Monolayer Cultures Of Pituitary Cells Research Articles

Effects of TGF‐β1 on Prolactin Synthesis and Secretion: an In‐Vitro Study

The hypothalamus exerts a predominantly inhibitory influence on prolactin secretion through dopamine. In addition, the expression of anterior pituitary hormone-gene products are regulated by intrapituitary growth factors. In particular, TGF-beta1 produced in the pituitary regulates lactotroph cell proliferation and prolactin gene-expression. This study characterized the regulation of in-vitro prolactin synthesis and secretion by TGF-beta1 using rat anterior pituitary cells in monolayer culture. Furthermore, we studied the interaction of TGF-beta1 with other signals involved in the neuroregulation of prolactin secretion, such as dopamine and TRH, as well as the importance of different signal transduction pathways in this response. TGF-beta1 inhibited prolactin secretion in a time- and concentration-dependent manner, with half-maximal inhibition occurring at the range of 15-30 pM. The inhibitory effect was observed after 4 h, being maximal after 4 days of exposure of the cells to the peptide. This inhibitory effect was mimicked by TGF-beta2 but not by inhibin, and was not influenced by oestrogens, being similar in male, normal female or oestradiol-treated rats. Prolonged pretreatment of the cells with TGF-beta1(4 days) did not modify GH or TSH secretion nor dopamine-induced inhibition of prolactin secretion, and blunted prolactin responses to TRH, Forskolin, But2-cAMP and to the calcium ionophore A23187. The effect observed after long-term treatment (24 h to 4 days) is essentially caused by a decrease in prolactin synthesis, since TGF-beta1 inhibited prolactin mRNA levels and de novo prolactin protein synthesis. However, in the short term (up to 12 h) TGF-beta1 inhibition of prolactin secretion was associated with an increase in intracellular prolactin content, dissecting a dual mechanism of action of TGF-beta1. The short-term TGF-beta1 effect did not modify Erk-2 phosphorylation, basal or TRH-induced increase in intracellular calcium concentration, but blunted basal and forskolin stimulated cAMP levels. But2-cAMP replacement did not revert the inhibition of prolactin secretion. However, pertussis toxin was able to recover a large percentage of TGF-beta1-induced inhibition of prolactin secretion. This study indicates that TGF-beta1 plays a crucial role as a modulator of lactotroph function, inhibiting prolactin biosynthesis after long-term treatment, as well as, after short-term exposure prolactin secretion at the level of the secretory process, through a mechanism pertussis toxin sensitive but independent of Erk-2 phosphorylation, calcium concentrations or intracellular cAMP.

Immortalized human pituitary cells express glycoprotein alpha-subunit and thyrotropin beta (TSH beta).

A major problem in the study of human pituitary cells is their lack of proliferative capacity in vitro. To address this issue, we have infected normal human, postmortem pituitary cells in monolayer culture with a temperature-sensitive (tsA58) mutant of SV40 large T antigen. Several epithelial-like colonies were isolated; and one, designated CHP2, has been studied in detail to identify its functional characteristics. CHP2 cells have undergone more than 150 culture passages and retain an epithelial morphology. They exhibit tight temperature-dependent growth, in the presence and absence of serum, with cell division at 33 C and growth inhibition at 39 C. CHP2 cells, at both temperatures, showed diffuse immunostaining for human alpha-subunit and focal staining for TSH beta. Gene expression was confirmed by RT-PCR and sequencing. TRH and GnRH receptors were not detectable, and their absence was confirmed by their lack of effects on intracellular calcium and inositol phospholipids. Cytogenetic analysis showed that the cells had a modal peak in the diploid range and a smaller peak in the tetraploid range. There was also a consistent loss of chromosome 22 and a normal chromosome 2 homologue, the latter being replaced by one of two chromosome 2 markers, M2A or M2B. In conclusion, we have immortalized human pituitary cells using SV40 tsT, from which we have cloned a cell line expressing alpha-subunit and TSH beta.

