Recombinant human erythropoietin reduces the need for erythrocyte and platelet transfusions in pediatric patients with sarcoma: a randomized, double-blind, placebo-controlled trial.

OBJECTIVE: To evaluate the effect of recombinant human erythropoietin (EPO) and iron supplementation on transfusion requirements in pediatric patients with sarcoma who were receiving chemotherapy, we performed a double-blind, placebo-controlled, randomized trial. METHODS: Twenty-four pediatric patients with malignant solid tumors were randomly assigned to receive either placebo (saline solution) or EPO for a 16-week study period. The starting dose was 150 IU/kg per dose three times a week and was escalated by 50 IU/kg per dose increments monthly until packed red blood cell (PRBC) transfusion independence was achieved or a dosage of 300 IU/kg per dose was reached. Iron supplementation was prescribed at a dose of 6 mg of elemental iron per kilogram daily. The primary study end point was the comparison of PRBC transfusion requirements in the two groups. RESULTS: Of 24 patients, 20 were evaluable for response. The median PRBC transfusion requirement during the 16-week period was 23 ml/kg in EPO-treated patients versus 80 ml/kg in placebo patients (p = 0.02). The median number of single-donor platelet units transfused was zero in the EPO-treated patients compared with four in the placebo group (p = 0.005). No statistical difference in the intensity of bone marrow suppression was seen, as measured by the median number of complete blood cell counts with an absolute neutrophil count of < 1000 cells/microliter. CONCLUSIONS: Treatment with EPO and iron significantly reduces PRBC transfusions in pediatric patients receiving concomitant chemotherapy for malignant sarcomas. A decrease in the number of platelet transfusions was also seen and deserves further study.

Expression of tyrosine hydroxylase gene in cultured hypothalamic cells: roles of protein kinase A and C.

In hypothalamic cells cultured in serum-free medium, the quantity of tyrosine hydroxylase mRNA increases after treatment with an activator of the protein kinase A pathway (8-bromoadenosine cyclic AMP, 3-isobutyl-1-methylxanthine, or forskolin) or an activator of protein kinase C (12-O-tetradecanoylphorbol 13-acetate or sn-1,2-diacylglycerol). The tyrosine hydroxylase mRNA level decreases in the cells after inhibition of protein kinase C with calphostin C or after depletion of protein kinase C by extended phorbol ester treatment. These data suggest that both protein kinase pathways regulate tyrosine hydroxylase gene expression in hypothalamic cells. As simultaneous activation of both pathways has less than an additive effect on the tyrosine hydroxylase mRNA level, they appear to be interrelated. Compared with the rapid and dramatic increase of the tyrosine hydroxylase mRNA level in pheochromocytoma cells, activation of the protein kinase A or protein kinase C pathway in the cultured hypothalamic cells induces slow changes of a small magnitude in the amount of tyrosine hydroxylase mRNA. The slow regulation of tyrosine hydroxylase gene expression in hypothalamic dopaminergic neurons corresponds to the relatively high stability of tyrosine hydroxylase mRNA (half-life = 14 +/- 1 h) in these cells.

Localization of a defect in hypothalamic dopaminergic neurons of the aged brain that results in impaired PKA-dependent activation of tyrosine hydroxylase.

Using an in vitro incubation system, the role of the cyclic AMP-dependent protein kinase A (PKA) pathway in the regulation of the in situ activity of tyrosine hydroxylase (TH) was studied in the hypothalamuses of young and aged ovariectomized rats. Hypothalamic tissue was incubated for 60 min in medium containing 3-hydroxybenzylhydrazine dihydrochloride, a dihydroxyphenylalanine (DOPA) decarboxylase inhibitor, and various agents that modify the activity of the PKA pathway. At the end of the incubation, the tissue was homogenized and analyzed for DOPA and TH mass. The in situ molar activity of TH was expressed as the moles of DOPA accumulating in the tissue per mole of TH per hour. Forskolin, an activator of adenylyl cyclase and the cyclic AMP agonist, (Sp)-cyclic adenosine 3',5'-monophosphothioate, significantly (P < .01) increased the in situ molar activity of TH in the hypothalamic dopaminergic (DAergic) neurons of both young and aged rats. Theophylline, a phosphodiesterase inhibitor, did not affect the TH molar activity in the hypothalamuses of aged animals but did significantly (P < .001) increase its activity in those of young rats. When vasoactive intestinal peptide was evaluated, the TH molar activity was significantly (P < .005) increased in the hypothalamuses of young rats but not in those of aged rats. It was suggested that the deficiency of DA secretion by hypothalamic DAergic neurons of aged rats may be the result of insufficient activation of PKA caused by failure of transduction of an extracellular signal to activate adenylyl cyclase and produce cyclic AMP.

