10(-7) M thymulin. Preincubation of thymulin with an antithymulin serum completely quenched the secretagogue activity of the hormone. Coincubation of thymulin with GHRH revealed a semiadditive release of GH in young and middle-aged AP cells and an additive effect in senescent cells. In middle-aged AP cells, the synthetic GH secretagogue GHRP-6 showed a synergistic effect with thymulin on GH release. The calcium chelator EGTA, but not the calcium ionophore A23187, blocked the GH-releasing activity of thymulin in AP cells. The cAMP enhancers, caffeine, NaF and forskolin significantly increased the thymulin-stimulated release of GH while trifluoperazine, a protein kinase C inhibitor, had no effect. The inositol phosphate enhancer LiCl potentiated the action of thymulin on GH release. The data suggest that the GH-releasing activity of thymulin is receptor-mediated and involves calcium, cAMP and inositol phosphates. In addition, senescence appears to impair somatotrope responsiveness to thymulin.

Thymulin stimulates prolactin and thyrotropin release in an age-related manner.

Thymulin is a Zn-bound nonapeptide produced by the thymic epithelial cells (TEC) whose secretion is modulated by prolactin (PRL) and thyroid hormones, among other hormones. We assessed the ability of thymulin to influence the release of PRL and thyroid stimulating hormone (TSH) from dispersed anterior pituitary (AP) cells from young, middle-aged and senescent Sprague-Dawley female rats. Perifused and incubated AP cells were used in different sets of experiments and PRL and TSH release was measured by radioimmunoassay. Perifusion of young and senescent AP cells with thymulin doses ranging from 10(-8) to 10(-5) M gave a logarithmic dose response pattern for both hormones. Supernatants from TEC lines also showed PRL and TSH secretagogue activity. Hormone release was always lower in the senescent cells. AP cells incubated with 10(-8) to 10(-3) M thymulin showed a time- and dose-dependent response for both hormones, the latter being bell-shaped with a maximum at 10(-7) M thymulin. Preincubation of thymulin with an anti-thymulin serum completely quenched the secretagogue activity of the thymic hormone. Coincubation of thymulin with TRH revealed a synergistic release of PRL and TSH in AP cells from all age groups. The calcium chelator EGTA but not the calcium ionophore A23187, blocked the hormone-releasing activity of thymulin in AP cells. The cAMP enhancers, caffeine, NaF and forskolin, significantly increased the thymulin-stimulated release of PRL and TSH, while trifluoperazine, a protein kinase C inhibitor, had no effect. The inositol phosphate enhancer LiCl, potentiated the action of thymulin on PRL and TSH. The present results suggest that the TRH-like activity documented here for thymulin is a receptor-mediated effect which appears to involve calcium, cAMP and inositol phosphates. Senescence but not middle age, appears to impair AP cell responsiveness to thymulin.

Use of recombinant herpes simplex virus type 1 vectors for gene transfer into tumour and normal anterior pituitary cells.

In this paper we demonstrate the use of recombinant viral vectors derived from herpes simplex virus type 1 (HSV1) to transfer reporter genes in vitro into rat anterior pituitary cells grown in primary cultures and the anterior pituitary tumour cell lines GH3 and AtT20. The three vectors used were, tsK/beta-galactosidase (beta-gal), tsK/CRH and tsK/TIMP, the corresponding transgene products respectively being E. coli beta-gal, pre-procorticotropin releasing hormone (ppCRH), and the chimeric protein TIMP/Thy1 (tissue inhibitor of metalloproteinases (TIMP)/linked to the carboxy terminus of Thy1 which confers the addition of a glycolipid glycosyl-phosphatidylinositol anchor in the ER). Double labelling immunofluorescence experiments to detect reporter proteins and transduced cell types indicated that the three vectors could transfer and express the reporter genes in normal and tumour anterior pituitary cells. Virus infection of pituitary cells was characterised, and it was shown that infection with tsK/beta-gal at multiplicities of infection (MOI)=10, 100% of tumour and non-endocrine anterior pituitary cells expressed beta-gal, whereas 75% endocrine anterior pituitary cells expressed the transgene. Long-term expression studies after infection with tsK/beta-gal indicated that anterior pituitary cells in primary cultures expressed the transgene for significant longer periods than tumour anterior pituitary cells. Growth arrest by serum starvation markedly decreased the frequency of transgene expression in anterior pituitary cells following infection with tsK/beta-gal. Transgenic products expressed from tsK were targeted to their correct intracellular domain in both anterior pituitary cells in primary cultures and in pituitary tumour cell lines. We conclude that transgenes can be delivered into anterior pituitary cells in primary culture and pituitary tumour cell lines using tsK derived HSV1 vectors. The prospect of employing viral vectors to transfer genes into endocrine cells opens up the potential exploration of various molecular aspects of pituitary cell function both in vitro and in vivo, as well as the use of gene transfer into the pituitary for potentially therapeutic applications, such as the treatment of pituitary tumours.

