Pituitary Proopiomelanocortin mRNA Research Articles

Placental restriction alters the functional development of the pituitary-adrenal axis in the sheep fetus during late gestation.

We have experimentally restricted placental growth in the sheep to investigate the impact of reduced substrate delivery on fetal pituitary proopiomelanocortin (POMC) mRNA levels and on circulating ACTH 1-39, immunoreactive ACTH, and cortisol concentrations during late gestation. Endometrial caruncles were removed in nine ewes before mating to reduce the number of placentomes formed [placental restriction group (PR)]. Fetal arterial PO2 and O2 saturation were reduced in the PR group (2.0 +/- 0.1 kPa and 42.8 +/- 1.1%, n = 9) when compared with control fetuses (3.1 +/- 0.1 kPa and 66.4 +/- 0.9%, n = 10). The ratio of anterior pituitary POMC mRNA:18 S ribosomal RNA was also lower (p < 0.05) in the PR group (0.49 +/- 0.05) when compared with the control group (0.80 +/- 0.12) after 140 d of gestation. In contrast, plasma concentrations of ACTH 1-39 and immunoreactive ACTH were similar in the PR and control groups throughout late gestation. Plasma ACTH 1-39 concentrations increased (p < 0.006) between 128 and 134 d of gestation, in both the PR (122-128 d: 2.70 +/- 0.34 pmol/L: 134-141 d; 7.07 +/- 1.57 pmol/L) and control (122-128 d; 3.36 +/- 0.56 pmol/L: 134-141 d; 10.78 +/- 2.88 pmol/L) groups. Combined adrenal weight was higher (p < 0.005) in the PR group (130 +/- 10 mg/kg) compared with controls (80 +/- 1 mg/kg) at 140 d of gestation, and plasma cortisol concentrations were also higher (p < 0.02) in PR than control fetuses between 127 and 141 d of gestation. These changes imply that the fetal hypothalamopituitary-adrenal axis is operating at a new central set point in the growth-restricted fetus.

Dietary sodium intake modulates pituitary proopiomelanocortin mRNA abundance.

The pituitary prohormone proopiomelanocortin gives rise to melanocortins of alpha, beta, and gamma primary structure in addition to corticotropin. Melanocortins have a variety of actions in mammals, and each is natriuretic. In particular, gamma-melanocyte-stimulating hormone has been shown to mediate reflex natriuresis after acute unilateral nephrectomy. We examined whether this peptide could play a role in longer term adjustments in sodium balance by measuring plasma gamma-melanocyte-stimulating hormone and corticotropin concentrations, as well as pituitary proopiomelanocortin mRNA abundance, in Sprague-Dawley rats ingesting either a low (0.07% NaCl) or high (7.5% NaCl) sodium diet. One week after the high sodium diet, plasma gamma-melanocyte-stimulating hormone concentration was double the value seen in rats on the low sodium diet (158 +/- 5 [SE] versus 76 +/- 9 fmol/mL, P < .001), a change that was accompanied by a fivefold increase in plasma atrial natriuretic peptide concentration but no change in plasma corticotropin. Whole pituitary proopiomelanocortin mRNA abundance, measured with a probe to exon 3 of the rat proopiomelanocortin gene, was significantly increased after 1 week of the high sodium diet compared with the low sodium diet and increased further at 2 and 3 weeks. This increase occurred primarily in the neurointermediate lobe as demonstrated by in situ hybridization; the content of gamma-melanocyte-stimulating hormone immunoreactivity was also increased in this lobe, but not the anterior lobe, after 1 week of the high sodium diet. These results demonstrate that high dietary sodium intake increases neurointermediate lobe proopiomelanocortin mRNA abundance compared with a very low sodium diet and also suggest that proopiomelanocortin is preferentially processed into gamma-melanocyte-stimulating hormone rather than corticotropin. These observations consequently raise the possibility of a role for this peptide hormone system in the adjustments to a high salt diet.

Activation of the baboon fetal pituitary-adrenocortical axis at midgestation by estrogen: responsivity of the fetal adrenal gland to adrenocorticotropic hormone in vitro.