Immunocytochemical Studies on the Cellular Origin of Ectopic Striated Muscle Fibers in Monolayer Cultures of Rat Anterior Pituitary Cells

We have previously reported that glia-like folliculo-stellate (FS) cells in the rat anterior pituitary may be involved in heterotopic differentiation of striated muscles which appear during pituitary cell culture. In the present immunocytochemical study, cytological alterations of FS cells in vitro were investigated by using antibodies to marker proteins (i.e., S-100 and vimentin) for FS cells. Expression of skeletal muscle-specific MyoD1 detected by immunocytochemistry, enabled identification of myogenic cells at a stage when they were still unicellular. Elongated myoblasts containing MyoD1-positive nuclei were found as early as the fifth day of monolayer culture of pituitary cells. The double immunostaining technique showed that some of these myoblasts reacted with antiserum to S-100. Although all of the myoblasts were immunoreac-tive to vimentin, this marker protein was unable to identify FS cells in vitro because rapidly proliferating fibroblasts were also immunoreactive to vimentin. Since the antiserum to S-100 that we used did not react with already differentiated striated muscle fibers, the demonstration of both MyoD1 and S-100-immunoreac-tive myoblasts in our pituitary cultures suggests that these myogenic cells are derived from FS cells. The present study does not rule out the possibility that fibroblasts, whose origin is presently unknown, are also involved in myogenesis in pituitary cultures.

Possible involvement of folliculo-stellate cells in the differentiation of muscle fibers during monolayer culture of pituitary cells.

A major objective of the present study was to examine the possibility that non-granular folliculo-stellate (FS) cells in the rat anterior pituitary are involved in the myogenesis that occurs during pituitary cell culture. Enzymatically dissociated anterior pituitary cells were fractionated by use of the Percoll gradient method. The proportion of FS cells was 5.8% on average before cell fractionation. After employing the Percoll gradient procedure, FS cells were enriched to a ratio of 12.2%. Three of five cell fractions were separately cultured, and the incidence of striated muscle fibers was quantitatively investigated. There was a good correlation between the numbers of muscle fibers and the proportions of FS cells in the fractions obtained from the Percoll gradient. These results suggest that FS cells are the cells that transform into striated muscles in pituitary monolayer cultures.

Regulation of the pituitary-specific transcription factor GHF-1/Pit-1 messenger ribonucleic acid levels by growth hormone-secretagogues in rat anterior pituitary cells in monolayer culture.

Pituitary-specific expression of the GH gene is dependent on a pituitary-specific transcription factor GH factor-1 (GHF-1), a homeodomain protein also known as pituitary-specific transcription factor-1 (Pit-1). The aim of this study was to investigate the regulation of GHF-1 messenger RNA (mRNA) levels in primary monolayer cultures of rat anterior pituitary cells. Specifically, in addition to direct activators of second messenger signaling systems, we studied the effects of different hormones, all of which are known to be involved in the regulation of somatotroph cell function. We found that GH-releasing hormone (GHRH) increased GHF-1 mRNA levels in a time- and dose-dependent fashion. GHF-1 mRNA levels were increased 2.5-fold (P < 0.01) after incubation for 2 h with 10(-8) M GHRH. Longer incubations (6, 12, or 24 h) with GHRH failed to show a similar stimulatory effect. A significant increase in GHF-1 mRNA concentration (1.7-fold, P < 0.01) was observed after a 2-h treatment with physiological concentrations (10(-11) M) of GHRH. The action of GHRH seems to occur at the transcriptional level without the need of protein synthesis. Thus, treatment of cells with actinomycin D (5 micrograms/ml) completely abolished GHRH-induced increase in GHF-1 mRNA levels. Cycloheximide (23 micrograms/ml) alone increased GHF-1 mRNA levels (6-fold increase after treatment for 12 h, P < 0.01), as well as potentiating GHRH-induced increase in GHF-1 mRNA concentration (9-fold increase after treatment with GHRH plus cycloheximide for 12 h, P < 0.01). The effect of GHRH on GHF-1 mRNA levels could be mimicked by direct activators of second messenger signaling systems such as forskolin (10(-5) M) or the phorbol ester tumor promoter tetradecanoyl phorbol acetate (TPA) (10(-6) M). Other peptides such as pituitary adenylate cyclase activating polypeptide-38 (10(-7) M) but not GHRP-6 (10(-10) to 10(-5) M), were also able to increase GHF-1 mRNA levels. Treatment of the cells with somatostatin (10(-6) M) for either 2 or 48 h failed to modify basal or GHRH-induced GHF-1 mRNA levels. In contrast, pretreatment of the cells with insulin-like growth factor-1 (5 nM) inhibited basal GHF-1 mRNA concentration as well as completely blunting the subsequent response to cells exposed to GHRH for 2 h. These data demonstrate that GHRH, acting at the transcriptional level and through a mechanism not dependent on protein synthesis, plays a stimulatory role on GHF-1 mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of the pituitary-specific transcription factor GHF-1/Pit-1 messenger ribonucleic acid levels by growth hormone-secretagogues in rat anterior pituitary cells in monolayer culture