Expression of transgenes in normal and neoplastic anterior pituitary cells using recombinant adenoviruses: long term expression, cell cycle dependency, and effects on hormone secretion.

Adenovirus vectors have recently been used to transfer genes into a variety of cell types, including neurons, glial cells, Schwann cells, and epithelial cells. To evaluate the efficiency of gene transfer into pituitary cells using viral vectors, we used replication-deficient recombinant adenovirus vectors (RAds) encoding beta-galactosidase driven by various viral promoters. We tested the ability of RAds to infect and express beta-galactosidase within the different identified cell populations of the anterior pituitary anterior pituitary gland and also in tumor cells of anterior pituitary origin, i.e. GH3 and AtT20 cells. Our results demonstrate that transgenes encoded by RAds are expressed within all cell types of the adenohypophysis in vitro and also within AtT20 and GH3 endocrine tumor cells. Our long term expression studies indicate that long term expression with low cytotoxicity can be achieved, but that the longevity of transgene expression from RAds depends on the proliferative status of the target cells. Slowly dividing cells (endocrine population) express transgenes for longer than actively dividing cells (tumor cells and nonendocrine anterior pituitary cells). The ability of anterior pituitary cells to secrete ACTH or LH through the regulated secretory pathway decreased after infection with RAds at high multiplicity of infection (> or = 20 plaque-forming units/target cell), whereas cell viability was not affected. We also demonstrate that a higher percentage of cells expressed the transgene beta-galactosidase when we infected actively dividing GH3 cells compared with the infection of growth-arrested GH3 cells. This could reflect differential virus entry or differential activity of the individual promoters during different stages of the cell cycle. This work demonstrates that high efficiency gene transfer into all pituitary cell types can be achieved with RAds, and that this system can be exploited to characterize and experimentally manipulate pituitary-specific gene expression. The higher efficiency of infection and transgene expression in actively dividing cells compared to that in their growth-arrested counterparts could also be exploited for the treatment of pituitary adenomas that do not respond to classical treatment strategies, using suicide or cytotoxic gene therapy.

Thyrotropin-releasing activity of histone H2A, H2B and peptide MB35.

Histones possess multiple hormone-like activities. We studied the specificity and signal transduction pathways involved in the thyrotrophin (TSH)-releasing activity of histones H2A, H2B and peptide MB35, a H2A fragment, using perifused and incubated dispersed rat pituitary cells and measuring TSH release by a specific R1A. Histones released TSH in a dose- and time-dependent fashion while peptide MB35 behaved as a weaker secretagogue. Trifluoperazine and EGTA blocked histone-stimulated TSH release while forskolin and other cAMP enhancers did not. We conclude that the TSH-releasing activity of histones H2A and H2B is mediated by calcium- and diacylglycerol-associated pathways.

Histones as extracellular messengers: effects on growth hormone secretion.

