GH mRNA Levels Research Articles

Regionalized Gene Expression of Prolactin and Growth Hormone in the Chicken Anterior Pituitary Gland

Messenger ribonucleic acid (mRNA) levers of PRL and GH in perifused cephalic and caudal lobes of the anterior pituitary gland of the chicken were determined by Northern blot analysis and are related to hypothalamic peptide perifusion. Perifusion of vasoactive intestinal peptide (VIP, 10 and 100 n M) significantly increased both PRL secretion and levels of PRI mRNA in the cephalic lobe, whereas no effects of VIP were observed in the caudal lobe. In contrast, growth hormone-releasing factor (GRF) (1-100 n M) did not induce an increase in either GH secretion or levels of GH mRNA in the cephalic lobe but did increase GH secretion and levels of GH mRNA in the caudal lobe. In addition, forskolin perifusion increased secretion and levels of PRL and GH mRNA in both cephalic and caudal lobes, respectively. These results indicate that gene expression of PRL and GH in response to VIP and GRF, respectively, is localized to specific lobes of the anterior pituitary gland, but that they may share, in part, a common second messenger pathway, namely, cAMP.

Changes in the growth hormone axis due to exercise training in male and female rats: secretory and molecular responses.

GH secretion is altered by exercise in humans. In an attempt to investigate the underlying mechanisms, we developed a rodent model. GH secretion was assayed in male and female rats that were sedentary (not exercised), acutely exercised, and chronically exercised. Sedentary males showed typical pulsatile GH secretion. The acutely exercised males had low GH concentrations during the exercise bout, but showed partial recovery of GH pulses during the 5.5-h postexercise period. GH secretion in the chronically exercised males was low during both the exercise and postexercise periods. Sedentary females displayed the typical pattern of GH secretion for this sex. The acutely exercised females had low GH concentrations during the exercise period; the pulsatile pattern of GH secretion did not return during the postexercise period. In contrast, the chronically exercising females had suppressed GH secretion during the exercise bout, but concentrations immediately returned to normal during the postexercise bout. The effects of exercise on GH, GH-releasing hormone (GHRH), and somatostatin messenger RNA (mRNA) levels using Northern and slot blot analyses were also determined. Acutely and chronically exercised male rats had decreased levels of GH mRNA compared to sedentary male rats. The acutely exercised female rats had increased levels of GH mRNA compared to the sedentary females, whereas the chronically exercised females had decreased levels. GHRH mRNA levels in acutely exercising male rats was decreased and in chronically exercising male rats was increased compared to those in the sedentary controls. The pattern of GHRH mRNA in female rats was the opposite of this. Somatostatin mRNA levels decreased in acutely exercised male rats and were not affected in chronically exercised male rats. This signal increased in both acute and chronically exercised female rats. These studies suggest that GH secretion is suppressed in response to exercise in the rat. This contrasts with the increase observed after exercise in humans.

Regulation of thyroid-stimulating hormone beta-subunit and growth hormone messenger ribonucleic acid levels in the rat: effect of vitamin A status.

T3 inhibits transcription of the rat TSH beta gene, and two T3 response elements have been identified that bind T3 receptors and that share sequence homology with the consensus sequence that is also recognized by retinoic acid receptors (RARs). We, therefore, asked whether RA was a regulator of TSH beta gene expression in vivo. Using RNase protection analysis, we found that vitamin A deficiency led to a 2-fold increase in rat pituitary TSH beta messenger RNA (mRNA) levels, which returned to normal 18 h after treatment with RA (20 micrograms/rat). Vitamin A deficiency had no effect on TSH beta mRNA levels in hypothyroid rats. Coadministration of RA and T3 (10 micrograms/100g body wt) to either vitamin A-deficient or vitamin A-deficient, hypothyroid animals caused decreases in TSH beta mRNA content that were indistinguishable from those seen with T3 alone. Surprisingly, vitamin A deficiency had no significant effect on GH mRNA levels in euthyroid or hypothyroid rats. Furthermore, treatment of vitamin A-deficient, hypothyroid animals with RA for either 18 or 72 h had no effect on GH mRNA levels, whereas T3 caused 11-fold and 18-fold increases in GH mRNA, respectively, at these times. We also used transient transfection to test for direct, retinoid receptor-mediated regulation of TSH beta gene transcription by RA. A plasmid TSH beta luciferase, containing 0.8 kilobases of rat TSH beta gene 5'-flanking sequences, exon 1, and 150 base pairs of intron 1, was transfected into CV-1 cells. Cotransfection with RAR alpha and retinoid X receptor-beta induced TSH beta expression by 3.5-fold, and treatment with RA suppressed this induction by 46%. These results show that vitamin A levels play a significant role in regulating the expression of the TSH beta gene, but not the GH gene, in vivo and suggest that RA may suppress TSH beta gene transcription directly by an RAR-retinoid X receptor heterodimer-mediated mechanism.

