Rat Pituitary Neurointermediate Lobe Research Articles

Synaptophysin immunoreactivity in the rat pituitary: alterations after 6-hydroxydopamine treatment.

Synaptophysin (SN) is a synaptic-vesicle-associated membrane protein whose presence is indicative of intact, functional synapses. This study examines the presence of SN in pituitary gland innervation after neurotoxin-induced denervation followed by reinnervation. Immunostaining of rat pituitary neurointermediate lobe tissue for SN reveals a pattern of dot-like densities in the intermediate lobe and intensely stained dispersed regions in the neural lobe of normal animals. In rats treated with 6-hydroxydopamine (6-OHDA), a catecholamine neurotoxin, by peripheral injection, there is a significant depletion of the SN immunostaining in the intermediate lobe, as well as a significant reduction of SN immunoreactivity in the neural lobe, in animals studied 1 wk after drug treatment, with computer analysis of the tissue sections. At 3 wk after 6-OHDA, there is a partial recovery of immunoreactivity for SN in the neural lobe in many tissue sections, and the intermediate lobe also contains only relatively sparse staining for the synaptic protein. Computer analysis revealed that at 3 wk after 6-OHDA, both lobes still had reduced SN immunoreactivity, but the difference in levels measured did not achieve statistical significance. These results contrast with the prior finding of significant recovery of immunoreactivity for GAP-43, a growth and regeneration-associated protein, in intermediate lobe innervation of rats treated with the same drug regimen. We suggest that 6-OHDA treatment damages synaptic vesicle integrity in both the intermediate and neural lobes of the pituitary, and that recovery is in progress, but not complete at 3 wk after the drug is administered.

Types and activities of voltage-operated calcium channels change during development of rat pituitary neurointermediate lobe

Cultures of pituitary neurointermediate lobe cells were established from rats aged 1, 12, and 42 days to identify the types and assess the activities of Ca2+ channels present in melanotropes, glial-like cells, and fibroblasts during development. Day 12 represents the time at which dopaminergic axons have become distributed throughout the lobe, glial cells begin to lose their radial orientation, and melanotropes robustly express the short isoform of the dopamine D2 receptor. Thus, we studied Ca2+ channels in relation to the event of innervation of melanotropes. Real-time fluorescence video microscopy, in the presence of pharmacological agents, which block L-, N-, P-, and T-type channels, was used as an indirect measurement of channel activity. Assessment of cell type was verified by triple-label fluorescence immunohistochemistry. In melanotropes, extracellular Ca2+ addition caused Ca2+ influx through ω-conotoxin GVIA-sensitive, N-type channels on days 1 and 12 but not on day 42. The K+ depolarization induced an increase in intracellular Ca2+ concentration in all age-groups. This effect was decreased by nifedipinc, an L-type channel blocker, at all ages, and by ω-agatoxin IVa, a P-type blocker, only on day 42. These results demonstrate that the predominance of N- or P-type channels on melanotropes is age-dependent and can be correlated with other developmental changes. The T-type blocker, NiSO4, had no effect. In glial-like cells of all ages, extracellular Ca2+ addition resulted in an increase in intracellular Ca2+ concentration, which was inhibited only by NiSO4. The percentage of responsive glial-like cells was equally high in days 1 and 12 cultures, then declined by day 42. The K+ depolarization had no effect on glial-like cells. Fibroblasts did not respond significantly to extracellular Ca2+ or K+ depolarization, indicating little detectable activity by this methodology from functional voltage-operated Ca2+ channels.