Pituitary-specific expression of the GH gene is dependent on a pituitary-specific transcription factor GH factor-1 (GHF-1), a homeodomain protein also known as pituitary-specific transcription factor-1 (Pit-1). The aim of this study was to investigate the regulation of GHF-1 messenger RNA (mRNA) levels in primary monolayer cultures of rat anterior pituitary cells. Specifically, in addition to direct activators of second messenger signaling systems, we studied the effects of different hormones, all of which are known to be involved in the regulation of somatotroph cell function. We found that GH-releasing hormone (GHRH) increased GHF-1 mRNA levels in a time- and dose-dependent fashion. GHF-1 mRNA levels were increased 2.5-fold (P < 0.01) after incubation for 2 h with 10(-8) M GHRH. Longer incubations (6, 12, or 24 h) with GHRH failed to show a similar stimulatory effect. A significant increase in GHF-1 mRNA concentration (1.7-fold, P < 0.01) was observed after a 2-h treatment with physiological concentrations (10(-11) M) of GHRH. The action of GHRH seems to occur at the transcriptional level without the need of protein synthesis. Thus, treatment of cells with actinomycin D (5 micrograms/ml) completely abolished GHRH-induced increase in GHF-1 mRNA levels. Cycloheximide (23 micrograms/ml) alone increased GHF-1 mRNA levels (6-fold increase after treatment for 12 h, P < 0.01), as well as potentiating GHRH-induced increase in GHF-1 mRNA concentration (9-fold increase after treatment with GHRH plus cycloheximide for 12 h, P < 0.01). The effect of GHRH on GHF-1 mRNA levels could be mimicked by direct activators of second messenger signaling systems such as forskolin (10(-5) M) or the phorbol ester tumor promoter tetradecanoyl phorbol acetate (TPA) (10(-6) M). Other peptides such as pituitary adenylate cyclase activating polypeptide-38 (10(-7) M) but not GHRP-6 (10(-10) to 10(-5) M), were also able to increase GHF-1 mRNA levels. Treatment of the cells with somatostatin (10(-6) M) for either 2 or 48 h failed to modify basal or GHRH-induced GHF-1 mRNA levels. In contrast, pretreatment of the cells with insulin-like growth factor-1 (5 nM) inhibited basal GHF-1 mRNA concentration as well as completely blunting the subsequent response to cells exposed to GHRH for 2 h. These data demonstrate that GHRH, acting at the transcriptional level and through a mechanism not dependent on protein synthesis, plays a stimulatory role on GHF-1 mRNA levels.

Hormonal regulation of human follicle-stimulating hormone-beta subunit gene expression: GnRH stimulation and GnRH-independent androgen inhibition.

