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.

DNA synthesis during hormone-dependent mammary tumor regression in rats.

A serial biopsy method was developed to study DNA synthesis in 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors during the regression process induced by bromocryptine (CB-154) administration in highly inbred female SD rats. With this technique the changes in tumor size could be correlated with those of DNA synthesis in single regressing tumors. DNA synthesis was estimated by the in vitro incorporation of tritiated thymidine into DNA which correlated well (correlation coefficient r = 0.95) with the in vivo mitotic activity of these neoplasms. Neither the biopsies themselves nor the estral status of the hosts affected significantly the rate of tumor DNA synthesis. DNA synthesis decreased sharply 4-8 days after the beginning of CB-154 treatment, whereas tumor shrinkage occurred more gradually. In conclusion, the serial biopsy method is a reliable technique for the estimation of changes in DNA synthesis in regressing DMBA-induced rat mammary tumors, whereas the measurement of the rate of tumor shrinkage is not.

Cognitive impairment and morphological changes in the dorsal hippocampus of very old female rats.

The hippocampus, a medial temporal lobe structure necessary for the formation of spatial memory, is particularly affected by both normal and pathologic aging. In previous studies, we observed a significant age-related increase in dopaminergic neuron loss in the hypothalamus and the substantia nigra of female rats, which becomes more conspicuous at extreme ages. Here, we extend our studies by assessing spatial memory in 4-6 month-old (young), 26-month-old (old) and 29-32-month-old (senile) Sprague-Dawley female rats as well as the age-related histopathological changes in their dorsal hippocampus. Age changes in spatial memory performance were assessed with a modified version of the Barnes maze test. We employed two probe trials (PTs), one and five days after training, respectively, in order to evaluate learning ability as well as short-term and longer-term spatial memory retention. A set of relevant hippocampal cell markers was also quantitated in the animals by means of an unbiased stereological approach. The results revealed that old rats perform better than senile rats in acquisition trials and young rats perform better than both aging groups. However, during short-term PT both aging groups showed a preserved spatial memory while in longer-term PT, spatial memory showed deterioration in both aged groups. Morphological analysis showed a marked decrease (94-97%) in doublecortin neuron number in the dentate gyrus in both aged groups and a reduction in glial fibrillary acidic protein-positive cell number in the stratum radiatum of aging rats. Astroglial process length and branching complexity decreased in aged rats. We conclude that while target-seeking activity and learning ability decrease in aged females, spatial memory only declines in the longer-term tests. The reduction in neuroblast number and astroglial arborescence complexity in the dorsal hippocampus are likely to play a role in the cognitive deficits of aging rats.

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.

Adenovirus-mediated herpes simplex virus type-1 thymidine kinase gene therapy suppresses oestrogen-induced pituitary prolactinomas.

We tested the hypothesis that gene transfer using recombinant adenovirus vectors (RAds) expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK) might offer an alternative therapeutic approach for the treatment of pituitary prolactinomas that do not respond to classical treatment strategies. HSV1-TK converts the prodrug ganciclovir (GCV) to GCV monophosphate, which is in turn further phosphorylated by cellular kinases to GCV triphosphate, which is toxic to proliferating cells. One attractive feature of this system is the bystander effect, whereby untransduced cells are also killed. Our results show that RAd/HSV1-TK in the presence of GCV is nontoxic for the normal anterior pituitary (AP) gland in vitro, but causes cell death in the pituitary tumor cell lines GH3, a PRL/GH-secreting cell line, and AtT20, a corticotrophic cell line. We have used sulpiride- and oestrogen-induced lactotroph hyperplasia within the rat AP gland as an in vivo animal model. Intrapituitary infection of rats bearing oestrogen-induced lactotroph hyperplasia, with RAd/ HSV1-TK and subsequent treatment with GCV, decreases plasma PRL levels and reduces the mass of the pituitary gland. More so, there were no deleterious effects on circulating levels of other AP hormones, suggesting that the treatment was nontoxic to the AP gland in situ. In summary, our results show that suicide gene therapy using the HSV1-TK transgene could be further developed as a useful treatment to complement current therapies for prolactinomas.

Changes in somatotropin and thyrotropin secretory patterns in aging rats.