Types and activities of voltage-operated calcium channels change during development of rat pituitary neurointermediate lobe

Cultures of pituitary neurointermediate lobe cells were established from rats aged 1, 12, and 42 days to identify the types and assess the activities of Ca2+ channels present in melanotropes, glial-like cells, and fibroblasts during development. Day 12 represents the time at which dopaminergic axons have become distributed throughout the lobe, glial cells begin to lose their radial orientation, and melanotropes robustly express the short isoform of the dopamine D2 receptor. Thus, we studied Ca2+ channels in relation to the event of innervation of melanotropes. Real-time fluorescence video microscopy, in the presence of pharmacological agents, which block L-, N-, P-, and T-type channels, was used as an indirect measurement of channel activity. Assessment of cell type was verified by triple-label fluorescence immunohistochemistry. In melanotropes, extracellular Ca2+ addition caused Ca2+ influx through ω-conotoxin GVIA-sensitive, N-type channels on days 1 and 12 but not on day 42. The K+ depolarization induced an increase in intracellular Ca2+ concentration in all age-groups. This effect was decreased by nifedipinc, an L-type channel blocker, at all ages, and by ω-agatoxin IVa, a P-type blocker, only on day 42. These results demonstrate that the predominance of N- or P-type channels on melanotropes is age-dependent and can be correlated with other developmental changes. The T-type blocker, NiSO4, had no effect. In glial-like cells of all ages, extracellular Ca2+ addition resulted in an increase in intracellular Ca2+ concentration, which was inhibited only by NiSO4. The percentage of responsive glial-like cells was equally high in days 1 and 12 cultures, then declined by day 42. The K+ depolarization had no effect on glial-like cells. Fibroblasts did not respond significantly to extracellular Ca2+ or K+ depolarization, indicating little detectable activity by this methodology from functional voltage-operated Ca2+ channels.

Immunocytochemical Studies of Tryptophan Hydroxylase, Tyrosine Hydroxylase, and Serotonin Innervation in the Aging Rat Neurointermediate Pituitary

Innervation within the aging male rat pituitary neurointermediate lobe was examined using immunocytochemical methods with antisera to rate-limiting enzymes for serotonin (tryptophan hydroxylase, TpOH) or catecholamine (tyrosine hydroxylase, TH) synthesis. Immunostaining for both enzymes was observed in both neural and intermediate lobes in younger rats (age 6 weeks or 7 months), but TpOH was absent from the intermediate lobe in older groups (10-12, 15-16, and 18-19 months). Colocalization of TpOH and TH was demonstrated in some nerve fibers, using immunofluorescence. The oldest age group showed a reduced staining pattern for TH. 5-hydroxytryptophan, the immediate precursor for serotonin (5-HT), was taken up and converted to immunoreactive 5-HT in all age groups. Overall, the pattern of staining for regulatory neurotransmitters or enzymes which ultimately affect pituitary intermediate lobe secretory function is significantly modulated during the aging process.

Isoproterenol-stimulated release of β-endorphin and related peptides from the rat pituitary neurointermediate lobe in vitro: Evidence for preferential release of certain molecular forms of β-endorphin

