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.

Gene therapy for long-term restoration of circulating thymulin in thymectomized mice and rats.

Thymulin is a thymic peptide possessing hypophysiotropic activity and antiinflammatory effects in the brain. We constructed a synthetic DNA sequence encoding met-FTS, a biologically active analog of thymulin, and subsequently cloned it into different expression vectors. A sequence optimized for expression of met-FTS in rodents, 5'-ATGCAGGCCAAGTCGCAGGGGGGGTCGAACTAGTAG-3', was cloned in the mammalian expression vectors pCDNA3.1(+) and phMGFP (which expresses the Monster Green Fluorescent Protein), thus obtaining pcDNA3.1-metFTS and p-metFTS-hMGFP, which express met-FTS and the fluorescent fusion protein metFTS-hMGFP, respectively. The synthetic sequence was also used to construct the adenoviral vector RAd-metFTS, which expresses met-FTS. Transfection of HEK293 and BHK cells with pcDNA3.1-metFTS (experimental groups) or pcDNA3.1 (control), led to high levels of thymulin bioactivity (>600 versus <0.1 pg/ml in experimental and control supernatants, respectively). Transfection of HEK293 and BHK cells with pmetFTS-hMGFP revealed a cytoplasmic and nuclear distribution of the fluorescent fusion protein. A single intramuscular (i.m.) injection (10(7) plaque forming units (PFU)/mouse or 10(8) PFU/rat) of RAd-metFTS in thymectomized animals (nondetectable serum thymulin) restored serum thymulin levels for at least 110 and 130 days post-injection in mice and rats, respectively. We conclude that RAd-metFTS constitutes a suitable biotechnological tool for the implementation of thymulin gene therapy in animal models of chronic brain inflammation.

Potential of gene therapy for the treatment of pituitary tumors.

Pituitary adenomas constitute the most frequent neuroendocrine pathology, comprising up to 15% of primary intracranial tumors. Current therapies for pituitary tumors include surgery and radiotherapy, as well as pharmacological approaches for some types. Although all of these approaches have shown a significant degree of success, they are not devoid of unwanted side effects, and in most cases do not offer a permanent cure. Gene therapy-the transfer of genetic material for therapeutic purposes-has undergone an explosive development in the last few years. Within this context, the development of gene therapy approaches for the treatment of pituitary tumors emerges as a promising area of research. We begin by presenting a brief account of the genesis of prolactinomas, with particular emphasis on how estradiol induces prolactinomas in animals. In so doing, we discuss the role of each of the recently discovered growth inhibitory and growth stimulatory substances and their interactions in estrogen action. We also evaluate the cell-cell communication that may govern these growth factor interactions and subsequently promote the growth and survival of prolactinomas. Current research efforts to implement gene therapy in pituitary tumors include the treatment of experimental prolactinomas or somatomammotropic tumors with adenoviral vector-mediated transfer of the suicide gene for the herpes simplex type 1 (HSV1) thymidine kinase, which converts the prodrug ganciclovir into a toxic metabolite. In some cases, the suicide transgene has been placed under the control of pituitary cell-type specific promoters, like the human prolactin or human growth hormone promoters. Also, regulatable adenoviral vector systems are being assessed in gene therapy approaches for experimental pituitary tumors. In a different type of approach, an adenoviral vector, encoding the human retinoblastoma suppressor oncogene, has been successfully used to rescue the phenotype of spontaneous pituitary tumors of the pars intermedia in mice. We close the article by discussing the future of molecular therapies. We point out that although, gene therapy represents a key step in the development of molecular medicine, it has inherent limitations. As a consequence, it is our view that at some point, genetic therapies will have to move from exogenous gene transfer (i.e. gene therapy) to endogenous gene repair. This approach will call for radically new technologies, such as nanotechnology, whose present state of development is outlined.

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.

The thymus-pituitary axis and its changes during aging.