Dopa accumulates in the hypothalamic-hypophysial portal vessels and is taken into the anterior pituitary of NSD-1015-treated rodents.

DOPA was measured in the anterior pituitary and hypothalamic-hypophysial portal blood after treatment with NSD-1015, a DOPA decarboxylase inhibitor. NSD-1015 caused DOPA to accumulate in the anterior pituitary of mice and rats, and increased DOPA in the hypothalamic-hypophysial portal blood of rat. Serum prolactin was also increased. Interruption of the anterior pituitary blood supply from the hypothalamic-hypophysial system by cannulation of the entire pituitary stalk eliminated the NSD-1015-induced DOPA accumulation in the rat pituitary. We conclude that DOPA can be taken into the anterior pituitary from the portal blood of NSD-1015-treated rodents and that the anterior pituitary lacks tyrosine hydroxylase activity in both mice and rats.

Tyrosine hydroxylase expression in hypothalamic cells: analysis of the roles of adenosine 3',5'-monophosphate- and Ca2+/calmodulin-dependent protein kinases in the action of pituitary cytotropic factor.

We investigated the involvement of second messenger systems in the control by pituitary cytotropic factor (CTF) of tyrosine hydroxylase (TH) expression in primary cultures of hypothalamic cells. Forskolin, an activator of adenylyl cyclase, as well as Sp-cAMP[S] [(Sp)-cyclic adenosine 3',5'-monophosphothioate], a cAMP agonist, and theophylline, an inhibitor of phosphodiesterase activity, stimulate the secretion of dihydroxyphenylalanine (DOPA) and dopamine (DA), suggesting a role for cAMP-dependent protein kinase in the secretion of catecholamines by hypothalamic dopaminergic cells. When cells were cultured with either CTF or forskolin for 14 days, a progressive increase in the secretion of DOPA and DA was observed throughout the period of incubation. At the end of the 2-week culture period, the amount of TH in the cells, determined by immunoblot analysis, was appreciably increased compared to controls. When the cells were analyzed immunocytochemically for TH, the TH-positive cells that had been incubated with CTF or forskolin for 2 weeks were found to have neurites that appeared larger than those of TH-positive cells in the controls. The diameters of the perikarya of TH-positive cells in cultures incubated with CTF also appeared larger than the controls. After incubation of hypothalamic cells with CTF for 96 h, the amount of TH mRNA in the cultures was significantly increased. When membranes isolated from PC12 cells were incubated for 10 min with 50 microM forskolin, the specific activity of adenylyl cyclase was increased 20-fold; CTF had no effect on adenylyl cyclase activity of PC12 cell membranes. Yet, CTF significantly (P less than 0.001) stimulated the secretion of DOPA and DA by PC12 cells. When hypothalamic cells were incubated with both forskolin and CTF, using doses of each that stimulated maximal secretion, the secretion of DOPA and DA was equal to sum of the secretions with each stimulant alone. These additive actions of forskolin and CTF and the failure of CTF to activate adenylyl cyclase in membranes of PC12 cells suggest that forskolin and CTF stimulate catecholamine secretion by hypothalamic dopaminergic cells through different mechanisms, perhaps through different protein kinases. When hypothalamic cells were incubated with CTF and W-7 [N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide], an inhibitor of calmodulin, the secretion of DOPA was significantly (P less than 0.001) less than that in cultures that were not incubated with W-7. The findings of this study suggest that TH expression in hypothalamic dopaminergic cells is controlled by redundant protein kinases, including cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase.

Incertohypothalamic A13 dopamine neurons: effect of gonadal steroids on tyrosine hydroxylase.

In this study the effect of gonadectomy and steroid treatment on the dorsal component of the incertohypothalamic dopamine system or nucleus A13 was assessed by immunocytochemistry using an antibody raised to tyrosine hydroxylase (TH). A computer graphic system interfaced to a microscope was used to count and measure the diameters of TH-positive neurons and display the data in the three-dimensional space of the nucleus. In males, castration resulted in a dramatic decrease in the reaction product representative of TH. The number of TH-positive cells in the A13 DA nucleus decreased to 25% of intact levels. In females, ovariectomy also caused an impressive loss in the TH-immunostainable material, but this was not indicated by a change in the total number of TH-positive neurons. In both sexes the loss in TH immunostain was confined mainly to the mid-portion of the nucleus. Hormone treatment restored the TH immunostain (cell number and size) to and/or above intact levels in both sexes. These data suggest that A13 TH immunostain is stimulated by gonadal steroids in male and female rats.