Interaction of LHRH with growth hormone-releasing factor-dependent and -independent postnatal development of somatotrophs in rat pituitary cell aggregates.

Addition of LHRH for 40 h to aggregate cell cultures of 14-day-old rat pituitary significantly decreased the number of [3H]thymidine ([3H]T)-incorporating cells which immunostained for GH protein as well as the number of [3H]T-labelled cells expressing GH mRNA detectable by in situ hybridization with a digoxigenin-labelled riboprobe. The effect at the level of GH protein was seen at a dose of 1 nM LHRH. However, the effect at the GH mRNA level required a higher dose of LHRH (10 nM) or a longer incubation time (64 h). Treatment of the cultures for 40 h with 0.1 nM GH-releasing factor (GRF) provoked a 54% increase in the number of [3H]T-labelled cells containing GH mRNA and a 30% increase in the number of cells immunostained for GH protein. The latter effects of GRF were completely blocked by simultaneous addition of LHRH (1 nM) to the cultures. In the absence of GRF, LHRH (1 nM) also had an inhibitory effect on the total number of cells containing GH mRNA and a comparable effect on the total number of cells stained for GH protein. The present data show that LHRH is capable of inhibiting the GRF-independent as well as the GRF-dependent development of somatotrophs in postnatal rat pituitary in culture.

Long-term changes of somatotrophic function induced by deprivation of growth hormone-releasing hormone during the fetal life of the rat

We have studied the effects of intra-amniotic administration of an anti-GH-releasing hormone serum (GHRH-Ab) on day 16 of fetal life in the rat, when the ontogenetic development of the GHRH neuronal system occurs. Control animals received normal rabbit serum. Following delivery, body weight was monitored for the next 30 days as an index of somatic growth, and the following indices of somatotrophic function were determined: plasma and pituitary GH, pituitary GH mRNA, hypothalamic GHRH and somatostatin mRNA, and the in vivo GH responsiveness to GHRH. At birth, GHRH-Ab-treated rats had a body weight that was equivalent to that of control rats but, starting from postnatal day 6 up to day 30, they had a significantly reduced body weight. Pituitary weight, the absolute pituitary GH content and GH mRNA levels were lower in experimental compared with control rats, while pituitary GH concentrations were similar in the two groups, thus implying that there was a defect, not only in GH synthesis, but also in GH release. In agreement with this theory, basal GH levels and GHRH-stimulated GH secretion were reduced in GHRH-Ab-treated rats but, in contrast, hypothalamic regulation of GH secretion appeared to be working in these rats as they were still able to respond to the low plasma GH by increasing GHRH and decreasing somatostatin mRNA levels. These findings indicate that deprivation of GHRH during fetal life induces long-lasting changes of growth rate and somatotrophic function.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular Genetics of Growth Hormone-Releasing Factors

To elucidate the effects of GRF on GH synthesis and of the GH-IGF-I axis on hypothalamic GRF synthesis, we measured the mRNA levels of hypothalamic GRF and pituitary GH in pubertal male rats treated for 3 weeks with antirat GRF gamma-globulin (GRF-ab), antisomatostatin gamma-globulin (SRIF-ab) or both. Immunoneutralization of circulating endogenous GRF resulted in a marked decrease in serum IGF-I and pituitary GH mRNA levels in Northern blot analysis, whereas it significantly increased GRF mRNA levels in the arcuate nucleus in both Northern blot and in situ hybridization analysis. Immunoneutralization of circulating endogenous SRIF failed to affect GRF mRNA levels but caused a slight reduction in pituitary GH mRNA. Then, we examined the effect of systemic replacement with rat GH or IGF-I for 1 week on GRF mRNA levels in the hypothalamus of rats treated with GRF-ab for 2 weeks. Replacement with either rat GH or IGF-I significantly suppressed the increased hypothalamic GRF mRNA levels. These data indicate that endogenous GRF is essential in normal synthesis of pituitary GH and that both GH and IGF-I negatively regulate the synthesis of hypothalamic GRF.