Previous studies from our laboratory demonstrated that chronic testosterone administration to castrated transgenic mice suppressed human follicle-stimulating hormone-beta (FSH beta) mRNA levels transcribed from a human transgene to approximately 20% of control values. In the present study we used primary pituitary cultures prepared from the transgenic mice and in vivo experiments in hypogonadal (hpg) mice carrying the human transgene to assess the role of hypothalamic gonadotropin-releasing hormone (GnRH) in this inhibitory action. The levels of human FSH beta mRNA in monolayer cultures of pituitary cells were decreased by 24-hour treatments with 10 nM testosterone propionate or 5 alpha-dihydrotestosterone to 13 and 26% of control values, respectively, in the absence of GnRH. For the in vivo experiments we introduced the 10-kb human FSH beta transgene into the hpg genetic background by selective crossbreeding. Daily injections of 1 microgram GnRH for 14 days induced expression of the human FSH beta gene in male and female mice. Maximal effects were obtained by GnRH treatment of gonadectomized, hpg transgenic mice. Human FSH beta mRNA levels rose to approximately 4- or 10-fold that of control males and females, respectively. The stimulation was blocked completely by simultaneous administration of testosterone propionate in males and partially by estradiol in females. Pituitary content of immunoreactive FSH paralleled the mRNA changes. These data suggest that testosterone feedback inhibits the human FSH beta subunit gene directly at the pituitary gland in addition to the indirect mechanism of GnRH suppression. Furthermore, the in vitro data indicate that the suppression of human FSH beta gene expression is at least partly a direct androgen effect that does not require aromatization of testosterone to estradiol.

Characterization of Rat Pituitary Cells by Their Responses to Hypothalamic Releasing Hormones

Rat pituitary cells in monolayer culture were characterized by their [Ca 2+] i responses to hypothalamic releasing hormones, growth hormone-releasing hormone (GHRH), thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH) and corticotropin-releasing hormone (CRH). The percentages of the cell population responding to GHRH, TRH, GnRH, CRH and non-responding cells were 27.3%, 47.6%, 13.8%, 6.2% and 35.3%, respectively. Some of the cells responded to two or more of those hormones. In the GHRH-responding cells, the population of TRH-responding cells was 51.4%, In the TRH-responding cells, the population of GHRH-responding cells was 30.8%. Some of the GHRH-responding cells also responded to CRH and GnRH. In the GnRH-responding cells, the population of TRH-responding cells was 61.8%. In summary, GHRH-responding cells have an especially close relationship with TRH-responding cells, and GnRH-responding cells also have close relationship with TRH-responding cells. There is also some relationship between the populations responding to other pairs of releasing hormones. These findings suggest functional overlapping among each population of pituitary cells.

Role of growth hormone-releasing hormone on pentagastrin-induced growth hormone release in normal subjects

In order to investigate the mechanisms by which gastrin cause GH release in humans we measured the GH response to pentagastrin alone (1.5 micrograms/kg/hour from 120 to 210 min) and following pretreatment with GHRH (GHRH 1-29,250 micrograms, iv at 0 min) in normal male subjects. Prior GHRH administration abolished the GH response to the second bolus of GHRH (1 micrograms/kg) administered two hours later. Pentagastrin infusion induced a rise in GH levels maximal at 60 min (9.1 + 0.6 ng/ml, mean + SE), but this rise was abolished by pretreatment with GHRH. Finally, we found that gastrin did not modify basal GH release or GH responses to GHRH by rat anterior pituitary cells in monolayer culture. Taken together, these data suggest that gastrin regulates GH secretion by acting at hypothalamic level.