We have previously demonstrated that increased expression of fetal pituitary proopiomelanocortin mRNA and the induction of enzymes catalyzing fetal adrenal cortisol formation at term are regulated by estrogen-induced changes in placental oxidation of maternal cortisol to cortisone. To test the hypothesis that induction of fetal pituitary adrenocorticotropic hormone (ACTH) production by estrogen-induced changes in placental cortisol oxidation results in increased responsivity of the fetal adrenal gland to ACTH, in the present study we compared fetal adrenal sensitivity to ACTH in vitro at midgestation in untreated controls and in animals treated at this time in gestation with estrogen. Fetal adrenals were obtained on Day 100 (n = 7) and Day 165 (n = 5; term = Day 184) from untreated baboons and on Day 100 following maternal treatment with estradiol (s.c.; Days 70-100; n = 10) or androgen precursor (n = 3). Adrenal slices (15-25 mg) were perifused (100 microliters/min; 37 degrees C) with Medium 199 (no phenol red); media were collected at 10-min intervals and assayed for cortisol and dehydroepiandrosterone. Secretion of cortisol and dehydroepiandrosterone reached equilibrium after 140 min of perifusion; therefore, basal release was calculated as the mean steroid concentrations during 190-240 min. Adrenal slices were then perifused for 20 min with saline or ACTH at 240 (0.001 nmol), 370 (0.01 nmol), and 490 (0.1 nmol) min, and an overall average cortisol/dehydroepiandrosterone secretion rate (pg/min/mg) between 240-600 min was calculated.(ABSTRACT TRUNCATED AT 250 WORDS)

Arcuate nucleus neurons that project to the hypothalamic paraventricular nucleus: neuropeptidergic identity and consequences of adrenalectomy on mRNA levels in the rat.

The possible role that the hypothalamic arcuate nucleus might play in mediating the increase in paraventricular nucleus corticotropin-releasing hormone mRNA levels following adrenalectomy was investigated in two series of experiments. In the first series in situ hybridization histochemistry was used to quantify levels of eight accurate nucleus neuropeptide and neurotransmitter mRNAs in neurons that potentially relay adrenal steroid feedback to the paraventricular nucleus. In the second series of experiments, arcuate neuropeptidergic projections to the hypothalamic paraventricular nucleus were characterized using retrograde tracing in combination with in situ hybridization histochemistry. Despite an increase in paraventricular nucleus corticotropin-releasing hormone (60%) and pituitary proopiomelanocortin mRNA levels (sixfold), arcuate mRNA levels for proopiomelanocortin, neuropeptide Y, somatostatin, galanin, dynorphin, tyrosine hydroxylase, glutamate decarboxylase, and the glucocorticoid receptor were unchanged 14 days following adrenalectomy. Neuropeptidergic characterization of arcuatoparaventricular projections was achieved by injection of the retrograde tracer fluorogold into the paraventricular nucleus; retrogradely labeled neurons were characterized with polyclonal antisera against fluorogold in combination with oligonucleotide probes directed against neuropeptide Y, proopiomelanocortin, or somatostatin. Out of these three arcuate neuropeptide Y mRNA was contained in 18% of the fluorogold-positive neurons in the arcuate, proopiomelanocortin mRNA was contained in 8%, and somatostatin mRNA was contained in 6%. Overall, the results from both experiments suggest that the arcuatoparaventricular neuropeptide Y, proopiomelanocortin, and somatostatin projections are not sensitive to a chronic (14 day) lack of adrenal steroids. These projections as well as the other arcuate neurotransmitter and neuropeptide systems appear not to contribute to the persistent elevations in paraventricular nucleus corticotropin-releasing hormone mRNA levels or pituitary proopiomelanocortin mRNA levels found in 14 day adrenalectomized rats.