The intermediate lobe of the pituitary gland synthesizes the multifactorial precursor molecule pro-opiomelanocortin (POMC), from which, through a process of post-translational enzymatic processing, β-endorphin-(1–31) (βE) and a variety of N α-acetylated and C-terminally shortened forms of this peptide are generated. Using an in vitro superfusion system, the release of these endorphins from intact rat neurointermediate lobes (NILs) was investigated under basal and isoproterenol (ISO) stimulated conditions. Superfusion of NILs with the β-adrenergic agonist ISO (30 min pulse) resulted in a rapid, sustained and concentration-dependent stimulation of the release of βE-like immunoreactivity (βE-IR) over basal as determined with an antiserum directed against the C-terminus of the βE-(1–31) sequence (10 −6M: +145%; 10 −7M: +73%; 10 −8m: +41%). The release of N α-acetylated-endorphin-like immunoreactivity (AcE-IR) was stimulated to a similar extent. These effects of ISO were antagonized by the competitive α-adrenoceptor antagonist propranolol in a concentration-dependent manner, indicating the involvement of α-adrenoceptors. The β-related peptides released from the NILs under basal and ISO-stimulated conditions were further characterized, based on their retention times in a reversed-phase HPLC system and their reactivity with specific antisera recognizing respectively the midportion of βE, the N-terminus of acetylated endorphines, the C-terminus of τ-endorphin (βE-(1–17); τE), or the C-terminus of α-endorphin (βE-(1–16); αE). In HPLC fractionated superfusates 10 peaks were resolved that reacted with the midportion βE antiserum. In superfusates collected under basal conditions, three major peaks possessed chromatographical and immunological characteristics of AcβE-(1–26), AcβE-(1–27) AcβE-(1–31). In addition, a prominent peak was found eluting around the retention time of βE-(1–31), that contained both acetylated and non-acetylated material. Six smaller peaks were observed, with the characteristics of βE-(1–26) and βE-(1–27) (these peptides were not resolved with the HPLC system used), AcτE, τE, AcαE, and des-tyrosine-αE (DTαE), respectively. In superfusates collected during superfusion of NILs with ISO (10 −6M) all peaks were increased. However, those eluting as βE-(1–31), βE-(1–26) βE-(1–27), AcβE-(1–26) and AcτE appeared to be preferentially stimulated. The results indicate, that the NIL in vitro simultaneously releases βE and a variety of highly processed βE-related peptides including τ- and α-type endorphins. In addition, the data suggest the existence of β-adrenoceptor coupled mechanisms, that govern the secretion of a preferred set of βE-related peptides from the pituitary intermediate lobe cell.

Chronic naltrexone infusion: Effects on innervation of rat neurointermediate lobe

The rat pituitary neurointermediate lobe contains nerve fibers which are sensitive to the neurotoxic effects of 6-hydroxydopamine and to acute injections of the opiate antagonist, naltrexone. In the present study, naltrexone hydrochloride was administered to adult male Sprague-Dawley rats in a chronic infusion over a period of one week, using Alza osmotic pumps to determine whether a low (10 −5 M) dose could, over a longer period, also induce toxic effects on pituitary innervation. Electron microscopy of pituitary intermediate lobe fibers revealed swollen neurovesicles and membranous structures in nerve terminals, and significantly fewer normal-appearing nerve terminals, after drug infusion. Light microscopic immunostaining of paraffin sections of pituitary glands illustrated some diminution of serotonin (5-HT)-immunofluorescence in the intermediate lobe after naltrexone treatment. Immunostaining of sections from the same animals for tyrosine hydroxylase (TH) revealed intensified fiber staining in the intermediate lobe, with some regions of terminals having a swollen appearance. High pressure liquid chromatographic-electrochemical detection (HPLC-EC) analysis of neurotransmitters in selected brain areas and pituitary indicated a significant increase in norepinephrine levels in the hypothalamus. The observations suggest that intermediate lobe innervation is sensitive to low, continuously infused doses of naltrexone, which acts as a neurotoxin to produce degenerative changes in nerve terminals. The changes appear to be reflected in alterations in staining patterns of neurotransmitters, and may also affect transmitter uptake in damaged terminals.

Differential Effects of Glucocorticoids on Corticotrophin-Releasing Factor in the Rat Pituitary Neurointermediate Lobe and Median Eminence.

We have investigated the effects of glucocorticoid manipulation on corticotrophin-releasing factor (CRF-41) in the neurointermediate lobe (NIL) and median eminence (ME) of the rat using a radioimmunoassay specific for CRF-41. CRF-41 content in the NIL (88+/-7 fmol, mean +/- SEM) was not significantly altered by administration of dexamethasone in drinking water for 12 days (67+/-9 fmol) or adrenalectomy for 12 days (96+/-17 fmol). The saline-stimulated increase in NIL CRF-41 content (154+/-24 fmol) was not affected by dexamethasone (152+/-24 fmol) or adrenalectomy (179+/-21 fmol). In contrast, the content of CRF-41 in the ME declined following dexamethasone treatment (1247+/-92 fmol to 864+/-26 fmol), or adrenalectomy (854+/-78 fmol). Our results provide further evidence that CRF-41 in the NIL and ME can be differentially regulated.