Negative regulation of hypothalamic growth hormone-releasing factor messenger ribonucleic acid by growth hormone and insulin-like growth factor I.

Increased growth hormone-releasing factor messenger ribonucleic acid (GRF mRNA) and decreased somatostatin (SRIF) mRNA levels have been reported in the hypothalamus of hypophysectomized rats as well as of dwarf mice. In order to elucidate the effect of the growth hormone-insulin-like growth factor I (GH-IGF-I) axis on hypothalamic GRF and SRIF synthesis, we measured levels of mRNA coding for GRF and SRIF and for pituitary GH in pubertal male rats treated for 3 weeks with antirat GRF gamma-globulin (GRF-ab), anti-SRIF gamma-globulin (SRIF-ab) or both. Immunoneutralization of circulating endogenous GRF resulted in a marked decrease in serum IGF-I and pituitary GH mRNA levels in Northern blot analysis, whereas it caused a significant increase in GRF mRNA levels in the arcuate nucleus as assessed by both Northern blot and in situ hybridization analysis. SRIF mRNA levels in the periventricular nucleus were slightly decreased by GRF-ab treatment when analyzed by in situ hybridization, but not significantly after Northern blot analysis. Immunoneutralization of circulating endogenous SRIF failed to affect mRNA levels of hypothalamic GRF and SRIF but caused a slight reduction in pituitary GH mRNA levels. Levels of mRNA coding for hypothalamic GRF and pituitary GH were also measured by Northern blot analysis in young male rats treated with rat GRF-ab for 2 weeks and replaced with rat GH or IGF-I for the second 1 week. Replacement with either rat GH or IGF-I suppressed the increased hypothalamic GRF mRNA levels. These data indicate that endogenous GRF is essential for normal synthesis of pituitary GH and that both GH and IGF-I negatively regulate the synthesis of hypothalamic GRF.

Effects of sex and pregnancy hormones on growth hormone and prolactin receptor gene expression in insulin-producing cells.

During pregnancy, marked hyperplasia of the pancreatic islet cells has been observed. This effect may be mediated by the pregnancy-associated peptide hormones, placental lactogen, PRL, and GH, which were previously shown to be mitogenic to beta-cells in vitro. To study whether the responsiveness of islet cells to these hormones is regulated on the receptor level, GH and PRL receptor gene expression was studied in pancreata from male rats and virgin, pregnant, and lactating female rats and in cultured islets and insulinoma cells (RIN-5AH) in response to various hormones. The mRNA levels were quantitated by ribonuclease protection assay, using probes specific for mRNA encoding, extracellular and intracellular domains of the GH receptor, and short and long forms of the PRL receptor, respectively. Specific transcripts for the GH receptor were present in pancreas, islets, and RIN-5AH cells. Furthermore, as previously observed in RIN-5AH cells, a predominant expression of the long form of PRL receptor vs. the short form was also found in pancreas and islet cells. Male and nonpregnant female pancreas did not differ significantly in their levels of GH and PRL receptor mRNAs. On day 14 of pregnancy, increases in both GH and PRL receptor mRNA levels were observed (1.7- and 2.4-fold, respectively), and a further increase occurred in late pregnancy (day 19), when GH and PRL receptor mRNA levels were 2.7- and 3.9-fold higher than those in the nonpregnant state. mRNA levels returned toward the basal level during lactation. In the cultured islets, PRL receptor mRNA levels were markedly increased by GH and PRL (3.5- and 6.5-fold, respectively) after exposure for 24 h, whereas estradiol and testosterone had modest stimulating effects (1.8- and 1.5-fold increases, respectively). Dexamethasone induced a 2.5-fold increase in GH receptor mRNA levels, and a weak stimulatory effect was also observed for progesterone. In RIN-5AH cells, the effect of dexamethasone on GH receptor mRNA was detectable after 2 h and maximal after 16 h. In contrast, the effects of GH and PRL on PRL receptor mRNA required 24-48 h of exposure. The effective doses were within the physiological ranges. In conclusion, these results show a differential hormonal regulation of GH and PRL receptor gene expression in the pancreatic islets, which may play a role in the adaptive beta-cell growth during pregnancy.