Interrelationship between the Actions of Testosterone and Primate Sertoli Cell Inhibin in the Control of Gonadotropin Secretion by Cultured Pituitary Cells*

There is accumulating evidence that the differential regulation of LH and FSH secretion in the male is partly accomplished by the direct actions of testosterone (T) and inhibin on the pituitary. The present study was designed to examine the interaction between T and inhibin, in the presence and absence of GnRH, using dispersed pituitary cells in monolayer culture and cells perifused with pulses of GnRH from intact, 2-week castrated, and castrated T-replaced young adult male rats. The effect of partially purified inhibin from primate Sertoli cell culture medium (pSCI) to suppress basal FSH secretion was similar with pituitary cells from intact and castrated rats. T increased basal FSH secretion in the presence or absence of pSCI but did not alter the dose-dependent suppression of FSH by pSCI with cells from either intact or castrate rats. Castration increased basal FSH and LH secretion, whereas only basal FSH release was increased with cells from T-replaced castrates. T pretreatment increased the action of pSCI to suppress GnRH-stimulated FSH and LH release from perifused pituitary cells. These data indicate that T and inhibin exert opposite but independent effects on basal FSH release. The action of inhibin to suppress basal FSH secretion is not impaired by the absence of T and inhibin subsequent to castration. By contrast, the actions of T and inhibin to suppress GnRH-stimulated gonadotropin secretion are coordinated and interrelated.

Effects of Gonadal Steroids on Follicle-Stimulating Hormone and Luteinizing Hormone Secretion by Pituitary Cells from Castrated and Intact Male Rats1

The effectiveness of androgens in suppressing gonadotropin secretion declines with time following orchidectomy; however, the mechanism for this acquired resistance to androgen action is unknown. The role of the pituitary was studied by use of perifused rat pituitary cells and cells in monolayer culture. Pituitary cells from 7-wk-old intact male rats and rats that had been castrated 2 wk previously were treated with 10 nM testosterone (T) for 24 h; cells were then packed into perifusion chambers and stimulated with 2.5 nM GnRH for 2 min every hour for 8 h during which time T treatment was continued. T suppressed GnRH-stimulated LH secretion and LH pulse amplitude equally in both groups to approximately 60% of control values. Interpulse LH secretion was unchanged by T in either group. GnRH-stimulated FSH release was suppressed more (p less than 0.05) by T with cells from castrated rats than with cells from intact rats (76 +/- 4% vs. 90 +/- 2% of control; mean +/- SEM). By contrast, the action of T to increase interpulse basal FSH secretion was less (p less than 0.05) with cells from castrated rats (115 +/- 10% of control) than with cells from intact rats (146 +/- 6% of control). T treatment for 72 h also increased basal FSH secretion by pituitary cells in monolayer culture to a lesser extent with cells from castrated rats than with cells from intact rats (151 +/- 14% vs. 191 +/- 16% of control, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of triiodothyronine, dexamethasone and estradiol-17β on GH mRNA in rat pituitary cells in culture as revealed by in situ hybridization

The GH lines of rat pituitary tumour cells have been largely used to study the regulation of GH mRNA. In order to investigate the role of T3, dexamethasone and estradiol-17 beta on GH expression in non-tumoural pituitary cells, we have used in situ hybridization techniques performed on rat anterior pituitary cells in monolayer culture. The amounts of mRNA encoding for GH, as evaluated by counting the number of grains per somatotrope, were markedly reduced after 4 days of culture in a steroid-free medium supplemented with an hypothyroid calf serum. Addition of T3 or dexamethasone for 3 days increased GH mRNA levels. The concomitant administration of the two hormones produced a synergistic effect on GH mRNA levels which became higher than those observed after T3 or dexamethasone administration alone. However, this effect did not restore GH mRNA levels to those measured in monolayer pituitary cells grown in medium containing 10% fetal calf serum. Moreover GH mRNA levels appeared higher in male than in female pituitary cells. The administration of E2 to pituitary cell cultures from both male and female rats produced an increase by 15, and 12.8% in GH mRNA levels in male and female, respectively. This stimulatory effect of E2 in cell culture was competitively blocked by simultaneous incubation with the antiestrogen LY156758 (Keoxifene). These results demonstrate that T3, dexamethasone as well as E2 act directly on somatotropic cells to regulate GH gene expression.