The pituitary-thymic axis constitutes a bidirectional circuit where the ascending feedback loop is effected by thymic factors of epithelial origin. The aim of the present article is to review the evidence demonstrating that aging brings about a progressive disruption in the integration of this network. In doing so, we briefly review the experimental evidence supporting the view that immune and neuroendocrine aging are interdependent processes. The advantages and limits of the nude mouse as a model of thymus-dependent accelerated aging is also discussed. Next, we review a number of studies which show that the endocrine thymus produces several bioactive molecules, generally called thymic hormones, which in addition to possessing immunoregulatory properties are also active on nervous and endocrine circuits. In particular, the reported activities of thymosin fraction 5 (TF5), thymosin alpha-1 and thymosin beta-4 on beta-endorphin, ACTH, glucocorticoids, LHRH and LH secretion in different animal and cell models are reviewed. The known hypophysiotropic actions of other thymic hormones like thymulin, homeostatic thymus hormone (HTH) and thymus factor are summarized. Aging has a significant impact on pituitary responsiveness to thymic hormones. Thus, it has been reported that TF5 and HTH have thyrotropin-inhibiting activity in young but not in old rats. Furthermore, intravenous administration of HTH was also able to reduce plasma GH and increase corticosterone levels in both young and old rats, although these responses were much weaker in the old animals. Further evidence on this topic is discussed. It is proposed that in addition to its central role in the regulation of the immune function, the thymus gland may extend its influence to nonimmunologic components of the body, including the neuroendocrine system. The early onset of thymus involution might therefore act as a triggering event which would initiate the gradual decline in homeostatic potential that characterizes the aging process.

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.

Procorticotrophin-releasing hormone: endoproteolytic processing and differential release of its derived peptides within AtT20 cells.

Procorticotrophin-releasing hormone (proCRH) is expressed mainly in the hypothalamus and in the placenta, where it undergoes tissue-specific endoproteolysis. Our results show that within stably transfected AtT20/D16V cells proCRH is cleaved to generate two fragments of approximately 8 and 3 kDa which could account for proCRH(125-194) and proCRH(125-151), respectively, and a 4.5 kDa product which could account for mature IR-CRH(1-41). The immunofluorescence staining patterns for IR-CRH and IR-ACTH and their response of secretagogues indicate targeting of proCRH and POMC to the secretory pathway in transfected AtT20 cells. In this work, we have used a unique set of specific RIAs and IRMAs to the full length POMC and proCRH molecules and several products of endoproteolytic processing to assess if they could be released differentially in response to stimulation. Although the release of both IR-ACTH and IR-CRH peptides from transfected AtT20 cells is stimulated in response to exposure to high potassium stimulation (51 mM KCl/SmM CaCl2), the sorting index (SI) suggests that mature ACTH is sorted to the regulated secretory pathway 2.1-fold more efficiently than mature CRH(1-41). Mature ACTH is also sorted to the regulated secretory pathway 9-fold more efficiently than IR-proCRH(125-151). Also, mature CRH(1-41) is sorted to the regulated secretory pathway 3-fold more efficiently than IR-proCRH(125-151). These results therefore indicate that the intracellular mechanisms for the storage and release of POMC, proCRH and their endoproteolytic products differ and would sustain the hypothesis that within mammalian peptidergic cells, different biologically active peptides originating from the same or different precursor molecules, could be differentially released in response to specific stimuli. This would give these cells the capacity to finely regulate neurotransmitter release in response to environmental and physiological demands.

Gonadotrophin-releasing activity of histones H2A and H2B.

We report that histones H2A and H2B possess gonadotrophin-releasing activity in vitro and assess the signal transduction pathways involved in these effects. Perifused and incubated rat anterior pituitary (AP) cells were used, and luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured by RIA. Perifusion of cells with histone H2A (30 muM) or histone H2B (30 muM), markedly stimulated LH release but failed to elicit any FSH response. Cells incubated with 6 or 30 muM histone H2A showed a dose- and time-dependent stimulatory effect on bot LH and FSH release which was blocked by 1 muM peptide MB35, an 86-120 amino acid fragment of histone H2A. Incubation of pituitary cells with gonadotrophin-releasing hormone (GnRH) and histones H2A or H2B showed a stimulatory effect on LH and FSH release which was similar to the sum of the separate effects. Trifluoperazine as well as ethylene glycol bis(b-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), alone or in the presence of the calcium ionophore A23187, significantly reduced the response of AP cells to histones. Various cyclic adenosine monophosphate (cAMP) enhancers had no effect on histone-stimulated release of gonadotrophins in incubated AP cells. Our results confirm previous evidence that histones may act as hypophysiotrophic signals. Calcium- and diacylglycerol-associated pathways, but not cAMP, appear to participate in these effects.