Effects of sex and pregnancy hormones on growth hormone and prolactin receptor gene expression in insulin-producing cells

During pregnancy, marked hyperplasia of the pancreatic islet cells has been observed. This effect may be mediated by the pregnancy-associated peptide hormones, placental lactogen, PRL, and GH, which were previously shown to be mitogenic to beta-cells in vitro. To study whether the responsiveness of islet cells to these hormones is regulated on the receptor level, GH and PRL receptor gene expression was studied in pancreata from male rats and virgin, pregnant, and lactating female rats and in cultured islets and insulinoma cells (RIN-5AH) in response to various hormones. The mRNA levels were quantitated by ribonuclease protection assay, using probes specific for mRNA encoding, extracellular and intracellular domains of the GH receptor, and short and long forms of the PRL receptor, respectively. Specific transcripts for the GH receptor were present in pancreas, islets, and RIN-5AH cells. Furthermore, as previously observed in RIN-5AH cells, a predominant expression of the long form of PRL receptor vs. the short form was also found in pancreas and islet cells. Male and nonpregnant female pancreas did not differ significantly in their levels of GH and PRL receptor mRNAs. On day 14 of pregnancy, increases in both GH and PRL receptor mRNA levels were observed (1.7- and 2.4-fold, respectively), and a further increase occurred in late pregnancy (day 19), when GH and PRL receptor mRNA levels were 2.7- and 3.9-fold higher than those in the nonpregnant state. mRNA levels returned toward the basal level during lactation. In the cultured islets, PRL receptor mRNA levels were markedly increased by GH and PRL (3.5- and 6.5-fold, respectively) after exposure for 24 h, whereas estradiol and testosterone had modest stimulating effects (1.8- and 1.5-fold increases, respectively). Dexamethasone induced a 2.5-fold increase in GH receptor mRNA levels, and a weak stimulatory effect was also observed for progesterone. In RIN-5AH cells, the effect of dexamethasone on GH receptor mRNA was detectable after 2 h and maximal after 16 h. In contrast, the effects of GH and PRL on PRL receptor mRNA required 24-48 h of exposure. The effective doses were within the physiological ranges. In conclusion, these results show a differential hormonal regulation of GH and PRL receptor gene expression in the pancreatic islets, which may play a role in the adaptive beta-cell growth during pregnancy.

Interaction between insulin and thyroid hormone in rat pituitary tumour cells: insulin attenuates tri-iodothyronine-induced growth hormone mRNA levels.

Insulin has previously been shown to inhibit basal and stimulated rat GH (rGH) secretion as well as basal GH transcription in rat pituitary cells. The effect of physiological doses of insulin on tri-iodothyronine (T3)-stimulated GH mRNA levels in rat pituitary tumour cells was therefore examined. Insulin (7 nmol/l) suppressed T3-stimulated GH mRNA levels in GC and GH3 rat pituitary tumour cells by 58%. This inhibitory effect of insulin on T3-stimulated GH mRNA levels was already present after 24 h of treatment, and persisted for at least 48 h after insulin treatment was withdrawn. The effect of insulin on GH mRNA was selective, as rat prolactin mRNA was stimulated by insulin and T3 in the same cells. Treatment of cells with cycloheximide (10 mumol/l) did not alter the attenuation of GH mRNA levels by insulin, indicating that the insulin effect is independent of new protein synthesis. When de-novo mRNA synthesis was blocked with actinomycin D (4 micrograms/ml) for up to 7 h, an additional decrease in the relative amount of GH mRNA levels was observed after 24, 48 and 72 h of insulin treatment, indicating that an effect of insulin on GH mRNA stability is likely. The results show that physiological doses of insulin selectively attenuate the stimulatory effect of T3 on GH mRNA levels. This suppressive effect of insulin occurs independently of protein synthesis and is presumably mediated both at a transcriptional and post-transcriptional level.