Histones display hormone-like properties when present in extracellular fluids. The authors report that histones H2A and H2B possess growth hormone (GH)-releasing activity in vitro and describe the specificity and signal transduction pathways involved in these effects. Perfused and incubated rat pituitary cells were used in different sets of experiments and GH release was measured by radio-immunoassay (RIA). Perfusion of cells with 30 microM histone H2A or H2B, generated significant GH secretory responses. Cells incubated with histone H2A showed a dose- and time-dependent stimulatory effect on GH release which was blocked by peptide MB35, a synthetic fragment of histone H2A. Incubation of pituitary cells with the GH secretagogue GHRP-6, and histones revealed an additive release of GH, whereas GHRH and histones revealed a synergistic effect. The basic peptide poly-Lys did not mimetize the action of histones. Both EGTA and the protein kinase C inhibitor trifluoperazine, but not the calcium ionophre A23187, were able to reduce significantly the GH response of somatotrophs to histones. Pituitary cell incubation with 30 microM forskolin alone or in the presence of H2A or H2B, stimulated GH release in the same magnitude. The results confirm previous evidence that histones may act as hypophysotropic signals and suggest, although do not prove, that this activity is receptor dependent. Calcium- and diacylglycerol-associated pathways participate in these effects.

Age-dependent prolactin-releasing activity of nucleoproteins.

In previous studies we demonstrated that histone preparations possess multiple effects in vivo on pituitary hormone secretion and that these effects tend to disappear with age. We have now evaluated the in vitro effects of histone and nucleohistone preparations on the secretion of prolactin (PRL) in perifused pituitary cells from young (4 months) and senescent (29-33 months) female rats. Freshly dispersed pituitary cells were packed into short columns and were continuously perifused with serum-free medium. The substances to be tested were pumped through the perifusion circuit, at the end of which perifusate fractions were collected and hormones measured by specific radioimmunoassay (RIA). Quantitative immunohistochemistry was carried out on the pituitary glands from seven young and six senescent females. In vitro basal PRL release was similar in both age groups. Perfusion of cells with median eminence extract (1/90 to 1/10), histone H2A (100 to 1000 micrograms/ml) or nucleohistone (200 to 1000 micrograms/ml), generated PRL responses which were higher in young than in senescent cells. The pituitaries of the senescent animals were characterized, in most cases, by the presence of chromophobic microprolactinomas against a background of diffuse prolactotroph hyperplasia. Our results confirm previous evidence that circulating nucleohistones and histones may act as hypophysotropic signals. The morphologic alterations in PRL cell populations found in the sencscent rats may play role in the desensitization of the pituitary gland to nucleoproteins, and possibly to other hypophysiotropic molecules, with age.

Reduced ability of hypothalamic and pituitary extracts from old mice to stimulate thymulin secretion in vitro.

There is substantial evidence that growth hormone (GH) is particularly important in the control of the age-related decline of thymus function. It was therefore of interest: (a) to assess the overall capacity of tissue extracts from mediobasal hypothalamus (MBH), anterior pituitary (AP) and testis, obtained from young (3 months, Yc), middle-aged (13 months, MAc) and old (18 months, Oc) intact C57BL/6 mice to stimulate in vitro the release of thymulin, a Zn-bound immunoregulatory thymic peptide, from pure cultures of mouse thymic epithelial cells (TEC); (b) to perform the same evaluation utilizing MBH, AP and testicular extracts from mice of the same age-range but treated for 45 days with a sc dose of ovine GH (2 micrograms/g body wt) known to stimulate thymulin secretion in vivo. Pituitary hormones were measured by heterologous rat RIAs, whereas thymulin release was estimated by a rosette assay. Untreated animals showed a significant age-dependent increase in the AP content of follicle stimulating hormone but not in other AP hormones. In both control and treated animals, pituitary GH content decreased significantly with age. MBH extracts from C57BL/6 males evidenced thymulin-releasing activity on mouse TEC lines. This activity was maximal in the MBH from young animals and declined with the age of the MBH donors. The thymulin-releasing activity of MBHs from GH-treated mice was higher than that of the control animals and showed a less pronounced decline with age. AP extracts from the same animals showed a higher thymulin-releasing activity than did MBH preparations. This activity showed a progressive age-associated reduction in the APs from untreated mice, whereas in the GH-treated group, an age-related decline was only seen in the old donors. Control testicular extracts had little effect on thymulin release whereas GH treatment induced a definite thymulin-release inhibiting activity in the testicular homogenates of our animals which increased progressively with the age of the testis donors. We conclude that the MBH, AP and testis of the young mouse contain factors able to affect directly the endocrine activity of the thymic epithelium. The amount of these substances declines with age and seems to be modulated by GH.