In order to clarify whether pituitary enlargement influences the secretory patterns of growth hormone (GH) and thyrotropin (TSH) in old rats, we studied the correlation between pituitary weight and plasma levels of GH and TSH in Sprague-Dawley rats of different age and sex. Young female (3-4 months; YF), old female (25 months; OF), and senescent female (33-35 months; SF) rats and young male (3-4 months; YM) and old male (24-26 months; OM) rats carrying chronic intraatrial cannulas were used. Sequential blood samples were removed through the cannulas while the animals remained conscious and undisturbed. Plasma TSH and GH as well as serum thyroxine (T4) and triiodothyronine (T3) were measured by radioimmunoassay. At two years of age, both males and females showed a consistent decline in GH pulse amplitude without change in trough levels. By 33-35 months of age, females showed a reversal in the previous pattern of change for GH secretion: pulse amplitude, trough levels, and mean plasma GH increased significantly with respect to the old females. The correlation between mean plasma GH and anterior pituitary (AP) weight was positive and significant (p less than 0.01) for females but nonsignificant for males. Old and senescent rats showed significantly lower serum T4, but not T3, than young animals while plasma TSH increased with age in both sexes.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

A comparison between hormone levels and T lymphocyte function in young and old rats.

There is growing evidence that changes in hormone secretion during aging can alter some functions of the thymus. In order to identify further relationships between changes in endocrine and thymic function during aging we measured the circulating levels of prolactin (PRL), growth hormone (GH) thyrotropin (TSH) thyroxine (T4) and triiodothyronine (T3), and determined their relation to a number of T lymphocyte functional indices in 20 young (4 months) and 20 old (28-30 months) Long-Evans male rats. Half of the young and old rats were chronically cannulated in order to obtain sequential plasma samples for measuring PRL, GH and TSH by RIA. Total T4 and T3 were measured by RIA in the trunk serum of all animals. Thymus and spleen cell cultures from each rat were established and used to assess the ability of thymocytes to respond to mitogens and lymphokines as well as to measure the levels of IL-2 production by splenic lymphocytes. Mean plasma GH and serum T4 were significantly lower in old as compared to young rats (P less than 0.05 and P less than 0.001 for GH and T4, respectively), while PRL, TSH and T3 did not show significant age changes. Thymic cell number showed a 100-fold decrease in old as compared to young animals (10.6 +/- 3.0) x 10(6) vs. (898 +/- 98) x 10(6) cells, respectively) whereas no age change was detected in spleen cell numbers. The proliferative response of thymocytes exposed to Con A, IL-1, Con A + IL-1 or Con A + IL-2, was consistently higher in young than in old animals while the capacity of splenocytes to release IL-2 in response to Con A was not statistically different in young and old rats. The age changes in serum T4 showed a significant correlation with those in thymocyte count as well as with those in IL-1 + Con A- and IL-2 + Con A-induced thymocyte proliferation. Plasma GH showed a significant inverse correlation with splenocyte IL-2 release. Our results suggest, although do not prove, that the lower T4 and GH secretion that typically develops in old rats may play a causal role in some of the changes that occur in thymic-dependent functions during aging.

Gonadal function in aging rats and its relation to pituitary and mammary pathology.