Dexamethasone induces advanced growth hormone expression in the fetal rat pituitary gland in vivo.

The effects of dexamethasone (DEX) on the onset of GH expression in the fetal rat pituitary gland were studied. Pregnant rats received oral administration of DEX dissolved in drinking water (25 mg/liter) for 40 h before killing on days 16-19 of pregnancy. The number of immunoreactive GH cells in the control fetus on day 18 was very small, whereas it was remarkably increased by DEX to a compatible value obtained from control fetuses on day 19. The effect of DEX on the induction of immunoreactive GH cells was found to be less potent on day 17 and not evident on day 16. The population of GH cells on day 19 was doubled, and they stained more intensely by GH antiserum after DEX treatment. In situ hybridization revealed that there were many GH messenger RNA (mRNA)-positive cells in the pituitary gland of a DEX-treated fetus at day 18, whereas they were negligible in number in the age-matched control. Northern blot analysis revealed that DEX induced accumulation of small amounts of GH mRNA in the fetal pituitary gland on day 17, whereas it induced a marked elevation of GH mRNA level on day 18. Reducing the levels of endogenous glucocorticoids by the administration of metyrapone (0.5 mg/ml in drinking water) to the pregnant rats from day 17-19 resulted in the significant reduction in the number of GH cells at day 19. Short-term treatment of DEX (duplicate sc injections at 18 h and 6 h before killing) also induced immunoreactive GH cells on day-18 fetuses but not on day-17 fetuses. Immunoreactive GH cells appeared on day 17 only after quadruplicate injections of DEX during last 40 h, suggesting that there is a difference in fetal sensitivity to DEX between days 17 and 18. These results indicate that immunoreactive GH cells and GH mRNA are inducible by DEX at an earlier stage of the fetal life than they normally appear. It is suggested that glucocorticoids play an important role in the development of GH cells in the fetal pituitary gland in vivo.

Dexamethasone induces advanced growth hormone expression in the fetal rat pituitary gland in vivo

The effects of dexamethasone (DEX) on the onset of GH expression in the fetal rat pituitary gland were studied. Pregnant rats received oral administration of DEX dissolved in drinking water (25 mg/liter) for 40 h before killing on days 16-19 of pregnancy. The number of immunoreactive GH cells in the control fetus on day 18 was very small, whereas it was remarkably increased by DEX to a compatible value obtained from control fetuses on day 19. The effect of DEX on the induction of immunoreactive GH cells was found to be less potent on day 17 and not evident on day 16. The population of GH cells on day 19 was doubled, and they stained more intensely by GH antiserum after DEX treatment. In situ hybridization revealed that there were many GH messenger RNA (mRNA)-positive cells in the pituitary gland of a DEX-treated fetus at day 18, whereas they were negligible in number in the age-matched control. Northern blot analysis revealed that DEX induced accumulation of small amounts of GH mRNA in the fetal pituitary gland on day 17, whereas it induced a marked elevation of GH mRNA level on day 18. Reducing the levels of endogenous glucocorticoids by the administration of metyrapone (0.5 mg/ml in drinking water) to the pregnant rats from day 17-19 resulted in the significant reduction in the number of GH cells at day 19. Short-term treatment of DEX (duplicate sc injections at 18 h and 6 h before killing) also induced immunoreactive GH cells on day-18 fetuses but not on day-17 fetuses. Immunoreactive GH cells appeared on day 17 only after quadruplicate injections of DEX during last 40 h, suggesting that there is a difference in fetal sensitivity to DEX between days 17 and 18. These results indicate that immunoreactive GH cells and GH mRNA are inducible by DEX at an earlier stage of the fetal life than they normally appear. It is suggested that glucocorticoids play an important role in the development of GH cells in the fetal pituitary gland in vivo.

Coculturing posterior pituitary and GH3 cells: dramatic stimulation of prolactin gene expression.