Altered thyrotropic and somatotropic responses to environmental challenges in congenitally athymic mice.

Neonatal thymectomy or congenital absence of the thymus induces morphologic alterations in pituitary somatotrophs as well as in thyroid epithelium. It was therefore of interest to assess somatotropic and thyrotropic cell morphology and the corresponding serum hormone levels in athymic nude mice under basal and stressful conditions, taking as a reference their haired counterparts. Normal (+/+), heterozygous nude (nul+) and homozygous (nu/nu) CD-1 mice were subjected to either 1-h immobilization stress or 2-h cold stress. Serum levels of growth hormone (GH), thyrotropin (TSH), thyroxine (T4), and triiodothyronine (T3) were assessed by RIA at 0, 30, and 60 min poststress. Athymic animals showed lower basal levels of serum TSH, GH, and T3, but not T4, than their heterozygous littermates. Immunohistochemical assessment of somatotropic and thyrotropic cell populations revealed a normal morphology in the athymic animals. Immobilization stress induced a marked reduction in GH and TSH levels in normal mice but had only a weak effect in athymic animals. Two hours of cold exposure caused a comparable increase in serum TSH in normal and athymic animals, whereas the serum T4 and T3 response to cold was greater in the athymic nudes. Cold exposure drastically reduced serum GH levels in normal animals but had only a weak effect in the athymic mice. We conclude that congenital athymia in the mouse is associated with decreased basal levels of serum TSH and GH in the presence of a normal somatotroph and thyrotroph morphology. The anomalous responses of athymic mice to stress do not appear to be due to primary hypopituitarism but rather, to an altered modulation of pituitary hormone secretion.

Effects of growth hormone and thyroxine on thymulin secretion in aging rats.

It is well-established that the activity of the endocrine thymus is under neuroendocrine control. In particular, growth hormone (GH) and thyroxine (T4) have been shown to be capable of reconstituting thymus function in hormone-deficient animals. It was therefore of interest to assess the effect of combined administration of ovine GH (0.1 mg/100 g BW/day) and T4 (10 micrograms/100 g BW/day) on serum thymulin levels in young (5 months), old (21 months) and senescent (29-30 months) male Sprague-Dawley rats. Age-matched controls received 0.1 mg bovine serum albumin/100 g BW daily during the same period (14 days). Prolactin (Prl), GH, T4 and triiodothyronine (T3) were measured in serum by radioimmunoassay, whereas serum thymulin was determined by rosette bioassay. As expected, GH and T4 were lower in the old and senescent controls whereas serum Prl displayed a slight age-related increase. No age changes were detected in serum T3. Hormone-treated animals showed supraphysiologic levels of both T4 and T3, but serum levels were comparable among the three treated age groups for each thyroid hormone. Endogenous GH levels were moderately elevated in the treated rats. In the control rats serum thymulin showed a marked reduction from 5 to 21 months of age but no further reduction was observed between 21 and 29-30 months. Hormone treatment induced a mean relative increase (% increase relative to age-matched controls) in serum thymulin of 44, 38 and 48% in young, old and senescent rats, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Histones and related preparations interfere with immunoassays for peptide hormones.

We report here that histones and certain related preparations generate a consistent interference with radioimmuno (RIA), immunoradiometric (IRMA), and enzyme-linked immunosorbent (ELISA) assays for a number of peptide hormones. Histones H1, H2A, H2B, H3, HIIA, HIIS, protamine, and the related preparations homeostatic thymus hormone and peptide MB35 generated a dose-dependent signal in both the human corticotropin-releasing hormone (CRH) and the human adrenocorticotropic hormone (ACTH) IRMA. This signal was not affected when the linker antiserum was removed from the IRMA reagent mixture, thus proving that the signal was not due to cross-reaction or sample contamination with CRH or ACTH. The above histone preparations, as well as protamine, but not ubiquitin, also generated a strong negative interference with RIAs for ACTH, CRH, rat growth hormone (rGH), and rat prolactin (rPRL). In an ELISA system for the thymic peptide facteur thymique sérique, histones and protamine again showed a strong interfering activity. When known amounts of rGH, rPRL, and hACTH were dissolved in charcoal-washed horse serum or supernatants from rat liver homogenates (centrifuged 1 h at 10,000 x g), and the corresponding RIAs and IRMA (for ACTH) were performed in the absence or presence of histones HIIA and HIIS (at 1 mg/ml level), an interfering activity of histones was again observed. We conclude that histones and some related peptide preparations have, when present in biologic fluids, a significant capacity to interfere with peptide immunoassays.