Effect of the corticotrophin releasing hormone precursor on interleukin-6 release by human mononuclear cells.

A growing body of evidence suggests that corticotrophin releasing hormone (CRH) may exert direct modulatory effects on immune cells. In the present study we assessed the effects of its precursor molecule, proCRH, on interleukin-6 (IL-6) release by human peripheral blood mononuclear cells (MNC). Human MNC were incubated with the corresponding stimuli for 24 hr. The supernatants were collected and IL-6 measured by ELISA. Conditioned medium from CHO-K1 cells stably transfected with the recombinant plasmid pEE14/rat pre-proCRH cDNA was used as the source of proCRH. Western blot analysis of this medium, using an antibody specific for the intact precursor, showed that no proCRH degradation products were present. The proCRH had an inhibitory effect on basal and LPS-stimulated release of IL-6. These results suggest that the full length CRH precursor may possess immunomodulatory properties.

Sexual dimorphism in the age changes of the pituitary lactotrophs in rats.

It is known that aging is associated with alterations in hypothalamic and pituitary functions. In the present study, we have undertaken a quantitative immunohistochemical assessment of the lactotroph cell population as well as prolactin (PRL) secretion, in male and female rats of different ages. Pituitaries from young (3 months), old (20 months) and senescent (29 months) male and female Sprague-Dawley rats were processed for the immunohistochemical detection of lactotrophs. Serum PRL was measured by a homologous RIA. Additionally, the in vitro PRL secretory activity was estimated by perifusion of pituitary cells from senescent animals. Analysis of morphometric parameters revealed age-related changes of PRL cell population in animals of both sexes. The cell density (CD), surface density (SD) and volume density (VD) decreased with age in both male and female rats. However, CD as well as SD appeared to have increased in females when compared to males, either in young or old animals, while VD was higher only in old females. The pituitaries of senescent females displayed chromophobic microadenomas on a background of diffuse PRL cell hyperplasia. Prolactin serum levels showed a marked increase with age in females, but only a modest elevation in males. In senescent females, PRL production per cell was reduced. We conclude that in rats, there exists a clear sexual dimorphism in the age-related changes of pituitary PRL cells.

Gene transfer into enteric neurons of the rat small intestine in organ culture using a replication defective recombinant herpes simplex virus type 1 (HSV1) vector, but not recombinant adenovirus vectors.

We have designed a system in which to test gene transfer into gut neurons consisting of an organ culture of neonatal rat small intestine. The tissue was exposed to herpes simplex- and adenovirus-derived vectors: (1) a temperature-sensitive herpes simplex virus-1 (HSV1) vector (tsK-beta gal) containing the lacZ gene encoding beta-galactosidase (beta-gal), under the transcriptional control of the HSV1 immediate-early 3 (IE3) promoter; (2) RAd35, an E1-/E3- replication-deficient adenovirus expressing lacZ under the control of a truncated HCMV major IE promoter; and (3) RAd122, an E1-/E3- replication-deficient adenovirus expressing the lacZ under the control of the RSV LTR. Forty-eight hours after the vector was added to the organ culture, we detected beta-gal using immunohistochemistry or X-gal histochemistry in tissue sections examined by light microscopy. We encountered a distinctive staining of cells arranged in two concentric circles corresponding in location to the myenteric and submucosal plexuses. Cells in these areas were of similar size and morphology to neonatal enteric neurons, as visualized by NADPH-diaphorase histochemistry and immunocytochemical staining with antibodies to the neuronally expressed proteins PGP 9.5, or neurofilaments. Double labelling with antibodies recognizing neurofilaments and beta-galactosidase revealed that most cells infected by tsK were neurons, while the RAd35 and 122 vectors only infected non-neuronal cells. We thus demonstrate that both HSV1- and adenovirus-derived vectors can be used to transfer genes to the gut in vitro, but they transduce different populations of target cells.

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.

Uptake of alpha-(L)-iduronidase produced by retrovirally transduced fibroblasts into neuronal and glial cells in vitro.