Expression of tyrosine hydroxylase in cultured brain cells: stimulation with an extractable pituitary cytotropic factor.

Expression of tyrosine hydroxylase (TH) in cultured cells of the ventral hypothalamus-midbrain of fetal rats has been investigated. TH mRNA and TH were quantified by an S1 nuclease protection assay and an immunoblot assay, respectively. Dihydroxyphenylalanine (DOPA) and dopamine secretion were evaluated using their rates of accumulation in the culture medium. The rate of accumulation of DOPA was 2-3 times that of dopamine. Inhibitors of TH activity caused a dose-dependent reduction in DOPA secretion. During an 11-week culture of dissociated cells, TH mRNA increased from 1.6 to 2.8 attomole/well between the first and fourth week of culture, remained steady to the ninth week, and then declined. TH increased from 12 to 105 fmol/well between the first and seventh week and then declined. DOPA secretion increased until the sixth week and then remained steady to the tenth week. An extract of rat pituitaries stimulated DOPA secretion by the cultures in a dose-dependent manner. This activity, attributed to a cytotropic factor (CTF), was inactivated by heating for 10 min in a boiling water bath, but was unaffected by trypsin digestion. Incubation with CTF for 24, 48, 72, and 96 h resulted in a day by day increase in the secretion of DOPA. After 96 h of culture with CTF, the amount per well of TH mRNA, but not TH, was significantly (P less than 0.01) greater than the control value. Pituitary CTF is probably not PRL, since rat PRL did not appreciably affect DOPA secretion or the amount of TH mRNA or TH in the cells. Withdrawal of CTF from CTF-stimulated cells resulted in a marked reduction in DOPA secretion as well as a decrease in TH mRNA. These results support the hypothesis that the pituitary gland contains a cytotropic factor that stimulates TH expression in fetal brain cells of the hypothalamus-midbrain.

Mass and in situ molar activity of tyrosine hydroxylase in the median eminence. Effect of thyroidectomy and thyroid hormone replacement.

The effects of thyroidectomy and thyroid hormone replacement on the mass and in situ molar activity of tyrosine hydroxylase (TH) in the median eminence (ME) and superior cervical ganglia (SCG) of male rats were investigated. The tissue specificity of these effects were evaluated by comparing the ME with the superior cervical ganglion (SCG). All animals were thyroparathyroidectomized (Tx) or sham Tx. Tx rats were treated daily for 3 weeks with 0.15 M NaCl (solvent vehicle) or L-thyroxine (T4). Two doses of T4, 10 and 100 micrograms/day/kg BW, were used. Sham Tx rats were treated with 0.15 M NaCl. All animals were studied on the day following the last treatment. The mass of TH was determined using an immunoblot assay, and the in situ activity of TH was calculated from the rate of intracellular accumulation of L-dihydroxyphenylalanine (DOPA) after administration of an inhibitor of DOPA decarboxylase activity. In the ME, thyro-parathyroidectomy resulted in a 40% increase in the mass and a 100% increase in the in situ molar activity of TH over that of sham Tx rats. Compared to Tx animals given 0.15 M NaCl, Tx rats treated with a low dose of T4 (10 micrograms/day/kg BW) had a reduced quantity of TH in the ME, but the molar activity of the enzyme was increased. Treatment of Tx rats with a high dose of T4 (100 micrograms/day/kg BW) restored TH mass but not the in situ activity of TH in the ME to the level seen in sham Tx rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Saline ingestion stimulates the in situ molar activity of tyrosine hydroxylase in the median eminence and superior cervical ganglion.