Thymus-pituitary interactions during ageing.

We had previously shown that homoeostatic thymus hormone (HTH), an H2A-H2B histone dimer, possesses an age-dependent adrenostimulatory activity in vivo. It was therefore of interest to investigate the adrenocorticotropin (ACTH)-releasing activity of histones and other related preparations on perifused pituitary cells from young (2-4 months) and mature (16-18 months) rats. Histones and protamines gave a positive interference with the immunoradiometric assays (IRMA) for ACTH and corticotropin releasing hormone (CRH) whereas the histone fragment MB35, as well as nucleohistone and nucleoprotamine complexes did not. The latter three substances induced a significant ACTH response in pituitary cells from young and, to a lesser extent, mature rats. Our results suggest that nucleoprotein complexes released into the extracellular milieu may act as hypophysiotropic signals. On the other hand, the responsiveness of pituitary cells to these signals seems to decrease with the age of the donor.

Corticotropin-releasing (CRH) activity of thymic peptides on CRH-insensitive corticotropic tumor cells.

A number of thymic preparations are known to stimulate corticotropin (ACTH) release from pituitary cells but it remains unclear whether this effect is mediated by the corticotropin-releasing hormone (CRH) receptor-associated pathway. We report here that thymosin fraction five (TF5), peptide MB-35 and possibly calf thymus histones can stimulate the release of ACTH from a CRH-insensitive variant of the mouse corticotropic cell line AtT20. The effective concentration range at which TF5 and MB-35 displayed their ACTH-releasing activity in a dose-dependent manner was 100 to 2,000 micrograms/ml and 10 to 100 ng/ml, respectively, whereas neither preparation induced a significant depletion of intracellular ACTH stores. Our data suggest that thymosin peptides can stimulate ACTH release from corticotrophs by a CRH receptor-independent mechanism.

Thymosin peptides stimulate corticotropin release by a calcium-dependent mechanism.

Thymosin fraction five (TF5), a well-characterized immunoregulatory thymic preparation, has been reported to stimulate corticotropin (ACTH) release from rat pituitary cells. Since a previous study in our laboratory had shown that TF5 was able to stimulate ACTH release from corticotropin-releasing hormone (CRH)-insensitive corticotropic tumor cells, it was of interest to assess the role of calcium in the mechanism of action of TF5 on corticotropic cells. A CRH-insensitive variant, denoted AtT-20(CI), of the wild-type corticotropic tumor cell line AtT-20 was used. Synthetic h/rCRH within a dose range of 0.1-100 nM was completely ineffective to stimulate basal ACTH release from AtT-20(CI) cells, although the same batch of neuropeptide displayed the expected ACTH-releasing activity on dispersed rat pituitary cells (for instance, 0.1 nM CRH induced a 3.7-fold increase in ACTH release in this cell system). Median eminence extracts (1/10) induced only a 12% increase in ACTH release from AtT-20(CI) cells as compared to the 395% stimulation induced in normal pituitary cells. As expected, TF5 induced a dose-dependent increase in ACTH release from AtT-20(CI) cells. However, this ACTH-releasing activity of TF5 was completely abolished when cells were incubated in Ca-free medium or Ca-free medium containing 0.5 mM EGTA. On the other hand, the presence of the Ca ionophore A23187 (5 microM) in medium containing normal Ca levels (2.5 mM) did not affect the ACTH-releasing activity of TF5 on AtT-20(CI) cells.(ABSTRACT TRUNCATED AT 250 WORDS)