Effect of Hypertonic Saline on the Corticotropin-Releasing Hormone and Arginine Vasopressin Content of the Rat Pituitary Neurointermediate Lobe.

The changes in the levels of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) in the neurointermediate lobe of the pituitary (NIL) following hypertonic saline administration were examined in rats. The plasma osmotic pressure in rats receiving 2% NaCl for 8 days was greatly increased. Plasma AVP concentration in rats receiving 2% NaCl for 8 days were significantly higher than in control rats (566% of the control level). Plasma corticosterone was significantly higher in the saline-treated rats than in controls, whereas plasma ACTH was not significantly different. The pituitary ACTH concentration was much higher in the saline-treated rats than in controls. CRH in the NIL was increased significantly by saline treatment (419% of the control concentration), whereas the CRH in the paraventricular nucleus and median eminence of control and saline-treated rats did not differ significantly. The AVP in the NIL fell greatly in saline treated rats. The extract from both control and saline-treated rats showed a major peak for immunoreactive CRH, with a retention time identical to that of rat CRH. However, the peak was much higher in the extract from saline-treated rats. The immunoreactive AVP peak was greatly reduced in saline-treated rats. These results suggest that hypertonic saline administration increases the CRH in the NIL and causes AVP hypersecretion and/or hyperfunction of magnocellular-NIL CRH might be responsible for pituitary-adrenal stimulation in saline-treated rats.

Lithium and haloperidol differentially alter the dynorphin A (1–8) and enkephalin levels in the neurointermediate lobe of rat pituitary

Repeated administration of the antimanic drug lithium (4 mEq/kg/day for 2, 4 or 6 days, i.p.) to rats produced a progressive decline and eventual depletion of dynorphin-A (1–8) (DYN) concentration whereas Met 5-enkephalin (ENK) was only marginally decreased in the neurointermediate lobe of the pituitary (NIL). Administration of a neuroleptic haloperidol neither affected ENK and DYN levels nor influenced lithium-induced changes. The study reveals that lithium produces a preferential perturbation in the dynorphin system relative to the enkephalin system. These results taken together with other evidence, indicate that dynorphin is possibly coreleased with vasopression following lithium administration and provide a pharmacological support to the previously described colocalization and corelease of these endogenous peptides in the NIL.

The size of enzymes acetylating alpha-melanocyte-stimulating hormone and beta-endorphin.

Acetylation of alpha-melanocyte-stimulating hormone and beta-endorphin in a granule fraction from the rat pituitary neurointermediate lobe was examined by radiation inactivation. Target sizes obtained were 71 +/- 12 and 76 +/- 9 kDa, respectively. These data suggest that a single enzyme species may be responsible for acetylation of the two substrates. A similar analysis of alpha-melanocyte-stimulating hormone acetylation in the rat lens revealed a complex inactivation curve which could be explained by the presence of at least two different enzyme molecules, one of 86 and the other greater than 200 kDa.

Dopaminergic agents differentially alter β-endorphin processing patterns in the rat pituitary neurointermediate lobe

We have established the content and molecular species of immunoreactive β-endorphin (ir-β-END) and immunoreactive N-acetyl-endorphin (ir-Nac-END) in rat neurointermediate lobe by specific radioimmunoassay (RIA) and high-performance liquid chromatography after chronic administration of dopamine (DA) agonists and antagonists. The DA agonist, bromocriptine, reduces tissue levels of all major immunoreactive species, in particular the C-terminally shortened N-acetylated forms. The DA antagonist, haloperidol, proportionally increases all immunoreactive forms, except Nac-β-END 1–27, thus altering the relative abundance of this species. These data indicate that DA is involved in the control of both tissue levels and processing of β-END-like peptides in the rat neurointermediate lobe.

Inhibition by prostaglandin E2 of the release of vasopressin and beta-endorphin from rat pituitary neurointermediate lobe or medial basal hypothalamus in vitro.

