Neurointermediate Pituitary Research Articles

Dopamine D 2-receptor messenger RNA is differentially regulated by dopaminergic agents in rat anterior and neurointermediate pituitary

Hypothalamic dopamine (DA), acting at DA D 2-receptors (D 2-R) on pituitary target cells, mediates peptide release and biosynthesis of rat pituitary anterior lobe (AL) prolactin, and neurointermediate lobe (NIL) pro-opiomelanocortin (POMC). We were interested in determining if dopamine agonists and antagonists were capable of modifying D 2-R gene expression in these pituitary cells. Utilizing the recently published sequence of the rat D 2-R, we isolated a rat D 2-R cDNA clone by polymerase chain reaction, and have synthesized RNA probes to quantitate levels of D 2-R mRNA by solution hybridization/nuclease protection assay. We report here that 5-day administration of the DA antagonist haloperidol led to significant increase in both D 2-R mRNA and POMC mRNA in the NIL; the DA agonist bromocriptine caused a significant decrease in NIL POMC mRNA with no parallel change in D 2-R mRNA. In contrast, no significant changes in D 2-R mRNA in AL were observed following treatment with either the DA agonist or antagonist. These data provide evidence for tissue-specific regulation of D 2-R mRNA in response to dopaminergic manipulation.

Purification of secretory vesicles from ovine neurointermediate pituitaries by agarose gel electrophoresis

Purification of secretory vesicles from ovine neurointermediate pituitaries by agarose gel electrophoresis

Particular processing of pro-opiomelanocortin in Xenopus laevis intermediate pituitary Sequencing of α- and β-melanocyte-stimulating hormones

α- and β-melanocyte-stimulating hormones (α-MSH and β-MSH) have been isolated from Xenopus laevis neurointermediate pituitary and microsequenced. Intracellular α-MSH is not N-acetylated after proteolytic processing of pro-opiomelanocortin in contrast to mammalian α-MSHs. There is a high preservation of the melanotropic amino acid sequence common to all MSHs although in Xenopus β-MSH a histidine residue replaces the glutamic acid residue found in position 8 of mammalian β-MSHs.

Nα‐Acetyl‐Vasopressin‐ and Nα‐Acetyl‐Oxytocin‐Like Substances: Isolation and Characterization in the Rat Neurointermediate Pituitary and Presence in the Brain

Abstract Post-translational modifications of vasopressin and oxytocin in pituitary and brain were investigated in view of recent evidence that oxytocin is partly N(alpha)-acetyfated in the bovine pineal gland. Two peptides were isolated from the neurointermediate lobe of the rat pituitary gland and characterized as N(alpha)-acetyl-vasopressin and N(alpha)-acetyl-oxytocin, based on chromatographic and immunological properties as well as the blocked N-terminus. In the neurointermediate pituitary the acetylated forms represented approximately 1% of the vasopressin and oxytocin contents. These two peptides were also detected in some, but not all, investigated brain areas. The highest degree of acetylation was found in the pineal gland. In all regions acetylation of oxytocin was more abundant than that of vasopressin. The data indicate that acetylation of vasopressin and oxytocin generally occurs as a post-translational modification. They support the concept that acetylation may represent a mechanism aimed to control bioactivity of the neurohypophyseal hormones.

Differential responses in vasopressin and oxytocin gene expression in distinct hypothalamic nuclei after hypothalamoneurohypophyseal disconnection and vasopressin substitution.

A possible feedback of circulating vasopressin (VP) on the expression of its gene in the hypothalamoneurohypophyseal system was studied in rats with depleted stores of neurohypophyseal hormones. The neural lobe of the pituitary gland was disconnected by anterolateral deafferentation of the basal hypothalamus using a Halasz knife cut. One group of lesioned animals was substituted continuously with VP using mini-osmopumps. After 1 week, the lesion had caused a complete disappearance of neurohypophyseal hormones from the neurointermediate pituitary and induced a 2-fold increase in water intake and an increase in plasma osmolality of approximately 7 m0sm/kg. VP mRNA levels of the supraoptic nucleus (SON), paraventricular nucleus (PVN) and suprachiasmatic nucleus (SCN) were measured in punched tissue by Northern blot analysis and dot-blot analysis. VP mRNA levels of the SON and PVN were reduced by 50% due to the deafferentiation, which might have been caused by degeneration. VP substitution of the lesioned animals normalized water intake but not plasma osmolality. In the VP-substituted animals, VP mRNA levels of the SON were 2-fold reduced compared to the nonsubstituted lesioned animals. The VP mRNA levels of the PVN were not affected by VP substitution. The VP mRNA levels of the SCN increased 2-fold after the lesion but were not influenced by VP substitution. Concomitant determination of oxytocin (OT) mRNA showed that OT mRNA levels in the PVN and SON were not affected by the deafferentiation and did not respond to VP substitution.(ABSTRACT TRUNCATED AT 250 WORDS)