Developmental changes in the distribution of pro-opiomelanocortin and prolactin mRNA in the pituitary of the ovine fetus and lamb

Ontogenic changes in pituitary pro-opiomelanocortin (POMC) mRNA and prolactin (PRL) mRNA were examined during gestation and early neonatal life using in situ hybridization histochemistry. Pituitaries were harvested from fetuses at days 60-80, 100-120, 135-140 and 142-143 of gestation and at term, and from lambs at days 1-7 and 30-60 of age and adults. POMC mRNA, present by day 60, rose during mid- and late gestation. Concurrently there was a change in corticotroph distribution, resulting in a relatively greater quantity of POMC mRNA at the base of the pars distalis. At term, there was a significant (P < 0.05) further elevation of POMC mRNA. POMC mRNA levels remained high in the newborn lamb but decreased in the adult. Cells in the pars intermedia expressed large amounts of POMC mRNA early in fetal life and this pattern persisted throughout gestation and into the neonatal period. Changes in the expression of the POMC gene correlated closely with the presence of immunoreactive (ir)ACTH in the pituitary; in fetuses the proportion of irACTH-positive cells rose to 10% of pars distalis cells by day 100 and did not change significantly thereafter. The lactotrophs contained PRL mRNA by day 60, and the quantity increased towards parturition (P < 0.05). PRL mRNA subsequently decreased in the neonate, but rose as the lamb matured. These results indicate that in the fetal pituitary: (1) the POMC gene is highly expressed during gestation in both the pars distalis and the pars intermedia, (2) changes in the amounts of POMC mRNA and PRL mRNA in the pars distalis correlate with the distribution of irACTH and irPRL respectively, and (3) POMC mRNA is distributed primarily in the inferior aspect of the pars distalis, and in this region its quantity is highest immediately prior to parturition.

The TRH neuronal phenotype forms embryonic cell clusters that go on to establish a regionalized cell fate in forebrain.

How neurons diversify in developing brain to produce discrete cell fates in their appropriate regions remains a fundamental question. Embryonic Xenopus was previously used to identify juxtaposed embryonic cells that first express proopiomelanocortin mRNA in forebrain and pituitary, supporting the idea that this neuropeptide phenotype is induced locally (Hayes and Loh, 1990, Development 110: 747-757). To begin to examine how a more widespread population of forebrain cells is set up, the present focus is on the thyrotropin-releasing hormone (TRH) phenotype. Serial section in situ hybridization histochemistry produced the unexpected finding that the adult-like TRH system spanning forebrain and comprising over six different telencephalic and diencephalic nuclei, is preceded by an embryonic TRH cell population that is initially localized and then highly regionalized in the area from which the adult pattern develops. Thus, the first TRH cells, detected in vivo after 35h (stage 29/30), were confined to discrete anterior or posterior bilateral clusters in embryonic forebrain or hindbrain. Thereafter, the TRH cell clusters in diencephalon, but not hindbrain, expanded to form rows, extending anteriorly into telencephalon and bifurcating posteriorly around the infundibulum. By 80 h (stage 42), after extensive brain morphogenesis, these forebrain rows showed regional differences in levels of TRH mRNA corresponding to the specific brain nuclei that have been shown to contain TRH cells in adult. These findings show that subsets of phenotype-specific forebrain cells first form a regionalized neuronal cell fate before distinct brain nuclei form. This in turn points to the testable hypothesis in Xenopus that certain neuronal cell fates in forebrain may be dictated by cell lineage or local induction.

Fast glucocorticoid feedback inhibition of ACTH secretion in the ovariectomized rat: effect of chronic estrogen and progesterone.