Recent evidence suggests that the posterior pituitary (PP), also called the neurointermediate lobe, regulates PRL release. We previously reported that cocultures of anterior pituitary and PP cells resulted in a 2- to 3-fold increase in PRL content and release. For this study we chose GH3 cells (a somatomammotroph tumor cell line) to determine whether coculturing GH3 with PP cells: 1) stimulates the release and cell content of PRL as compared with GH; 2) increases GH3 cell proliferation; and 3) affects PRL messenger RNA (mRNA) levels. Exp 1. GH3 cells (25,000 cells per well; 25K) were cocultured with PP cells (0K, 12.5K, or 25K) from male rats in serum-free media for 1, 2, 4, and 7 days; hormones were measured by RIA. Coculturing resulted in 5- to 10-fold increases in both media and cell PRL that were linear with time and dependent on the number of PP cells. In contrast, media GH increased only 1.5- to 2-fold, and GH cell content reduced by 75%. Exp 2. GH3, PP, and GH3 + PP cells were cultured for 1, 2, and 4 days and then incubated with [3H]thymidine for 5 h. The incorporation of [3H]thymidine in GH3 cells remained constant over time and showed a small, early increase in cocultures. In contrast, incubation of PP cells alone resulted in a 50- to 60-fold rise in [3H]thymidine incorporation from days 1-4 in culture. Exp 3. Cytoplasmic mRNA was determined by slot blot hybridization with 32P-labeled complementary DNA probes for PRL and GH. After coculturing 25K GH3 cells with 12.5K and 25K PP cells for 4 days, PRL mRNA levels increased 15- and 30-fold, respectively, whereas GH mRNA levels rose less than 2-fold. Neither PRL nor GH mRNA were detected in PP cells. 1) coculturing GH3 with PP cells dramatically stimulates PRL gene expression, synthesis, and release; 2) this response is specific for PRL, has little effect on GH, and is not due to increased GH3 cell proliferation; and 3) we speculate that a subpopulation of intermediate lobe cells, possibly with a proliferative capacity, is responsible for inducing these effects.

The effect of "binge" ethanol exposure on growth hormone and prolactin gene expression and secretion.

The effects of ethanol (EtOH) on GH and PRL have been previously explored, and a dicotomy in results noted. While serum GH levels appear to fall after EtOH exposure, PRL levels rise. We have attempted to expand these studies by examining the impact of acute or "binge" EtOH in vivo on GH and PRL synthesis and secretion. At 0.5, 1.5, and 3.0 h after one dose of ip EtOH, serum GH levels fell significantly compared with those seen in saline-injected controls. This correlated with a fall in GH mRNA levels, but no change in pituitary GH content. Conversely, serum PRL levels rose significantly, while the mRNA for PRL decreased by approximately 20%. There was no change in pituitary PRL content. Interestingly, the mRNA for pit-1 (GHF-1), a transcription factor important to both GH and PRL gene expression, was unchanged at any time point. Despite the fall in GH and PRL mRNA levels, the pituitary cAMP content was markedly elevated at 0.5 h, with no change at any other time point. In summary, acute EtOH exposure in vivo appears to dampen both GH and PRL synthesis, while serum levels behave dissimilarily. Possible explanations for these findings are discussed.

Restoration of juvenile baseline growth hormone secretion with preservation of the ultradian growth hormone rhythm by continuous delivery of growth hormone-releasing factor.

The ability of continuously delivered GH-releasing factor (GRF) to enhance GH secretion while maintaining the normal ultradian GH rhythm was investigated. Synthetic human GH-releasing factor (hGRF(1-44)NH2) was continuously infused for 4 days by means of i.v. catheters to 11-week-old broiler chickens. At this age, overall endogenous GH secretion is low, and baseline GH is barely detectable. Six birds per treatment received vehicle (control), 0.324 mg hGRF(1-44)NH2/kg body weight per day (low dose) or 3.24 mg hGRF(1-44)NH2/kg body weight per day (high dose). After 4 days of GRF conditioning, concurrent with continued GRF infusion, serial blood samples were removed via atrial catheters at 15-min intervals for 6 h and GH plasma profiles determined. High dose GRF significantly increased GH plasma concentrations over tenfold compared with controls; however, most of this increase reflected an increase in basal GH, which was reinstated to juvenile baseline levels. Augmentation of pulse amplitude above this increased baseline was not proportionately as high, and failed to reach juvenile levels. The ultradian rhythm of GH was not altered by continuous GRF administration. Both low and high dose GRF treatments resulted in significant enlargement of the anterior pituitary gland. Total pituitary GH mRNA levels, although elevated over twofold by GRF treatment, were not significantly different from controls. Measures of plasma GH magnitude (overall and baseline mean, and peak amplitude) were significantly correlated with pituitary GH mRNA for control birds, but were not correlated for GRF treatments. Feed intake was markedly depressed (33%) on the high dose GRF treatment, in conjunction with total inhibition of body weight gain over the 4-day period of administration. Longitudinal bone growth and width of the epiphyseal growth plate were also significantly reduced by high dose GRF treatment, probably reflecting the reduced level of nutrient intake, despite high circulating concentrations of GH.