The present study was performed to examine the effect of the cyclo-oxygenase inhibitor, indomethacin, and that of various prostaglandins on the release of vasopressin and beta-endorphin-like immunoreactivity (beta-EI) from the rat neurointermediate lobe of the hypophysis, which was superfused in vitro. Indomethacin (2.8 and 28 mumol/l) changed neither basal secretion of vasopressin nor that evoked by electrical stimulation, whereas the resting release of beta-EI was enhanced by indomethacin (28 mumol/l). Prostaglandin (PG) E2 did not influence resting release of vasopressin but markedly inhibited (by about 50%) electrically induced release of vasopressin (least effective concentration: 300 nmol/l) as well as spontaneous secretion of beta-EI (least effective concentration: 100 nmol/l) in the presence of indomethacin (28 mumol/l). Prostaglandin F2 alpha (5 mumol/l) also inhibited the evoked release of vasopressin, whereas PGD2 (5 mumol/l) did not. Prostaglandin F2 alpha (5 mumol/l), D2 and I2 (1.5 mumol/l each) produced no effects on beta-EI release. As observed in the neurohypophysis, PGE2 inhibited the electrically induced release of vasopressin from the medial basal hypothalamus in vitro. We conclude that prostaglandins (especially PGE2) can inhibit (1) the stimulated release of vasopressin when acting on vasopressin-containing nerve terminals of either neurosecretory system (neurohypophysis, median eminence region), and (2) the secretion of beta-EI and, as can be inferred, alpha-MSH, by a direct action on intermediate lobe cells.

Dopamine receptors regulate Met-enkephalin content in pituitary

Met-enkephalin-like immunoreactive material (ME-LI) in the neurointermediate lobe of rat pituitary was dramatically altered by the long-term administration of drugs altering dopamine receptor activity. Treatment with the dopamine receptor antagonist haloperidol resulted in significant increases of ME-LI and treatment with the dopamine receptor agonist bromocriptine resulted in significant decreases of ME-LI in the neurointermediate pituitary. The treatments had considerably less effect on ME-LI in the anterior lobe of the pituitary. These results suggest that dopamine receptor activation or inhibition influences the content of ME-LI in the rat pituitary gland.

Localization and actions of cholecystokinin in the rat pituitary neurointermediate lobe.

Rat pituitary neural lobe contained high concentrations of cholecystokinin-like immunoreactivity (CCK-LI). Section of the pituitary stalk resulted in loss of CCK-LI, and both lactation and replacement of drinking water with 2% saline resulted in marked depletion of CCK-LI. Rats with congenital diabetes insipidus (Brattleboro strain) had a 73% reduction in CCK-LI below the levels of hooded Long-Evans controls, where as levels in the brain were unchanged. Release of CCK-LI, labeled dopamine, and gamma-amino butyric acid in response to potassium depolarization was studied. There was a low fractional release of CCK-LI. Addition of sulfated CCK-8 (CCK-8s) to the medium enhanced the calcium-dependent potassium-stimulated release of dopamine, but basal release was unaffected. gamma-Amino butyric acid release was only poorly calcium dependent and not effected by extracellular CCK-8s. Vasopressin and oxytocin release were stimulated by electrical stimulation of the pituitary stalk, and were unaffected by the addition of CCK-8s to the medium. In vivo, however, the injection of 5 micrograms CCK-8s into the third ventricle resulted in increased plasma vasopressin concentrations.

Neuropeptides derived from pro-opiocortin: behavioral, physiological, and neurochemical effects.

Neuropeptides derived from pro-opiocortin: behavioral, physiological, and neurochemical effects.