The purpose of this study was to determine whether estrogen and progesterone influence fast glucocorticoid negative feedback regulation of the ACTH and corticosterone (CORT) responses to stress. Mature rats were ovariectomized and 6 weeks later implanted with 17 beta-estradiol (E2, 0.5 mg), E2 and progesterone (P, 100 mg; E2 + P group) or placebo pellets (OVX). Seven days later rats were subjected to a single or repeated intermittent footshock stress (0.2 mA, 15 s duration, 0.5 s on). The repeated stress was of the same intensity and duration, and was applied either during the time domain of the rate-sensitive fast glucocorticoid feedback when plasma CORT levels are rising (5 min after the onset of the first stress), or at the time of peak CORT response (15 min) to the initial stress. Plasma ACTH and CORT were measured from serial samples. Estrogen replacement alone or in combination with progesterone lowered the immediate (t = 5) ACTH and CORT response to a single stress in ovariectomized animals. The second stress applied 5 min after the initial stress produced net ACTH responses similar to those obtained after a single stress in the OVX and E2 + P-replaced hormone groups, while total ACTH responses were lower in the E2-treated group. In ovariectomized animals, a facilitation of ACTH response by a prior stress is apparent in response to a footshock 15 min later, when the integrated ACTH secretion is significantly greater than the response measured after a single shock, or after a repeated shock 5 min apart. Anterior pituitary proopiomelanocortin (POMC) mRNA levels were lower in groups with E2 or E2 + P replacement compared to OVX animals. In contrast, hypothalamic corticotropin-releasing factor (CRF) mRNA levels did not increase significantly. However, hypothalamic glucocorticoid receptor (GR) mRNA levels increased after 17 beta-estradiol treatment, and this increase was reversed by progesterone. These results suggest that prior stress leads to both a fast-feedback inhibition and a facilitation of the subsequent stress response. In the absence of gonadal hormones this facilitation is balanced by fast-feedback inhibition during the glucocorticoid fast-feedback time domain, and is unmasked outside of this time domain. Estrogen suppresses POMC mRNA synthesis leading to a decrease in the availability of releasable ACTH, thereby reducing the facilitation. Progesterone may counter this effect of estrogen by decreasing the efficacy of the fast rate-sensitive glucocorticoid negative feedback.

Sexually Dimorphic Effects of Alcohol Exposure in Utero on Neuroendocrine and Immune Functions in Chronic Alcohol-Exposed Adult Rats

Maternal ethanol consumption has deleterious effects on the offspring's neuroendocrine and T-cell-dependent functions. Chronic alcohol consumption in adulthood has also been associated with activated hypothalamic-pituitary-adrenal (HPA) function and immunosuppression which is demonstrable at the T-cell level. Our aim was to establish whether prenatal alcohol exposure alters the neuroendocrine and immune responses to chronic alcohol challenge in adult male and female offspring. Adult male rats placed on a liquid alcohol diet for 5 weeks had significantly decreased thymus weight, hypertrophied adrenals, and elevated plasma ACTH and corticosterone levels. Splenic lymphocyte Concanavalin A (Con A)-stimulated proliferation in the ethanol-treated rats was decreased compared to that in pair-fed controls. Thus, prolonged alcohol exposure activated the HPA axis and suppressed T-cell function. The effects of prenatal alcohol exposure, as a predispositional factor, on the HPA axis and on the T-cell functions of adult chronic alcohol-exposed rats were examined in the offspring of dams fed ethanol (FAE) or an isocaloric liquid (PF) diet during the last 2 weeks of gestation. The adult offspring of both sexes and prenatal treatment groups were then placed on an alcohol-containing liquid diet for 5 weeks. Fetal alcohol exposure decreased anterior pituitary proopiomelanocortin mRNA levels and increased glucocorticoid receptor (GR) mRNA levels in males and decreased GR mRNA levels in females. There were no differences in hypothalamic CRF mRNA and GR mRNA levels between the prenatal treatment groups. There was no significant difference in Con A-stimulated lymphocyte proliferation between FAE and PF males. However, FAE females showed Con A-stimulated lymphocyte proliferation significantly higher than those of all other groups, including pair-fed females. Prostaglandin E2 (PGE2) suppressed lymphocyte proliferation to a lesser degree in FAE males than in any other group. Furthermore, T-cell response to Con A was enhanced by indomethacin, a prostaglandin biosynthesis inhibitor, in FAE males suggesting that increased prostaglandin synthesis may occur in FAE males after chronic alcohol exposure. Increased levels of endogenous PGE2 could also be inferred from the enhanced levels of interleukin-2 receptor α (IL-2Rα) mRNA in activated lymphocytes of male but not of female FAE offspring compared to PF. In summary, the results of this study demonstrate that prenatal alcohol exposure leads to a specific HPA-related vulnerability in males to the deleterious effects of ethanol in adulthood. Although prenatal alcohol did not further aggravate the effects of chronic alcohol exposure on lymphocyte proliferation response to Con A in adult male offspring, altered T-cell responses could be unmasked.

