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Biomedical subjects

R G Goya

Publications and source records attributed to R G Goya.

At least 37 records · Page 2Linked to original sources
PubMed1998

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.

Aging↗
PubMed1998

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.

Animals↗
PubMed1998

Age changes in the activity of liver 3-hydroxy-3-methylglutaryl-CoA reductase in female rats: influence of mammary pathology.

Hydroxymethylglutaryl-Coenzyme A (HMG-CoA) reductase is a highly regulated enzyme which shows a marked circadian rhythmicity. We studied the impact of aging on this rhythm as well as the degree of correlation between age changes in circulating pituitary hormone levels and liver reductase activity in young (4 months) and old (33 months) Sprague-Dawley female rats. Lipid composition was also assessed in plasma and liver microsomes. The maximal activity (midnight) of HMG-CoA reductase fell from 864 +/- 28 pmol mevalonate/min/mg protein in the young rats to 552 +/- 45 pmol/min/mg protein in the old animals, whereas significant change was not observed in the basal (noon) activity levels of the enzyme. Noon serum cholesterol, but not midnight values, was significantly higher in the old rats. Liver cholesterol levels were similar in young and old rats. In old rats, fatty acid composition of liver microsomes revealed an increase in linoleic acid concurrently with a significant decrease in arachidonic acid (AA). A significant correlation was not detected between the age changes in pituitary hormone (GH, PRL, TSH, FSH) serum levels and those in reductase activity. On the other hand, a significant positive correlation was found in the old rats between hepatic reductase activity and the severity of mammary pathology. We conclude that, like most biological rhythms, HMG-CoA reductase circadian fluctuation decreases in amplitude with age. This change does not seem to be linked to the alterations of neuroendocrine function associated with the aging process. The presence of growing mammary tumors seems to stimulate liver reductase activity, which may constitute an adaptive response of the enzyme to cholesterol demand by the growing neoplastic tissue.

Aging↗
PubMed1998

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.

Animals↗
PubMed1998

Modulation of rat liver lipid metabolism by prolactin.

The effect of chronic hyperprolactinemia on the delta6- and delta5-desaturation activity, total lipid and fatty acid composition, as well as fluorescence anisotropy, was studied in liver microsomes from anterior pituitary-grafted rats. We observed a depression in delta6-desaturation activity but no changes in the delta5-desaturation activity in the grafted animals. The microsomal fraction from the hyperprolactinemic rats contained significantly less amount of linoleic acid and a higher content of 20:4 n-6, 22:5 n-6 and 22:6 n-3 acids. Lipid rotational mobility was increased in microsomes as well as in liposomes obtained from the microsomes of transplanted animals. The fluidifying effect induced by PRL was located in the deepest zone of the membrane. The results obtained indicate that high levels of prolactin induce changes in polyunsaturated fatty acid distribution in liver microsomes, which regulates the lipid rotational mobility and hence membrane fluidity.

Animals↗
PubMed1997

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.

Animals↗
PubMed1997

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.

Adjuvants, Immunologic↗
PubMed1997

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.

Aging↗
PubMed1997

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.

Analysis of Variance↗
PubMed1997

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.

Adenoviridae↗
PubMed1996

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.

Aging↗
PubMed1995

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.

Aging↗
PubMed1995

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.

Animals↗
PubMed1995

Quantitative immunohistochemical changes in the endocrine pancreas of nonobese diabetic (NOD) mice.

The nonobese diabetic (NOD) mouse is an animal model that shares a number of clinical, genetic, and immunologic characteristics with human insulin-dependent diabetes mellitus. Since little is known about the morphometric cell profiles in the endocrine pancreas of these NOD animals, it was of interest to assess their changes in morphometry within the pancreatic islet cell types during two stages of this syndrome. Prediabetic (6-week-old) and diabetic (16-week-old) NOD female mice, as well as normal C57BL/6 female mice (15 weeks old), were used. Light microscopic immunocytochemical and morphometric methods were employed to study the endocrine cell populations. The immunoperoxidase technique for the identification of insulin, glucagon, somatostatin, and pancreatic polypeptide, as well as the point-counting method, was used on serial sections of pancreas tissue. Compared to those of normal and prediabetic mice, pancreata from diabetic animals showed a decrease in both the number of islets and the volume density of the endocrine component. Analysis of islet tissue revealed a significant diminution of B-cell volume density, as well as an increased A-, D-, and PP-cell volume density. A parallel variation in the number of B and non-B cells was also found. In addition, when the total pancreatic tissue surface was taken as reference, the fractional area occupied by all the different types of islet cells was seen to be diminished in a variable fashion. We conclude that the diabetic syndrome of NOD mice not only severely affects the B-cell mass, but also causes marked changes in the non-B endocrine-cell populations.