Pre-exposure of rat anterior pituitary cells to somatostatin enhances subsequent growth hormone secretion.

The precise roles of GH-releasing factor (GRF) and somatostatin (SRIF) in the orchestration of pulsatile GH secretion have not yet been fully determined. We examined the interactions of rat GRF and SRIF in the concentration ranges present in rat hypophysial-portal blood, on the secretion of GH from dispersed male rat anterior pituitary cells in monolayer culture. The effects of exposing cells to GRF and/or SRIF (0.01-1.nmol/l) for 1 h were compared with the effects of preincubation of cells with SRIF before experimental incubations. As anticipated, the stimulatory effects of 0.1-1 nmol GRF/1 were abolished by concurrent incubation with SRIF at an equimolar concentration, although SRIF, at these concentrations, did not significantly inhibit basal GH secretion. Conversely, pre-exposure to 0.1 nmol SRIF/1 for 30 or 60 min, resulted in an increase in GH secretion during a subsequent 60-min incubation period, both in the absence or in the presence of GRF (0.01-1 nmol/l). Pretreatment with GRF caused increased responsivity to GRF rather than significant sensitization of the GH response to GRF. These observations demonstrate actions of SRIF, at low and probably physiological concentrations, which are more complex than those of a pure inhibitor of GH secretion. Pre-exposure of the pituitary to SRIF enhances subsequent GH secretion, suggesting that SRIF may play an additional physiological role in amplifying the GRF signal.

Isolation of bovine ovarian inhibin, its immunoneutralization in vitro and immunolocalization in bovine ovary

A purification scheme involving gel permeation chromatography, anion exchange chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) was used to isolate from bovine follicular fluid (FF) biologically-active inhibin of molecular weight 32 kDa. Chromatographic fractions were monitored for inhibin-like biological activity (ILA) using a simplified bioassay procedure in which a suppression of total basal FSH production by rat pituitary cells in monolayer culture indicates the presence of ILA. Approximately 3 mg protein having an ILA potency (ED 50 value in in vitro bioassay) of 1.7 ng/ml was obtained from 4 l crude bovine FF (260 g protein; ILA potency 3750 ng/ml) reflecting an approximate 2200-fold purification factor with an overall recovery of about 3%. The isolated material appeared as a single major UV absorbance peak on RP-HPLC and as a single band (32 kDa) when subjected to SDS-PAGE (15% gel) under non-reducing conditions. Under reducing conditions the molecule dissociated into 2 subunits of apparent molecular weight 22 and 14 kDa confirming that it is probably identical to the 31 32 kDa form of bovine ovarian inhibin previously reported by two other independent research groups. An antiserum raised in a chicken against the isolated material completely neutralized the suppressive effects of both 32 kDa inhibin and bovine FF on basal production of FSH by rat pituitary cells in vitro but only partially reversed the suppressive effects of both porcine and human FF. Immunohistochemical staining of sections of bovine ovary and of isolated preparations of bovine granulosa cells using this antiserum confirmed that granulosa cells are a major source of inhibin. The observation that specific immunostaining was not confined to these cells, however, suggests that they may not be the exclusive source of immunoreactive inhibin in the bovine ovary.

Effects of Castration on Luteinizing Hormone and Follicle-Stimulating Hormone Secretion by Pituitary Cells from Male Rats*