The effects of drinking saline for 7 days on the mass and in situ activity of tyrosine hydroxylase (TH) in the median eminence (ME) and superior cervical ganglion (SCG) of rats were investigated. TH mass was quantified by immunoblot assay. In situ TH activity was calculated from the rate of intracellular accumulation of L-dihydroxyphenylalanine (DOPA). In rats that drank 10 mM, 30 mM, and 100 mM NaCl for 7 days, TH activity in the ME was 34 +/- 4, 36 +/- 5, and 45 +/- 3 (mean and S.E.M.) mol of DOPA.h-1.mol of TH-1, respectively, compared to 30 +/- 2 for rats that drank water. The activity of TH in the SCG of animals that drank 10 mM, 30 mM, and 100 mM NaCl was 143 +/- 24, 167 +/- 12, and 272 +/- 13 mol DOPA.h-1.mol TH-1, respectively, compared to 119 +/- 10 for animals that drank water. The mass of TH in the ME and SCG decreased as a function of the concentration of NaCl in the drinking water. In animals that drank water, 10 mM, 30 mM, and 100 mM NaCl, the amounts (pmol) of TH were, respectively, 0.28 +/- 0.03, 0.31 +/- 0.04, 0.23 +/- 0.02, and 0.21 +/- 0.01 per ME and 0.67 +/- 0.06, 0.72 +/- 0.11, 0.37 +/- 0.01, and 0.34 +/- 0.02 per SCG. TH activity in the ME or SCG was unaffected by treatment for 7 days with arginine vasopressin.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of hypothalamic dopamine in the regulation of prolactin secretion.

The neuroendocrine control of prolactin (PRL) secretion is known to be a multifactorial process, but dopamine (DA) secreted by the tuberoinfundibular dopaminergic (TIDA) neurons of the hypothalamus is believed to exert a predominant inhibitory control on the secretion of PRL. The secretory activity of the TIDA neurons, including the rate of biosynthesis of DA and the rate of release of the neurohormone into hypophysial portal blood, can be readily evaluated in the rat. In most conditions in which an altered secretion of PRL has been documented, an altered secretory activity of the TIDA neurons has been found. When an acute reduction in the secretion of DA is observed, an increased secretion of PRL is associated, with an inverse relationship between DA and PRL concentrations in hypophysial portal and systemic blood, respectively. However, the secretion of PRL can be regulated by PRL itself through stimulation of the secretory activity of the TIDA neurons, and consequently hyperprolactinemia can be observed concomitantly with a sustained high secretion of DA, as seen after treatment with estrogen. The short loop feedback of PRL secretion seems to be impaired in the aging rat, since a sustained reduced hypothalamic secretion of DA is observed in spite of long-term hyperprolactinemia.

Dopaminergic neurons in the mediobasal hypothalamus of old rats: evidence for decreased affinity of tyrosine hydroxylase for substrate and cofactor.

The effect of aging on the activity of tyrosine hydroxylase (TH) and on the number of TH-positive perikarya in the hypothalamus was studied in old and young female rats. The activity of TH in the mediobasal hypothalamus (MBH) of old rats was significantly (P less than 0.025) less than that in young rats. In old rats, the Km of TH for tyrosine as well as cofactor, 6-methyl-5,6,7,8-tetrahydropterine (6MPH4), was markedly greater than the Km in young rats. The maximal velocity was only slightly reduced in old animals. Contiguous coronal sections of the brain of an old and a young female rat were immunocytochemically stained for TH, and the TH-positive perikarya in the hypothalamus were counted. In the circumventricular region, 6793 TH-positive perikarya were present in the young brain and 6632 in the old brain. In the arcuate region, 2868 and 2760 TH-positive perikarya were counted in the young and old brain, respectively. It is concluded that the reduced TH activity in the MBH of old rats is not a consequence of a reduction in the number of TH-positive perikarya in the arcuate or circumventricular regions of the hypothalamus but is due to a reduction in the affinity of TH for its substrate and cofactor.

Luteinizing hormone-releasing hormone and thyrotropin-releasing hormone in the hypothalamus of women: effects of age and reproductive status.

In the study of the effects of age and reproductive status on LHRH and TRH content in the hypothalamus of women, we found that the amount of LHRH (58 +/- 5.5 ng; mean +/- SE) in the hypothalamus of young women (16-29 yr) was significantly greater (P less than 0.001) than that (28 +/- 3.0 ng) in postmenopausal women (50-78 yr). The hypothalamic content of LHRH (18 +/- 2.4 ng) of bilaterally ovariectomized women (39-47 yr) was significantly less (P less than 0.001) than that (60 +/- 12.6 ng) in younger ovulatory women (30-39 yr) or that (56 +/- 13.5 ng) in ovulatory women of comparable age (40-49 yr). In contrast, the hypothalamic content of TRH (121.4 +/- 32.8 ng) in postmenopausal women were similar to that (122.3 +/- 12.5 ng) in young women. Although aging in women is associated with a significant reduction in the amount of LHRH in the hypothalamus, such a reduction appears to be a consequence of ovarian failure and not of aging per se.