Evidence for an opiomelanotropin acetyltransferase in the rat pituitary neurointermediate lobe

Results of this study demonstrate two distinct forms of acetyltransferase activity which will acetylate α-MSH. These acetyltransferases are distinguished by pH optima, subcellular distribution and sensitivity to magnesium and several solubilizing detergents. A general acetyltransferase, as characterized in the rat pituitary anterior lobe and lens, has a pH optima of 7.4 and is inhibited by magnesium. Subcellular fractionation of anterior and neurointermediate lobes revealed that this acetyltransferase is primarily localized in the cytosol fraction of these tissues. An α-MSH acetyltransferase (MAT) and a β-endorphin acetyltransferase (EAT) have a pH opitma of 6.0–6.6, are inhibited by detergents, and are specifically localized in the secretory granules of the neurointermediate lobe. Comparative studies of MAT and EAT suggest a single enzyme responsible for the acetylation of opioid and melanotropin peptides, and we term this enzyme opiomelanotropin acetyltransferase (OMAT).

Immunoreactive gamma-melanotropin in rat pituitary and plasma: a partial characterization.

A RIA for the gamma MSH region of proopiomelanocortin has been established and validated. The antiserum was raised to synthetic bovine gamma MSH, and it cross-reacts well with larger polypeptides, including gamma MSH and 16K fragment, which contain the gamma MSH sequence. Gel filtration chromatography of extracts prepared from the anterior and neurointermediate lobes of rat pituitary and from rat plasma reveal two heterogeneous immunoreactive (IR-) forms of gamma MSH in each venue which elute in molecular weight peaks of approximately 11,000 (11K) and 6,000 (6K). Addition experiments indicate that the smaller material is not an artifact generated from the larger form during preparation. By the criterion of retention on affinity columns of Concanavalin A-agarose, these peptides are glycosylated. The 6K form represents 8-17% of the total IR-gamma MSH in the anterior lobe and about 30% of that in the neurointermediate lobe. IR-gamma MSH are released by dispersed rat pituitary cells in response to several of the same secretagogues which modulate the secretion of other proopiomelanocortin-derived peptides. Changes in the plasma and anterior lobe content of IR-gamma MSHs and ACTH resulting from perturbation of the pituitary-adrenal axis are concordant. In particular, the plasma concentration of 6K IR-gamma MSH increases dramatically after imposed stress.

Characterization of pro-opiocortin-converting activity in purified secretory granules from rat pituitary neurointermediate lobe.

Lysates of secretory granules from rat pituitary neurointermediate lobes were incubated with [3H]arginine- or [3H]phenylalanine-labeled toad pro-opiocortin. The processed products formed were identified by immunoprecipitation with adrenocorticotropin (ACTH) and endorphin antisera and by migration behavior on acid/urea/polyacrylamide gels. Pro-opiocortin was cleaved by the proteolytic activity in the secretory granule fraction to approximately 21,000 Mr ACTH, approximately 13,000 Mr ACTH, alpha-melanotropin, 16,000 Mr NH2-terminal glycopeptide, beta-lipotropin, and an endorphin-related peptide. Characterization of this pro-opiocortin-converting activity shows that it (i) is present in membrane and soluble fractions of the granule lysates, (ii) has a pH optimum of 5.0, (iii) appears to cleave at pairs of basic amino acid residues in the precursor, and (iv) is inhibited by leupeptin, pepstatin A, and p-chloromercuribenzoate but not diisopropyl fluorophosphate, N alpha-p-tosyl-L-lysine chloromethyl ketone hydrochloride, chloroquine, or EDTA. These inhibitor studies suggest that the converting-enzyme activity is due to an acid thiol, arginyl protease, distinct from any known cathepsin B-like activity.