Because the role of the pituitary in the testicular control of gonadotropin secretion remains controversial, we examined the effects of castration on the release of LH and FSH under basal conditions and in response to GnRH stimulation by dispersed pituitary cells in monolayer culture as well as by cells perifused with pulses of GnRH. These effects were compared to changes in LH beta, FHS beta, and alpha-subunit mRNA levels determined by Northern blot analysis. Pituitary cells were prepared from 7-week-old intact rats and rats orchidectomized 2 weeks previously. Castration increased basal FSH secretion from monolayer cultures, interpulse FSH release from perifused pituitary cells, FSH beta mRNA concentrations and serum FSH levels each approximately 2-fold, whereas pituitary FSH contents were similar in cells from intact and castrated rats. Pituitary LH content rose 3-fold, LH beta mRNA rose 5.6-fold, and basal LH secretion increased 6-fold, but serum LH levels increased 22-fold. Thus, the change in FSH synthesis inferred from the increase in FSH beta mRNA was proportional to the increase in FSH secretion both in vitro and in vivo. Whereas the basal release of LH in vitro was also proportional to the change in LH beta mRNA, secretion of LH in vivo exceeded these changes, underscoring the importance of increased GnRH to the serum LH castration response. Castration resulted in an increase in the sum of FSH content and secretion during 10 days in culture in the absence of GnRH, indicating ongoing FSH synthesis. Total LH declined in cells from intact rats, and this decline was prevented by castration; this effect may be due to a castration-related decrease in intracellular LH degradation or increased LH synthesis in the absence of GnRH. Castration also augmented the GnRH-stimulated release of LH and FSH from monolayer cultures 4.5- and 1.8-fold, respectively, and increased the amplitude of GnRH-stimulated LH and FSH pulses 5- and 2-fold in experiments with perifused pituitary cells. The EC50 for GnRH was unaffected by castration.(ABSTRACT TRUNCATED AT 400 WORDS)

Rat Spleen Lymphocytes Contain an Immunoactive and Bioactive Luteinizing Hormone-Releasing Hormone

An interaction between the immune and endocrine systems has been long known. This association is further strengthened by the finding that splenic lymphocytes have the capacity to produce molecules similar to if not the same as classical hormones, including several members of the opiate family, PRL, GH, and neuropeptide Y. Because of such findings and because of information from other laboratories suggesting that LHRH might have direct effects upon the immune system, we hypothesized that immune cells themselves might contain LHRH. Lymphocytes were purified from spleens of intact adult male Sprague-Dawley rats and the cells were lysed with sodium hydroxide. The concentration of immunoreactive LHRH was 403 +/- 184 pg/20 X 10(6) lymphocytes. Increasing amounts of lymphocyte lysate displaced [125-I]LHRH from LHRH antibody in a manner parallel to that produced by synthetic hypothalamic LHRH, suggesting immunologic similarity between lymphocyte and hypothalamic LHRH. Lymphocyte LHRH-like immunoactivity coeluted from Nova-Pak C18 columns with synthetic hypothalamic LHRH. When lymphocyte lysates were applied to rat anterior pituitary cells in monolayer culture, significant stimulation of LHRH secretion was seen, from 2,144 +/- 54 pg LH/ml.4 h to 15,364 +/- 587 pg LH/ml.4 h (P less than 0.001), a finding verified in five additional experiments. In other studies, this LH response evoked by lymphocyte lysates was found to be dose dependent and could be significantly inhibited by an LHRH-antagonist. Furthermore, when lymphocyte lysate and identically treated synthetic LHRH were HPLC fractionated, there was coelution of lysate and hypothalamic LHRH bioactivity. The lysate itself contained no substantial LH immunoreactivity. Thus, lymphocytes from spleens of adult male rats contain an immunoactive and bioactive LHRH, a finding further strengthening an association between the endocrine and immune systems.

Synthetic analogs of growth hormone-releasing factor with antagonistic activity [formula omitted

Analogs of human and rat growth hormone-releasing factor (hGRF and rGRF), related to [D-Arg 2]hGRF(1–29)NH 2, were synthesized by solid phase methodology. Their capacity to inhibit growth hormone secretion stimulated by hGRF(1–44)NH 2 was tested on rat anterior pituitary cells in monolayer culture. Among the analogs of hGRF, [D-Arg 2,29,Arg 30]hGRF(1–30)NH 2 showed the highest antagonistic potency of 3.64 relative to [D-Arg 2]hGRF(1–29)NH 2 = 1. However, the most potent analog synthesized thus far was [N-Ac-His 1, D-Arg 2,Ala 15]rGRF(1–29)NH 2, which showed a relative potency of 27.7.