Impaired Generation of Cyclic Adenosine 3′,5′- Monophosphate in a Somatomammotroph Cell Line Derived from Dwarf (dw) Rat Anterior Pituitaries

The dwarf (dw) mutation in rats results in 40-50% growth retardation associated with a selective reduction in pituitary somatotroph number, GH content, and GH mRNA levels and a decreased GH secretory response to GH-releasing factor (GRF). Recent studies in freshly dispersed pituitary cells have provided evidence for a defect in adenylate cyclase-linked GRF signal transduction in dw somatotrophs. To further examine this defect in a more specific cell population, we developed a somatomammotroph cell line (DP) derived from anterior pituitaries of male dw rats. A similar cell line from normal rats (Po) was used as control. We studied acute GH (4-h release) and cAMP (30-min intracellular accumulation) responses to GH secretagogues known to interact with the adenylate cyclase system. Basal GH release in both cell lines was 80-130% of the cell content, thus limiting the capacity for further GH responses. GRF (10(-8) M) produced a doubling of cAMP levels in Po and DP cells (P less than 0.01), but inconsistent effects on GH release. (Bu)2cAMP (5 x 10(-3) M) increased GH secretion by 50-100% in both groups (P less than 0.01). Cholera toxin (10(-9) M) increased GH release by 50% in both Po and DP (P less than 0.01), but the cAMP response in DP cells was only half that in Po cells (P less than 0.01). Forskolin (10(-5) M), a direct stimulator of adenylate cyclase, doubled GH release in both groups (P less than 0.01). However, cAMP generation was impaired in DP, with a maximal response to forskolin less than one third that in Po (P less than 0.01). In somatotrophs, cAMP mediates not only GRF-stimulated GH release, but also GH synthesis and mitogenesis. The impairment in maximal cAMP generation in DP cells, while not affecting acute GH release, may underlie the defect in somatotroph cell number and GH content in the dw pituitary gland.

Sequence and Expression of Hamster Prolactin and Growth Hormone Messenger RNAs*

Complementary DNAs encompassing the complete protein-encoding regions for PRL and GH of the Syrian Golden hamster were sequenced and used as probes to examine the expression of hamster PRL and GH messenger RNA (mRNA)s. The complementary DNA (cDNA) for hamster PRL encodes a 226 amino acid preprotein which, by analogy to rat and mouse PRLs, is predicted to be processed to yield a 197 amino acid secreted protein. The hamster GH cDNA codes for a 216 amino acid preprotein predicted to yield a 190 amino acid secreted protein. Both hamster proteins are highly homologous to the corresponding rat and mouse hormones. For the secreted proteins, hamster PRL has 82% amino acid identity with rat PRL and 72% identity with mouse PRL. The rodent GH sequences are more strongly conserved, with 97-98% sequence identity between hamster, rat, and mouse GHs. The hamster hormones contain the highly conserved cysteine residues (six in hamster PRL and four in hamster GH) present in other mammalian PRLs and GHs. Neither hamster PRL nor hamster GH contains cysteine residues corresponding to the unique pair of cysteines present in hamster placental lactogen-II. The hamster PRL and GH cDNAs each hybridized to pituitary mRNAs of approximately 1 kilobase. Expression of hamster PRL and GH mRNAs was compared between 2 days of the estrous cycle (proestrus and estrus) and early, mid, and late pregnancy (days 5, 10, and 15). PRL mRNA levels in cycling hamsters were approximately 50% of those in pregnant hamsters. No other significant differences in PRL or GH mRNA levels were observed, suggesting that differences in circulating PRL and GH protein levels during the estrous cycle and pregnancy in the hamster are the result largely of factors other than changes in mRNA levels.