The uptake of recombinant alpha-(L)-iduronidase into glial and neuronal cells, produced by retrovirally transduced NIH3T3 fibroblasts, was studied. We demonstrate that: (1) neuronal and glial cells take up alpha-(L)-iduronidase released into the medium by retrovirally transduced fibroblasts expressing high levels of alpha-(L)-iduronidase; (2) both glial and neuronal cells express the cation independent mannose-6-phosphate receptor responsible for lysosomal enzyme uptake; and (3) uptake of the lysosomal enzyme can be blocked by excess free mannose-6-phosphate, but not glucose-6-phosphate. Thus, various brain cells take up alpha-(L)-iduronidase, possibly through a cation independent mannose-6-phosphate receptor mediated pathway, and this uptake is higher in actively dividing or immature brain cells. Consequently, (1) neuronal metabolism ought to be capable of cross correction by enzyme provided by genetically engineered and transplanted cells provided by bone marrow transplantation (BMT); (2) that BMT could have a more beneficial effect on neurological function if performed as early as possible; and (3) given that the uptake mechanism of glial cells has a higher capacity, it might be easier to target diseases like the leukodystrophies in which lysosomal enzymes are needed in glial cells, compared to diseases where lysosomal enzymes ought to be delivered into neurons.

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.

Corticotrophin-releasing hormone receptor type 1: generation and characterization of polyclonal antipeptide antibodies and their localization in pituitary cells and cortical neurones in vitro.

Corticotrophin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays a major role in regulating the endocrine response to stress. CRH acts by first binding to specific receptors on the plasma membrane of target cells. A CRH receptor from a human corticotroph adenoma and rat brain has recently been cloned (CRH-R1). In this paper, we have chosen three different peptide sequences within the CRH-R1 molecule which bear no similarity to other members of this receptor subfamily (or indeed any known protein) and which are likely to be exposed on the surface of the native protein, for antibody production. Some of these fragments produced antipeptide antibodies of good titre which cross-reacted with the CRH-R1 receptor expressed in transiently transfected COS-7 cells and in tissue extracts from rat cerebellum, cortex, pituitary gland and human myometrium, both in Western blots and in liquid-phase radioimmunoassay. We used immunofluorescence techniques to localize the CRH receptor in transiently transfected COS-7 cells, primary cultures of rat anterior pituitary (AP) cells, the corticotroph-tumour cells AtT20 D16-16 and cortical neurons in primary culture. Our results indicate IR-CRH-R1 receptors have a punctate distribution on the plasma membrane of AP cells and AtT20 D16-16 cells. Whilst in AP cells their appearance is a fine punctate pattern, in AtT20 cells, they appear as large patches which could account for receptor clusters. Within primary cortical neurons, their distribution does not appear to be polarized. Our results suggest that distribution of CRH-R1 receptors within the different cell-types investigated depends not only on the amino acid sequence but also on cellular factors.

Hormonal modulation of antioxidant enzyme activities in young and old rats.

The correlation between serum levels of pituitary and thyroid hormones and the activity of two antioxidant enzymes (AOE), catalase (CAT) and superoxide dismutase (SOD), in different immune and nonimmune organs of young and old rats was investigated. Serum levels of growth hormone (GH), prolactin (Prl), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroxine (T4), and triiodothyronine (T3) as well as the activity of AOE in liver (CAT and SOD), spleen (CAT and SOD), thymus (SOD) and mammary tissue (SOD) were determined in 7- and 29-month-old Sprague-Dawley female rats. A marked hyperprolactinemia and lower levels of TSH, LH, FSH and GH were observed in the old rats when compared to the young ones (p < 0.05). Old animals showed a higher SOD activity in liver and thymus but a lower activity in spleen than their young counterparts (p < 0.05). In addition, CAT activity in spleen was lower in old than in young rats (p < 0.05). The Spearman's test revealed a positive correlation between: 1) serum levels of TSH and the activity of SOD in liver and 2) serum levels of Prl and GH and the activity of SOD in mammary tissue of young and old animals (p < 0.05). Likewise, CAT in liver showed a highly significant correlation with serum TSH (but not T4 and T3) levels in young (p < 0.01) but not in old rats. Our results confirm that aging is associated with alterations in the endocrine balance and the activity of AOE in lymphoid as well as in nonimmune organs. In addition, our findings suggest that Prl and GH may be physiological modulators of mammary SOD activity, and that TSH can possibly influence the activity of both CAT and SOD in liver via a thyroid-independent pathway. Furthermore, our data show that, when significant, the correlation between hormone levels and AOE activity deteriorates with age.