Corticotropin-releasing factor (CRF) and neurotransmitters modulate melanotropic peptide release from rat neurointermediate pituitary in vitro

Isolated pituitary neurointermediate lobes from adult male Sprague-Dawley rats were incubated for up to 90 minutes in media containing stimulatory or inhibitory agents, to study the potential effects on secretion of alpha-melanocyte stimulating hormone (α-MSH) from the pars intermedia. Dopamine (DA), as expected, inhibited the release of α-MSH, as measured by radioimmunoassay (RIA), while corticotropin-releasing factor (CRF) and serotonin (5-HT) stimulated peptide release to varying degrees over the incubation period. A combination of DA with CRF produced an inhibition of peptide release, suggesting that DA suppresses the stimulatory effects of the hypothalamic peptide. Immune staining of pituitary tissue for beta endorphin or α-MSH, and quantified for staining intensity, supported the specific effects of stimulation or inhibition of the respective treatments. In addition, electron microscopy of incubated tissues demonstrated alterations in secretory vesicles and membranous organelles which were consistent with peptide secretion patterns. Our in vitro findings suggest that neurotransmitters and hypothalamic peptide, which are present together in vivo in the pituitary intermediate lobe, interact at the level of the endocrine cells to modulate pro-opiomelanocortin peptide secretion.

Purification of secretory vesicles from ovine adrenal medulla and neurointermediate pituitary

Purification of secretory vesicles from ovine adrenal medulla and neurointermediate pituitary

Isolation of neurosecretory granules containing vasopressin and MSEL-neurophysin from guinea pig neurointermediate pituitary

Neurosecretory granules have been isolated from rat and guinea pig neurointermediate pituitaries and their contents have been analyzed by reverse-phase high-pressure liquid chromatography and polyacrylamide gel electrophoresis. Granule components have been compared with synthetic neurohypophysial hormones and chemically characterized neurophysins. In rat granules, oxytocin, arginine vasopressin and MSEL- and VLDV-neurophysins have been identified. In isolated guinea pig granules, only arginine vasopressin and mature MSEL-neurophysin have been found. From these results it can be concluded that both the “dibasic” cleavage between vasopressin and MSEL-neurophysin and the “monobasic” cleavage between MSEL-neurophysin and copeptin occur within the granule compartment. Previous isolation from frozen guinea pig glands of a partially processed precursor encompassing MSEL-neurophysin and copeptin suggests a two-step processing of the three-domain vasopressin precursor, each involving a distinct enzymic system.

Functional topography of the rat hypothalamic dopamine neuron systems: Retrograde tracing and immunohistochemical study

The origins of the dopamine (DA)-containing nerve terminals in the external layer of the median eminence and in the neurointermediate pituitary were determined in rats by a combination of retrograde labeling with wheat germ agglutinin (WGA) and immunohistochemistry for tyrosine hydroxylase (TH). Biotinylated WGA (b-WGA) was injected into the posterior pituitary (group 1) and into the median eminence (group 2). In group 1 animals, all the magnocellular neurons of the paraventricular, supraoptic, and accessory nuclei, and many parvicellular neurons in the rostral periventricular region (RPR) were labeled with WGA. In group 2 animals, many neurons were labeled in the arcuate nucleus and the RPR, and in a small population of the preoptic-septal region. In group 1 animals, about 39% of TH neurons in the RPR were labeled with WGA, whereas only a few TH neurons (1%) in the arcuate nucleus were labeled with WGA. In group 2 animals, on the contrary, almost all TH neurons (73%) in the arcuate nucleus carried WGA, whereas in the RPR, only some of the TH neurons (19%) were labeled with WGA. It is concluded that DA neurons involved in the hypothalamic-anterior pituitary axis are located in the arcuate nucleus; those involved in neuro-intermediate lobe function in the RPR.