Changes in hypothalamic corticotrophin-releasing factor and anterior pituitary pro-opiomelanocortin mRNA during the course of experimental allergic encephalomyelitis

The pituitary-adrenal axis is activated during the course of experimental allergic encephalomyelitis (EAE), a paralytic disease resulting from an immunological reaction against central nervous system myelin. The magnitude of the adrenal response not only correlates with the severity of disease, but also serves an important functional role in recovery. We have shown that in EAE there are short-term changes in anterior pituitary pro-opiomelanocortin (POMC) mRNA as well as plasma corticosterone which can be detected before the development of clinical disease. At peak clinical signs when corticosterone and POMC mRNA are maximal, hypothalamic corticotrophin-releasing factor (CRF) mRNA is suppressed, suggesting that pituitary-adrenal activation is not mediated by CRF. Following recovery all parameters return to normal.

Direct Stimulation of the Pituitary by Transfer of Activated Leucocytes

Some strains of rat develop arthritis, and have profoundly elevated adrenocorticotropin (ACTH) and corticosterone, following intradermal injection of an adjuvant containing heat-killed mycobacteria. Transfer to syngeneic recipients of immune spleen (IS) cells taken from arthritic rats 14 days after injection of the adjuvant, but not of non-immune cells, causes increased circulating ACTH and increased pituitary proopiomelanocortin (POMC) mRNA. Transfer of immune cells does not transfer the disease, but does protect recipients from subsequent challenge with the adjuvant. In these immune-protected rats, the secondary immune response raises ACTH and POMC to levels similar to those seen in arthritic rats. These data show that endogenous inflammatory mediators have direct actions on the neuroendocrine system.

Levels of corticotropin-releasing hormone messenger ribonucleic acid (mRNA) in the hypothalamic paraventricular nucleus and proopiomelanocortin mRNA in the anterior pituitary during late gestation in fetal sheep

Levels of corticotropin-releasing hormone messenger ribonucleic acid (mRNA) in the hypothalamic paraventricular nucleus and proopiomelanocortin mRNA in the anterior pituitary during late gestation in fetal sheep

Effects of cyclosporine A on the hypothalamic-pituitary-adrenal axis and anterior pituitary interleukin-6 mRNA expression during chronic inflammatory stress in the rat

In the rat, adjuvant arthritis (AA) is an inflammatory joint disease associated with chronic stimulation of the hypothalamic-pituitary-adrenal (HPA) axis. We have investigated the effects of the immunosuppressive agent cyclosporine A (CsA) on plasma levels of adrenocorticotropin (ACTH) and corticosterone (B), as well as on anterior pituitary proopiomelanocortin (POMC) and interleukin (IL)-6 mRNA accumulation in control and adjuvant-injected animals. In control animals, CsA reduced basal anterior pituitary POMC and IL-6 mRNA and decreased plasma levels of ACTH and B. Adjuvant-injected animals that were treated with CsA showed no clinical signs of AA. Moreover, CsA inhibited the arthritis-induced increases in pituitary POMC and IL-6 mRNA levels and in circulating ACTH and B. In vitro, CsA reduced the POMC mRNA content of cultured anterior pituitary cells and diminished the stimulatory effects of corticotropin-releasing hormone (CRH) on POMC mRNA expression and ACTH secretion from these cells. These data indicate that CsA has a direct action on the HPA axis and also reduces the activation of the HPA axis seen in chronic inflammatory arthritis.

Glucocorticoid-mediated responses of plasma ACTH and anterior pituitary pro-opiomelanocortin, growth hormone and prolactin mRNAs during adjuvant-induced arthritis in the rat.