Dopamine in plasma of lateral and medial hypophysial portal vessels: evidence for regional variation in the release of hypothalamic dopamine into hypophysial portal blood.

The plasma concentrations of dopamine in blood from hypophysial portal vessels in various locations on the pituitary stalk were evaluated in diestrous rats. It was found that the mean concentration of dopamine in blood from lateral hypophysial portal vessels, which contain the venous effluent of the lateral median eminence, was significantly less (P less than 0.005) than that in blood from medial portal vessels, which contain the venous effluent of the medial median eminence [1.59 +/- (SE) 0.23 ng/ml vs. 3.12 +/- 0.48 ng/ml]. The mean plasma concentration of dopamine in blood of lateral portal vessels and of medial portal vessels was at least 20-40 times greater than that in arterial blood of these animals. It was calculated that the rate of release of hypothalamic dopamine was 174 +/- 38 pg/h into a medial portal vessel and 73 +/- 15 pg/h into a lateral portal vessel. The mean plasma concentration of norepinephrine or epinephrine in blood from a medial portal vessel was not different from that from a lateral portal vessel. To address the issue of whether the rate of release of dopamine into a medial portal vessel and into a lateral portal vessel was correlated with the rate of synthesis of dopamine in discrete regions of the median eminence, the concentration of L-dihydroxyphenylalanine (DOPA), the precursor of dopamine, was evaluated in lateral and medial segments of the median eminence of diestrous rats treated with 3-hydroxybenzylhydrazine, an inhibitor of DOPA decarboxylase activity. The concentration of DOPA was similar in the medial and lateral segments of the median eminence, suggesting that the rate of synthesis of dopamine did not account for the difference in the rate of release of dopamine into portal blood. The finding of different concentrations of dopamine in blood from various hypophysial portal vessels may be important in view of the heterogenous perfusion of the pars distalis with hypophysial portal blood. We suggest that topographic differences may exist in the release of PRL by cells of the pituitary gland as a consequence of uneven concentrations of dopamine in portal blood perfusing the lactotropes.

An inhibitory role for morphine on the release of dopamine into hypophysial portal blood and on the synthesis of dopamine in tuberoinfundibular neurons.

The intracerebroventricular administration of morphine to ovariectomized rats resulted in a marked decrease in the concentration of dopamine in plasma of hypophysial portal blood. A 90% reduction in the rate of release of hypothalamic dopamine into hypophysial portal blood occurred during the 60 min following the intraventricular administration of 60 ng of morphine sulfate. A dose-related decrease in the rate of release of dopamine into the portal vasculature was observed between 7.5 ng and 60 ng of morphine sulfate. Regardless of the quantity of morphine sulfate (1-500 ng) given to the animals, the concentrations of norepinephrine and epinephrine in hypophysial portal plasma and femoral arterial plasma remained unchanged. The efficacy of morphine on the release of dopamine into hypophysial portal blood was not associated with an equal efficacy of the drug on the synthesis of dopamine in tuberoinfundibular neurons, as evaluated by the accumulation of dihydroxyphenylalanine (DOPA) in the median eminence of rats given 3-hydroxybenzylhydrazine (NSD 1015). No effect of morphine was observed on DOPA accumulation in the median eminence of NSD-treated rats that had received 50 ng of morphine sulfate intracerebroventricularly, and only a 50% reduction was observed in the accumulation of DOPA in the median eminence of rats given 500 ng of morphine sulfate. These findings are supportive of the view that morphine inhibits both the release and synthesis of dopamine but is more effective in inhibiting the release than synthesis of dopamine.

Molecular-weight profiles of immunoreactive corticotropin in the hypothalamus of the aging rat.