The separation of neuropeptides by high performance liquid chromatography and its application to the analysis of peptides in the rat pituitary neurointermediate lobe (author's transl)

High performance liquid chromatography (HPLC) was used for the separation of many neuropeptides. Chromatography was carried out using a Hitachi Model 638 high performance liquid chromatograph. Peptides and samples from tissue dissolved in an aqueous buffer were injected into a stainless-steel column (4 X 250mm) packed with Hitachi #3053 (octadecylsilane). The aqueous buffer consisted of NaH2PO4 and H3PO4. After a loading phase (0% organic solvent) of 1 min, the peptides were sequentially eluted at room temperature using a gradient of organic solvent (acetonitrile or methanol, 0-60%). The eluted polypeptides were detected by UV absorbance at 220nm, and then they were collected for subsequent bio and radioimmunoassay using a fraction collector. The gradient of methanol or acetonitrile in 0.02M NaH2PO4, 0.1% H2PO4 was useful for separating small molecular peptides. The gradient of acetonitrile in 0.05-0.1M NaH2PO4, 0.1% H2PO4 was useful for separating many neuropeptides including ACTH related peptides. Retention times of chromatographed polypeptides showed good reproducibility. Good reproducibility was also found in peak areas of these peptides. A linear relationship was observed between the doses of peptides and their peak areas. The extracts of rat pituitary neurointermediate lobe showed several peaks of UV absorbance on PHLC; some of them coincided with AVP, oxytocin, alph-MSH, CLIP and beta-endorphin but others were unidentified. AVP immunoreactivity showed one peak which coincided with the AVP peak of UV absorbance, but ACTH immunoreactivity showed 5-6 peaks. Thus, many polypeptides were well separated using HPLC by changing the eluting condition. The simplicity, speed, good reproducibility and good quality of the separations render this technique suitable for purification and quantitative analysis of neuropeptides, and the combination of HPLC, radioimmunoassay and bioassay gives very fine analysis of neuropeptides.

Biosynthesis and release of beta-endorphin-, N-acetyl beta-endorphin-, beta-endorphin-(1-27)-, and N-acetyl beta-endorphin-(1-27)-like peptides by rat pituitary neurointermediate lobe: beta-endorphin is not further processed by anterior lobe

Continuous labeling and pulse-chase techniques were employed to study the synthesis and secretion of multiple forms of immunoreactive beta-endorphin by cultured dispersed rat anterior lobe cells and intact neurointermediate pituitary lobe. Cell and medium extract immunoreactive beta-endorphin (specific immunoprecipitation and radioimmunoassay) exhibiting a Kav similar to authentic beta-endorphin upon gel filtration was characterized further by nonequilibrium isoelectric focusing, cation exchange chromatography, reverse phase high pressure liquid chromatography, and partial tryptic and chymotryptic mapping. Intact neurointermediate lobes incorporated radiolabeled amino acids into four to six forms of immunoreactive beta-endorphin. Four of these forms were physicochemically similar to authentic beta-endorphin, N-acetylated beta-endorphin, beta-endorphin-(1-27), and N-acetylated beta-endorphin-(1-27). Pulse-chase studies indicated that a beta-lipotropin-like molecule served as a metabolic intermediate for a beta-endorphin-like molecule. As beta-endorphin-like material accumulated in the cell, some of it was N-acetylated (approximately 18% at 2 hr chase and approximately 65% at 18 hr chase). At later chase times, beta-endorphin-(1-27)- and N-acetylated beta-endorphin-(1-27)-like peptides were the predominant molecular species detected. All endorphin forms were detected in unlabeled tissue maintained in culture or tissue continuously labeled for 72 hr and were released into the medium under basal, stimulatory (10(-8) M norepinephrine), or inhibitory (10(-7) M dopamine) incubation conditions. In all cases, beta-endorphin-(1-27)-like species were the predominant forms (more than 70% of total) present in the cells and released into the medium. In contrast, approximately 90% of radiolabeled immunoreactive beta-endorphin extracted from anterior lobe cells and medium similarly incubated appeared to represent the authentic beta-endorphin molecule. Continuous labeling (72 hr) revealed the beta-lipotropin/beta-endorphin molar ratio to be approximately 4. We conclude that, in anterior lobe, most of the beta-endorphin is not processed further and is released intact, while in neurointermediate lobe, it serves as a biosynthetic intermediate.