In the female rat, aging is characterized by a high incidence of prolactin (Prl)-secreting pituitary adenomas and mammary tumors. In contrast to this, old males show only a moderate to low incidence of pituitary and mammary pathology. Since gonadal steroids and Prl are thought to be key factors in the genesis of the above neoplastic pathologies, it was of interest to compare the serum levels of progesterone (P), estradiol (E2), testosterone (T) and Prl with the incidence of pituitary and mammary tumors in aging male and female rats. Young (3-4-month; YF), old (25-month; OF) and senescent (33-35-month; SF) female and young (3-4-month; YM) and old (24-26-month; OM) male Sprague-Dawley rats were killed by decapitation and their pituitaries weighed. Serum sex steroids and Prl were measured by RIA. The average life span of females but not males was markedly extended by systematic removal of mammary tumors. Females showed a rising incidence of mammary tumors after 14 months of age. In males, this pathology which began to appear at 16 months, had a much lower incidence than in females at all ages. Serum levels of E2 were (means +/- S.E.M.) 22.0 +/- 1.6; 18.9 +/- 0.8; 32.9 +/- 2.5; 37.3 +/- 2.0 and 32.2 +/- 3.0 pg/ml for YM, OM, YF, OF and SF, respectively. Serum P was 1.4 +/- 0.3; 1.6 +/- 0.2; 10.4 +/- 2.2; 9.7 +/- 3.3 and 6.8 +/- 0.8 ng/ml for YM, OM, YF, OF and SF, respectively. Serum T was 1578.9 +/- 188.7; 807.6 +/- 103.0; 197.5 +/- 11.8; 223.7 +/- 25.5 and 176.9 +/- 20.7 pg/ml for YM, OM, YF, OF and SF, respectively. Finally, serum Prl was 14.9 +/- 1.7; 21.9 +/- 4.0; 15.9 +/- 1.4; 52.4 +/- 9.4 and 170.8 +/- 31.1 ng/ml for YM, OM, YF, OF and SF, respectively. A strong correlation was found between serum Prl and anterior pituitary weight in OM, OF and SF, but not between serum Prl and sex steroid levels or sex steroid ratios. We conclude that, although the sex-related differences in mammary and pituitary tumor incidence during aging in rats can be partially accounted for by the different serum profiles of Prl and gonadal steroids in each sex, sex-associated differences in target tissue susceptibility should also be considered as an important determinant of the level of tumor incidence.

Impact of aging on the morphology and function of the somatotroph cell population in rats.

There is little information regarding the impact of aging on pituitary somatotroph (STH) cell population in rats. We therefore undertook a quantitative immunocytochemical assessment of this cell type in young (3 months), old (20 months) and senescent (29 months) male rats. An attempt was also made to correlate morphological parameters with serum levels of growth hormone (GH). Since thyroid status is highly influential on somatotropic function, serum levels of thyroxine (T4) and triiodothyronine (T3) were also measured in the three age groups. We found a marked age-related reduction in STH cell number, volume density, and surface density, as well as a milder but significant decline in STH cell area and perimeter. Basal serum levels of GH remained unchanged with age, whereas the estimated number and amplitude of GH pulses declined from young to old animals. Thyroxine but not T3 levels also declined with age. We conclude that in rats, aging causes a marked reduction in somatotroph number and, to a lesser extent, cell size. These alterations do not affect trough levels of circulating GH. Our data also suggest that the progressive hypothyroidism associated with aging in this species may contribute to the promotion of the above changes. The present study emphasizes the convenience of combining hormone measurements with quantitative morphological analysis of cell populations for the study of pituitary function during aging.

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.

Degradation of immunoreactive albumin in young and old conscious female rats.

The kinetics of inactivation of plasma albumin was studied in young (3-4 months) and old (25-28 months) Sprague-Dawley female rats. Conscious, free-moving animals carrying indwelling atrial and carotid cannulas received a single injection of [125I]-albumin (rat) via the carotid cannula. Sequential blood samples were removed at intervals during the following 120 min, and total (TR) and immunoprecipitable radioactivity (IPR) were determined in the corresponding plasmas. TR disappearance curves for young and old animals were almost identical but IPR disappearance curves showed a significantly faster decline in the young rats. The absolute plasma volumes for young and old rats were (mean +/- S.E.M.), 10.8 +/- 1.1 and 14.4 +/- 1.5 ml, respectively (P less than 0.05). The IPR/TR ratio, an estimate of albumin inactivation within the plasma space, showed a monoexponential decrease in vivo with a t 1/2 of 11.4 +/- 5.1 and 39.3 +/- 10.8 h (P less than 0.05) for young and old rats, respectively. The in vitro t 1/2s for albumin were 5.25 +/- 1.02 and 3.42 +/- 0.91 days (NS) for young and old rats, respectively. It is concluded that: the rate of albumin catabolism declines with age in the female rat; albumin is mainly inactivated in the extravascular space; and total plasma volume increases significantly with age in this species.

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.

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.

Immune-neuroendocrine interactions during aging: age-dependent thyrotropin-inhibiting activity of thymosin peptides.