Extracts of the medial basal hypothalamus (MBH), the preoptic anterior hypothalamus (POA), or the pituitary gland of young (4-month-old) and old (18-month-old) female rats were fractionated on columns of Sephadex G-75 superfine. Five forms of immunoreactive corticotropin (ACTHi) were found in the MBH or POA: greater than 40K, 30-40K, 20-30K, 5.7K, and 4.5K. In contrast, 4 forms of ACTHi were found in the pituitary gland: 30-40K, 20-30K, 5.7K, and 4.5K. Thus, hypothalamic tissue contains a large form of ACTH (greater than 40K ACTH) which is not present in the pituitary gland. We tentatively identified greater than 40K ACTH as a large form of pro-opiocortin, 30-40K ACTH as pro-opiocortin, 20-30K ACTH as ACTH biosynthetic intermediate, 5.7K ACTH as glycosylated ACTH 1-39, and 4.5K ACTH as ACTH 1-39. The content of ACTHi in the MBH and POA of old rats was lower than that of young rats. Nevertheless, regardless of the age of the animals, the fractional amount of 30-40K ACTHi was high in the MBH (a region that includes the presumed site of biosynthesis of pro-opiocortin) compared to that in the POA (a region that is distant to the site of biosynthesis of pro-opiocortin). Moreover, the reduced fractional amount of 30-40K ACTHi in the POA was associated with an increased fractional amount of greater than 40K, 20-30K, 5.7K, and 4.5K ACTHi. These findings are consistent with a precursor-product relationship between the 30-40K ACTHi and 20-30K ACTHi, 5.7K ACTHi, and 4.5K ACTHi.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-dependent extinction of thyrotropin-releasing hormone in the human cerebellum.

Immunoreactive TRH was quantified in eight regions of the cerebellum as well as in the medulla, pons, and hypothalamus of the fetal and adult human brain. High levels of TRH were detected in the fetal cerebellum, ranging from 216 +/- 103 pg/mg protein (mean +/- SD) in the deep cerebellar nuclei to 591 +/- 153 pg/mg protein in the anterior vermis. The concentrations of TRH were significantly greater (P less than 0.001) in each of the eight regions of the cerebellum of the fetal brain than in the corresponding regions of the adult brain. The magnitude of the difference between adult and fetal cerebellar levels ranged from an 18-fold difference in the deep cerebellar nuclei to more than a 100-fold difference in the anterior hemisphere. However, the TRH levels in pons and medulla were similar among fetuses and adults, and the TRH concentration in the adult hypothalamus was significantly higher (P less than 0.01) than that in the fetal hypothalamus. The TRH levels in adult rat hypothalami were extremely stable for several hours post mortem. We, therefore, conclude that the differences in cerebellar TRH concentrations of the fetal compared to those of the adult human are not related to a difference in the extent of postmortem degradation of TRH. Rather, we postulate that the decline in cerebellar TRH during maturation is a normal developmental process, and speculate that TRH, which has been found to have diverse effects on the central nervous system, may also influence the development of the human cerebellum.

Disproportionate accumulation of immunoreactive corticotropin, melanotropin, and lipotropin in the brain of the maturing rat.

The accumulation of immunoreactive ACTH (ACTHi), alpha MSH (alpha MSHi), and gamma-lipotropin (gamma LPHi) as a function of age (10-120 days) was determined in three regions of the brain of male rats: the medial basal hypothalamus (MBH), the preoptic anterior hypothalamus (POA), and the thalamus. In each region of the brain, the concentrations of ACTHi, alpha MSHi, and gamma LPHi increased with age. In the MBH, the increase occurred in such a manner that the molar ratio of alpha MSHi to ACTHi remained constant regardless of the age of the animals. In contrast, in the POA and thalamus, the increase occurred disproportionately in favor of alpha MSHi, and thus the molar ratio of alpha MSHi to ACTHi was 3 times higher in the adult (120 days old) than in the young (10 or 21 days old) animals. Nevertheless, the ratio of (ACTHi plus alpha MSHi) to gamma LPHi was constant at a level of about 2 regardless of the age of the animal or the region of the brain. Extracts of the MBH or POA were fractionated on columns of Sephadex G-75 superfine. The gel filtration profiles of ACTHi were indicative of the presence of five molecular weight forms of ACTH: greater than 40K, 30-40K, 20-30K, 5.7K, and 4.5K. We tentatively identified greater than 40K ACTH as a large form of proopiocortin, 30-40K ACTH as proopiocortin, 20-30K ACTH as ACTH biosynthetic intermediate, 5.7K as glycosylated ACTH(1-39), and 4.5K ACTH as ACTH-(1-39). Regardless of the age of the animals, the fractional amount of 30-40K ACTH the age of the animals, the fractional amount of 30-40K ACTH was high in the MBH compared to that in the POA. Moreover, the small fractional amount of 30-40K ACTH in the POA was associated with a large fractional amount of small molecular weight forms of ACTHi. However, the predominant form of ACTHi in the POA changed with age: 20-30K ACTH was the major form in the young, whereas 4.5K ACTH was the major form in the adult. These results support the proposal that the production of proopiocortin increases with age, and there is enhanced processing of proopiocortin to ACTH-(1-39) and alpha MSH in the brain of the maturing rat.