Rapid release of multiple hormones from rat pituitaries perifused with recombinant interleukin-1

Previous studies demonstrated a direct action of interleukin-1 (IL-1) on release of hormones from rat anterior pituitary cells in monolayer culture. To rule out any possibility of a paracrine effect from the elevated hormones in the static monolayer system, and to examine further the dynamics of hormone release elicited by IL-1, studies were conducted with rat anterior pituitary tissue in a computer-controlled automated perifusion system. In experiments performed on the same day as sacrifice, IL-1 stimulated the release of adrenocorticotrophic hormone (ACTH), luteinizing hormone (LH), thyroid stimulating hormone (TSH), growth hormone (GH) and prolactin (PRL) in a dose-related manner. Peak levels were achieved within 6 minutes of exposure to IL-1. However, PRL was not increased over the baseline fluctuations when pituitaries were perifused with IL-1 after 72 hours of incubation. Hormone release did not appear to undergo desensitization after multiple short pulses of IL-1. Heat-denatured IL-1 had no effect on hormone release. The rapid response suggests that IL-1 acts acutely to release preformed hormone stores.

Differential Regulation of Rat Luteinizing Hormone α- and β-Subunits During the Stimulatory and Down-Regulatory Phases of Gonadotropin-Releasing Hormone Action*

Chronic treatment with agonist analogs of GnRH or long term continuous administration of GnRH results in down-regulation of pituitary LH secretion. We investigated the changes in the LH subunits during the stimulatory and down-regulatory phases of GnRH action in rat pituitary cell monolayer culture. The rat pituitary cells in culture, pretreated with medium alone or GnRH agonist for 48 h, were incubated with graded doses of GnRH for 4 h, and LH, LH alpha, and LH beta concentrations in the media and cell pellets were measured by specific and sensitive RIAs. Cells pretreated with medium alone responded to GnRH with a dose-dependent increase in LH, LH alpha, and LH beta immunoreactivity in the medium. However, rat LH, LH alpha, and LH beta concentrations in the cell pellets showed a dose-dependent decrease with GnRH treatment. Pretreatment with GnRH agonist led to a marked decrease in the LH response of cells to graded doses of GnRH. During this down-regulatory phase, the concentrations of LH beta in the medium remained undetectable even though LH alpha immunoreactivity remained persistently and disproportionately elevated. These data suggest that the alpha- and beta-subunits of rat LH are both coordinately and differentially regulated. During the stimulatory phase of GnRH action, both subunits rise concordantly, but during the down-regulatory phase the secretion of the two subunits becomes unbalanced. The changes in the beta-subunit closely parallel the changes in the concentrations of LH dimer, pointing to the key role of beta-subunit in regulation of LH secretion.

Inhibins and Activins

Publisher Summary This chapter discusses the isolation, structure, characterization, and biosynthesis of inhibins and activins. The two pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) which regulate the development and maturation of the gonad, are identified. The ability to quantitate FSH and LH in serum revives the inhibin concept, which offers an explanation for the differential regulation of FSH and LH release often observed in humans and experimental animals. Inhibin activity is monitored by an in vitro bioassay using rat anterior pituitary cells in monolayer culture. The message encoding each of the subunits of the inhibins is cloned from porcine and bovine ovarian complementary DNA (cDNA) libraries. DNA sequence analysis shows that the inhibin a subunit is initially synthesized as a large precursor protein with potential N-linked glycosylation sites. The two β subunits are also found to be derived from large polypeptide precursors and cleavage at the processing sites to yield the mature β subunits at the carboxy termini of the precursors. Using the porcine cDNAs as hybridization probes, the human and murine inhibin subunit precursors are also cloned and sequenced.