Thymosin fraction 5 (TF-5), a partially purified thymic preparation, has been previously shown to have luteinizing hormone-releasing hormone (LH-RH)-releasing activity in perfused rat hypothalamus as well as in vivo stimulatory effect on the pituitary-adrenal axis in prepubertal monkeys. We report here the effect of TF-5 on the TSH-thyroid axis in young (3 months) and old (25 months) Sprague-Dawley male rats. Conscious free-moving animals carrying an indwelling atrial cannula received a single dose of 5 mg/kg body wt. of either bovine serum albumin (BSA) or TF-5 via the cannula. In the young rats, TF-5 induced a marked reduction of plasma thyrotropin (TSH) which was significantly greater than the normal circadian decline observed in the BSA-treated controls. The old males displayed high basal levels of TSH which showed no circadian rhythmicity, and did not respond to TF-5. Thyroxine (T4), triiodothyronine (T3), corticosterone, and prolactin levels were not affected by TF-5 at the dose levels tested. The old rats had significantly lower basal levels of T4, but not T3, than their young counterparts. The synthetic peptides thymosin alpha-1 and serum thymic factor, which are components of TF-5, had no effect on the above hormones when injected in doses up to 5 micrograms/kg body wt. Acute thymectomy in 3-month-old males induced a significant increase in basal levels of TSH without affecting plasma T4 or T3. These results suggest that the thymus has an inhibitory action on TSH in the rat, which is not mediated by the thyroid gland.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effect of homeostatic thymus hormone on plasma thyrotropin and growth hormone in young and old rats.

There is increasing evidence that the neuroendocrine system is responsive to hormonal signals generated by the immune system. Thus, interleukin-1, hepatocyte stimulating factor and thymosin have been shown to stimulate adrenocorticotropin, beta-endorphin and luteinizing hormone secretion. We report here that homeostatic thymus hormone (HTH), a well-characterized thymic preparation, reduces plasma thyrotropin (TSH) and growth hormone (GH) in young (3 months) Sprague-Dawley male rats, but fails to do so (TSH) or has a significantly weaker effect (GH) in old (26 months) animals. Young and old conscious, free-moving rats carrying an indwelling atrial cannula received the substances to be tested via the cannulas. Plasma samples were taken every 30 min for 5 h and hormones were measured by RIA. In the young rats, HTH (8 mg/kg body wt) induced a marked reduction in plasma TSH which was significantly greater than the normal circadian decline observed in saline-injected young controls. The old rats displayed high basal levels of TSH which showed no circadian rhythmicity and did not respond to HTH. Plasma thyroxine (T4) showed a significant age-related reduction but was not affected by HTH. The above dose of HTH significantly reduced plasma GH in young and old rats, but the effect was greater in the young animals. Mean basal levels of plasma GH were significantly lower in old than in young rats. The present results suggest that HTH, whose production by the thymus is known to be stimulated by TSH and GH, is involved in an inhibitory feedback loop regulating plasma TSH and GH in young rats. Our data also suggest an age-related desensitization of the TSH and GH systems to thymic influence in this species.

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.

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.

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.

Half-life of plasma growth hormone in young and old conscious female rats.

The kinetics of disappearance of plasma GH was studied in young (3-4 months) and old (24-27 months) Sprague-Dawley female rats. Conscious, free moving animals carrying indwelling atrial and carotid cannulas received a single injection of 125I-rGH via the carotid cannula. Sequential blood samples were removed at intervals during the following hour, and total (TR) and immunoprecipitable radioactivity (IPR) were determined in the corresponding plasmas. Both TR and IPR displayed biexponential kinetics in vivo which did not differ significantly, for each variable, between young and old animals. The volumes of distribution of GH were also similar in both age-groups. The IPR/TR ratio, an estimate of GH inactivation within the plasma space, showed a decreasing sigmoid-shaped kinetics in vivo with a time of semi-inactivation (ti1/2) of 23.8 +/- 1.2 and 29.0 +/- 1.0 min (mean +/- SE) for young and old rats, respectively (P less than 0.02). The estrous status did not significantly affect ti1/2 values in vivo. The in vitro t1/2 was estimated by incubating plasma from the young and old animals at 37 degrees C with 125I-rGH for several hours. The IPR/TR ratio displayed a linear kinetics in vitro with t1/2 values of 23.7 +/- 1.7 and 25.8 +/- 1.9 h (NS) for young and old animals, respectively. The above results show that GH catabolism decreases slightly with age in the female rat, although it is unlikely that this change has a significant effect on plasma levels of GH. The data also suggest that GH is physiologically inactivated in the extravascular space.