Adjuvant arthritis (AA) in the rat leads to chronic stimulation of the hypothalamic-pituitary-adrenal (HPA) axis and the loss of its diurnal rhythmicity. We have investigated the effects of adrenalectomy (ADX) and different levels of corticosterone replacement upon plasma ACTH levels and anterior pituitary pro-opiomelanocortin (POMC), GH and prolactin mRNAs during the development of AA. In control ADX animals, we observed the negative feedback effects of exogenous corticosterone on plasma ACTH and anterior pituitary POMC mRNA. In the ADX animal with AA, however, the increased POMC mRNA which was observed was not reduced by exogenous corticosterone on day 7 of AA, although the negative feedback effect of corticosterone on plasma ACTH was intact. On day 14, however, even high dose corticosterone replacement failed to have a significant feedback effect on the raised levels of plasma ACTH. In control ADX animals, corticosterone replacement resulted in increased anterior pituitary GH mRNA and reduced prolactin mRNA. In contrast, in ADX animals with AA, GH mRNA was reduced and there was a further decrease in prolactin mRNA. In these animals, corticosterone replacement did not affect GH or prolactin mRNA expression. These data demonstrate a disruption of the normal mechanisms underlying feedback inhibition of the HPA axis by glucocorticoids during AA. Similarly, the glucocorticoid-dependent regulation of GH and prolactin mRNA expression is altered in AA.

Steroid hormones regulate the release of immunoreactive beta-endorphin from the Ishikawa human endometrial cell line.

Immunoreactive beta-endorphin (IR-beta END) is present in human endometrium. Several indirect lines of evidence suggest that endometrial beta END is under steroid hormone control, i.e. IR-beta END is detectable in the secretory, but not the proliferative, endometrium, and progesterone administration increases the concentration of IR-beta END in uterine secretions of ovariectomized gilts. To study the effect of steroid hormones on endometrial beta END, we first questioned whether Ishikawa human endometrial adenocarcinoma cells (which respond to steroid hormones) express the proopiomelanocortin (POMC) gene. Indeed, on Northern blot analysis, a RNA similar or identical in size to pituitary POMC mRNA was present in Ishikawa cell RNA extracts. IR-beta END was also present in Ishikawa cell extracts and culture medium, which coeluted with synthetic human beta END in a Sephadex G-50 column. Ishikawa cells released most of their IR-beta END into the culture medium. Estradiol decreased the release of IR-beta END from Ishikawa cells, an effect that was dependent upon dose and time. The maximal effect was observed after a 4-day exposure to 10 nM estradiol (44 +/- 6% of the control value; n = 6; P less than 0.001). This effect was almost completely counteracted by a 100-fold excess of the antiestrogen 4-hydroxytamoxifen. Progesterone and dihydrotestosterone did not have a statistically significant effect on IR-beta END release. Dexamethasone had effects similar to those of estradiol, i.e. decreased the release of IR-beta END in a time- and dose-dependent manner. The maximal effect was detected after a 4-day exposure to 10 nM dexamethasone (53 +/- 6% of the control value; n = 6; P less than 0.001). Interestingly, the antiprogestin-antiglucocorticoid RU486 exhibited agonistic properties, i.e. diminished the release of IR-beta END in a time- and dose-dependent fashion, possibly via the glucocorticoid receptor. Its maximal effect was reached after a 4-day exposure to 10 nM RU486 (55 +/- 6% of the control value; n = 6; P less than 0.001). In conclusion, our data demonstrate that the release of IR-beta END from Ishikawa cells in culture is inhibited by estradiol and dexamethasone, suggesting that endometrial beta END is under estrogen and glucocorticoid regulation, as is the case with hypothalamic and pituitary POMC-derived peptides. This is the first time that the in vitro release of a peripheral-extracranial POMC-derived peptide has been found to be under the direct control of estrogens and glucocorticoids.