Expression of the Growth Hormone Gene and the Pituitary-Specific Transcription Factor GHF-1 in Diabetic Rats

Diabetes in the rat is associated with poor growth and decreased GH in the pituitary. In this study we have examined whether this reduction reflects an impairment of GH gene expression. Diabetes was induced by the administration of streptozotocin (7 mg/100 g BW), and 18 days later, GH content, GH mRNA, and GH transcription rate were determined. GH mRNA levels were reduced by more than 80% in the pituitaries of diabetic rats, which had a similarly reduced GH content. The differences observed in transcription fully account for the changes in mRNA concentration, since the transcription rate of the gene was also reduced by a factor of 10 in the diabetic pituitaries. Insulin therapy (3 U/15 days) partially restored these parameters. The expression of the specific transcription factor GHF-1/Pit-1 in diabetic rats was also analyzed. Both GHF-1 mRNA levels and the binding of nuclear proteins to an oligodeoxynucleotide conforming to the GHF-1 proximal binding site in the promoter of the GH gene were normal in the diabetic pituitaries, thus excluding the possibility that decreased availability of this factor could be responsible for the decreased GH transcription. Since diabetes produced an approximately 3-fold reduction of circulating T3, the potential role of thyroid hormones on GH gene expression was also evaluated in thyroidectomized and thyroidectomized diabetic rats. Thyroidectomy decreased GH and GH mRNA to less than 5% of the values found in intact animals, and a single saturating injection of T3 (250 micrograms/100 g BW) resulted in a 8- to 10-fold induction of GH mRNA after 6 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth Hormone and Prolactin Gene Expression and Release in the Pituitary of Rainbow Trout in Serum-Free Culture*

The trout pituitary continuously released a large amount of GH for over a week in serum-free culture, but PRL secretion declined by day 2 in culture. The total amount of GH released during the 8 days exceeded the initial content. The pituitary content of PRL remained unchanged after 8 days of culture. The number of weakly stained GH-immunoreactive (ir) cells was increased by day 4 in culture. The GH mRNA levels in individual cells as revealed by in situ hybridization were maintained at around the same level throughout culture. In contrast, the stainability of PRL-ir cells remained unchanged during culture, although the levels of PRL mRNA decreased by day 8. Electron microscopic immunocytochemistry showed a decrease in the numbers of secretory granules and dilation of rough endoplasmic reticulum in GH-ir cells. GH synthesis, measured by incorporated [3H]leucine after GH immunoprecipitation, was elevated 120% on day 8 over day 0. The dominant control of GH and PRL secretion in rainbow trout seems to be the converse of those seen in mammals; inhibition for GH and stimulation for PRL.

The production of growth hormone by subpopulations of rat mononuclear leukocytes

In this study we analyzed the production of GH mRNA and secretion of GH by purified subpopulations of rat lymphoid cells. The data demonstrate that mononuclear leukocytes from various tissues, including spleen, thymus, bone marrow, Peyer's patches, and peripheral blood, all have the ability to produce GH mRNA and secrete GH. Data obtained with cells separated by adherence, nylon wool columns, and positive and negative sorting with monoclonal antibodies that define B, monocyte, T helper and T cytotoxic cells show that several different cell types have the ability to produce GH mRNA. The results suggest that B cells, macrophages, and T helper cells produce more GH mRNA and protein than that of T cytotoxic cells. Natural killer (NK) cells also produce detectable levels of GH mRNA and protein. To validate that leukocyte GH RNA produced in vitro was similar in structure to pituitary GH RNA, we studied the RNA by reverse transcription and the polymerase chain reaction. A sample of the PCR reaction products, analyzed by gel electrophoresis, showed a single major DNA band corresponding in length (600 base pairs) to the distance between the 5′ ends of the two GH-specific primers. The DNA band was specifically labeled with a GH-specific probe after Southern transfer to nitrocellulose. Leukocyte GH purified by immunoaffinity chromatography from culture fluids was shown to be bioactive based on its ability to stimulate the incorporation of tritiated thymidine in primary rat spleen cell cultures. The bioactivity could be blocked with specific antibodies to rat GH. Taken together, the data suggest that there is heterogeneity within lymphocytes regarding their ability to produce GH and are consistent with the idea that GH may be active in local immune responses.