Animals↗
PubMed1995

Assessment of cardiovascular risk factors in menopausal Argentinian women.

The cardiovascular risk factor profile was assessed in a population sample consisting of 60 nonmenopausal (control) and 100 menopausal women from different cities in Buenos Aires Province, Argentina. Each subject was individually interviewed and asked to complete a specially designed questionnaire aimed at identifying cardiovascular risk factors. A clinical general and gynecological examination including blood pressure and anthropometric measurements as well as a Papanicolaou smear were performed. The most prevalent risk factor in the menopausal group was low physical activity (87% of the subjects), followed by nervous complaints (67%), obesity (64%), familial antecedents of cardiovascular disease (CVD; 38%) and hypertension (33%). Other risk factors assessed showed a level of prevalence below 10%. In the control group, a tobacco smoking habit was the CVD risk factor with the highest prevalence (47%). Nervous complaints also showed a high prevalence (48%). Most menopausal patients (77%) had a cardiovascular risk index (RI) level between 1.5 and 4.0, whereas 17% of these subjects had an RI greater than 4.0 (high-risk patients). The present study reveals that, in the studied community, the menopause is associated with increased levels of both estrogen-dependent and psychosocial risk factors for CVD.

Adult↗
PubMed1995

Immunohistochemical and radioimmunological assessment of thyrotrophs in the pituitary of aging rats.

Thyrotrophs were studied by quantitative immunohistochemistry in the pituitary gland of young (4 months), old (20 months) and very old (29 months) male rats. An attempt was also made to correlate morphometric parameters with serum levels of thyrotropin (TSH), thyroxine (T4) and triiodothyronine (T3). Cells were immunostained by the peroxidase-antiperoxidase method and hormones were measured in serum by specific radioimmunoassays. There was a marked age-related reduction in TSH cell number, volume density and surface density but a significant increase in TSH cell area and perimeter. Basal serum levels of TSH increased, T4 decreased and T3 remained unchanged with age. There was a highly significant (p < 0.001) negative correlation between serum TSH and T4, but no significant correlation was found between TSH and morphometric parameters. The present results suggest that aged rats possess a reduced but functionally preserved thyrotrophic cell population. The coexistence of high circulating levels of TSH with reduced serum T4 suggests that aging brings about a progressive desensitization of the thyroid to TSH.

Aging↗
PubMed1994

Immunohistochemical and radioimmunological study of pituitary gonadotrophs during aging in male rats.

The impact of aging on pituitary gonadotrophs in rats is little known. We therefore undertook a quantitative immunohistochemical assessment of the follicle-stimulating hormone (FSH) and luteinizing hormone (LH) immunoreactive cell population in the pituitaries of young (4 months), old (20 months) and senescent (29 months) male rats. In addition, an attempt was made to correlate morphometric parameters with serum levels of FSH and LH. Gonadotrophs were immunostained by the peroxidase-antiperoxidase method using anti-rat LH and anti-rat FSH rabbit sera as primary antibodies. Hormones were measured in serum by specific radioimmunoassays. Analysis of morphological parameters revealed, for both types of gonadotrophs, a marked age-related reduction in cell density, volume density and surface density. Gonadotroph cell perimeter and area showed a trend towards an increase from 5 to 20 months of age but appeared drastically reduced in the 29-month-old rats. Basal serum levels of LH and FSH showed a significant increase from 4 to 20 months of age but remained at the same level in the senescent rats. The correlation between serum levels of gonadotropins and gonadotroph morphometric parameters was low in all cases. We conclude that in male rats aging brings about a significant reduction in both the number and size of pituitary gonadotrophs which, nevertheless, seem to be still capable of keeping appropriate basal levels of circulating LH and FSH.

Aging↗