Rat hypothalamic proopiomelanocortin messenger RNA is unaffected by adrenalectomy

The negative feedback control of hypothalamic cortocotrophin releasing factor (CRF) and anterior pituitary proopiomelanocortin (POMC) by corticosteroids is well understood. However, less is known about the mechanisms that regulate POMC gene expression in the arcuate nuclei in the medial basal hypothalamus (MBH). Using a sensitive and specific S1 endonuclease protection assay, we have examined the effect of adrenalectomy on POMC mRNA in the rat MBH and pituitary. Our results show that adrenalectomy does not change POMC mRNA levels in the MBH at 7 or 14 days post surgery. The neurointermediate lobe of the pituitary was similarly unaffected by adrenalectomy, while in the anterior lobe, POMC mRNA increased 7-10 fold at both time points, effects that were prevented by dexamethasone treatment. We conclude that while POMC mRNA in the anterior lobe of the pituitary is regulated by plasma glucocorticoids, in the MBH and neurointermediate lobe, it is not.

Response of pituitary and spleen pro-opiomelanocortin mRNA, and spleen and thymus interleukin-1β mRNA to adjuvant arthritis in the rat

During development of adjuvant-induced arthritis (AA) in the rat, pituitary pro-opiomelanocortin (POMC) mRNA expression was increased. Pituitary POMC mRNA was much higher following adrenalectomy and AA. Spleen POMC mRNA also increased with a similar time kinetics, although the levels in the spleen were much lower than those in the pituitary. In control animals, spleen interleukin-1β (IL-1β mRNA) was undetectable, whereas AA led to the accumulation of IL-1β mRNA and the highest levels were seen in the adrenalectomised AA group. Thymic IL-1β expression was also increased in AA animals. These results suggest that AA leads to the activation of both the neuroendocrine and the immune systems and the interaction between these systems may play a role in this disease state.

Hormonal Tolerance to Ethanol is Associated with Decreased Expression of the GTP‐Binding Protein, Gsα, and Adenylyl Cyclase Activity in Ethanol‐Treated LS Mice

Using the proopiomelanocortin (POMC) system as a marker, long sleep (LS) and short sleep (SS) lines of mice were investigated to explore the cellular events that occur during the acquisition of hormonal tolerance to ethanol. Four-day ethanol exposure (1.8 g/kg/24 hr) increased anterior pituitary POMC mRNA levels 4-fold in the LS line and 2-fold in the SS line. Following 7 days of ethanol exposure (1.8 g/kg/24 hr), anterior pituitary POMC levels returned to basal values in the LS line but remained elevated (2-fold) in the SS line. In this setting, the loss of ethanol's ability to sustain elevated POMC mRNA levels in the LS line is defined as hormonal tolerance. Since POMC biosynthesis is primarily regulated through adenylyl cyclase, ethanol-induced alterations in this signal transduction system were explored. Paralleling the effects of ethanol on POMC mRNA levels, ethanol exposure reduced GTP-gamma-S, AIF3-, and MnCl2-stimulated adenylyl cyclase activity by 35%, 21%, and 24%, respectively, in the LS line without effecting adenylyl cyclase activity in the SS line. To determine whether ethanol-induced changes in adenylyl cyclase activity in LS mice could result from alterations in G proteins, protein levels of G, alpha and Gi alpha were determined by western analysis before and after ethanol exposure. Paralleling the effect on POMC mRNA levels and adenylyl cyclase activity, ethanol induced a 35% reduction in Gs alpha protein levels in LS mice but did not alter Gi alpha levels. Neither Gs alpha nor Gi alpha levels were altered in the SS line.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in rat pituitary POMC mRNA after exposure to cold or a novel environment, detected by in situ hybridization.

Plasma adrenocorticotropin (ACTH) levels increase after acute cold exposure. The purpose of this study was to determine if there were parallel changes in pituitary proopiomelanocortin (POMC) mRNA. Male rats were exposed to cold (3-5 degrees C) or a novel environment for 15 or 30 min. Others were unstressed. POMC mRNAs in frozen sections or dissociated cells were hybridized with a photobiotinylated oligonucleotide probe which was detected in situ by streptavidin-alkaline phosphatase. The percentage of area labeled for POMC mRNA was quantified by the Cue-3 color image analysis system. In frozen sections, 24-fold increases in the percentage of area labeled for POMC mRNA were evident in intermediate lobes (IL) 30 min after stress. No change was seen in anterior lobes (AL). If the ALs were dissociated, a 66-99% increase in percentage of labeled cells was detected 2-3 hr after the cold exposure. Fifteen min of cold stress (CS) also caused a 117% increase in the area of label for POMC mRNA per corticotrope. No change was seen after 30 min. Exposure to a novel environment caused a 73% increase in the percentage of area labeled for POMC mRNA per AL corticotrope and an 11-fold increase in the IL. These results indicate that both AL corticotropes and IL melanotropes are stimulated by acute exposure to cold and novel environments.

Stress responsiveness of hypothalamic corticotrophin-releasing factor and pituitary pro-opiomelanocortin mRNAs following high-dose glucocorticoid treatment and withdrawal in the rat

In-situ hybridization with synthetic oligonucleotide probes was used to determine the mRNA content of corticotrophin-releasing factor (CRF) and proenkephalin A mRNA in the paraventricular nucleus, and of pro-opiomelanocortin (POMC) mRNA in the anterior pituitary gland of male rats immediately after, and during recovery from, chronic high-dose prednisolone treatment. Levels of transcripts for mRNA for both CRF and POMC were markedly reduced after the treatment, but there was a rapid return to control values for CRF mRNA within 18 h of steroid withdrawal. In untreated animals, the stressful stimulus of i.p. hypertonic saline increased CRF and proenkephalin A mRNA within 4 h with no significant difference in response seen whether the tissues were removed at 13.00 or 20.00 h. The increase in POMC mRNA did not reach statistical significance in these animals. Although prednisolone resulted in a marked reduction of basal CRF mRNA, the stress-induced increment of CRF mRNA remained comparable with that found in untreated animals. On the day following cessation of prednisolone treatment at 09.00 h, basal and stress levels of CRF mRNA were significantly higher in rats killed at 20.00 h than at 13.00 h. Proenkephalin A mRNA transcripts were below quantifiable levels of detection in control or non-stressed prednisolone-treated animals at all the time-points studied. Stress, however, resulted in the accumulation of proenkephalin A mRNA in control animals. This response was inhibited by prednisolone treatment and only returned 18 h after withdrawal. Prednisolone treatment reduced POMC mRNA below the levels detected in untreated animals, with no detectable response to stress.(ABSTRACT TRUNCATED AT 250 WORDS)

Optimization of non-radioactive in situ hybridization: image analysis of varying pretreatment, hybridization and probe labelling conditions

Using detection of proopiomelanocortin (POMC) mRNA in rat pituitary as a model, varying conditions of tissue pretreatment, hybridization and probe labelling have been tested. Results were evaluated both by visual assessment and by image analysis of coded specimens. Good correlations between visual gradation, optical densities and cell area percentages were obtained. However, determinations of optical densities (or pixel values) provided most detailed information. The data obtained emphasize the interdependence of fixation and permeabilization conditions and clearly show that the stronger the primary fixation, the more efficient the permeabilization by proteinase K must be. The hybridization temperature is also of importance and temperatures between 40-45 degrees C produced the best signal to noise ratio. The POMC-directed 24-mer probe had a theoretical melting point (Tm) of 49.4 degrees C (in the absence of formamide) and four individual experimental determinations of Tm produced a mean value of 48.9 degrees C. Detection of the biotinylated probe was best accomplished with monoclonal antibiotin antibodies and the alkaline phosphatase-anti-alkaline phosphatase (APAAP) system. Short washes at high-stringency (0.1 x SSC, 45 degrees C) produced an optimal signal to noise ratio. Inclusion of 50% formamide in the hybridization buffer produced an enhanced signal to noise ratio, in spite of a higher background staining. The probe employed for most studies was a synthetic 24-mer oligodeoxynucleotide, complementary to the MSH[4-11]-coding region of POMC mRNA. It was labelled with biotinylated dUTP and unlabelled dCTP using terminal transferase. Chromatographical analyses revealed the labelled probe to be heterogeneous in tail length.(ABSTRACT TRUNCATED AT 250 WORDS)