Structure of the precursor to an enzyme mediating COOH-terminal amidation in peptide biosynthesis.

Many bioactive peptides terminate with an amino acid alpha-amide at their COOH terminus. The enzyme responsible for this essential posttranslational modification is known as peptidyl-glycine alpha-amidating monooxygenase or PAM. We identified cDNAs encoding the enzyme by using antibodies to screen a bovine intermediate pituitary lambda gt11 expression library. Antibodies to a beta-galactosidase/PAM fusion protein removed PAM activity from bovine pituitary homogenates. The 108,207 dalton protein predicted by the complete cDNA is approximately twice the size of purified PAM. An NH2-terminal signal sequence and short propeptide precede the NH2 terminus of purified PAM. The sequences of several PAM cyanogen bromide peptides were localized in the NH2-terminal half of the predicted protein. The cDNA encodes an additional 430 amino acid intragranular domain followed by a putative membrane spanning domain and a hydrophilic cytoplasmic domain. The forms of PAM purified from bovine neurointermediate pituitary may be generated by endoproteolytic cleavage at a subset of the 10 pairs of basic amino acids in the precursor. High levels of PAM mRNA were found in bovine pituitary and cerebral cortex. In corticotropic tumor cells, levels of PAM mRNA and pro-ACTH/endorphin mRNA were regulated in parallel by glucocorticoids and CRF.

Tissue kallikrein in rat brain and pituitary: regional distribution and estrogen induction in the anterior pituitary.

We have detected tissue kallikrein and kallikrein mRNA in various brain regions with a kallikrein direct RIA and with nucleic acid hybridization using a kallikrein cDNA probe. In the direct RIA, rat urinary kallikrein-like activity was found in the pituitary and pineal glands, hypothalamus, cerebral cortex, cerebellum, and brain stem. Pituitary and pineal gland kallikrein concentrations were significantly higher than those in other regions. Only in pituitary was there a significant difference in tissue kallikrein concentration according to sex, with glands from female rats showing levels 4-fold higher than those from male rats. Kallikrein mRNAs were detected in all of the regions and were about 4-fold higher in female than in male pituitary gland. Northern blot analyses show sex dimorphism of pituitary kallikrein mRNA, similar in size to submandibular gland and kidney mRNA. In castrated male rats, whole pituitary kallikrein content was reduced to 50% of the control value and increased 1.7-fold with testosterone replacement and 18-fold with 17 beta-estradiol treatment. Neither T4 nor cortisol affected whole pituitary kallikrein levels in the castrated male rat, but testosterone decreased pituitary kallikrein in normal female rats by 35%. When anterior pituitary or neurointermediate lobe extracts were separately examined, immunoreactive kallikrein was 10.2- and 1.3-fold higher respectively, in female than in male rat lobes. Estradiol benzoate (30 micrograms/kg) administration increased kallikrein levels 90- and 22-fold, respectively, in the anterior pituitary of gonadectomized male and female rats, while it increased by only 40-50% kallikrein levels in the male and female neurointermediate lobe. In dot blot analysis, kallikrein mRNA levels were increased 5-fold by 17 beta-estradiol in the whole pituitary of castrated male rats. In the cytoplasmic dot hybridization analysis, estradiol benzoate treatment increased kallikrein mRNA levels 54-fold in the anterior pituitary of ovariectomized rats. The data show that a tissue kallikrein indistinguishable thus far from a urinary kallikrein is widely distributed in brain and pituitary and that levels of enzyme and mRNA are comparable in certain central sites. Kallikrein levels in the anterior and neurointermediate pituitaries are differentially regulated by estrogen.

Structure and bioactivity of the amino-terminal fragment of pro-opiomelanocortin

The primary sequence of the amino-terminal or 16 K fragment (16 K) of pro-opiomelanocortin (POMC) is highly conserved throughout the mammals. This suggests an important biological role for this peptide. We have performed studies to determine the structure, biosynthetic origin and bioactivity of this pituitary peptide. A comprehensive study of all the biosynthetic derivatives of POMC in the neurointermediate lobe of the rat and mouse pituitary was undertaken. Inspection of the amino acid composition of these peptides indicated that cleavage at all available dibasic processing sites within POMC is essentially complete except for Arg 49-Lys 50 within the 1–74 16 K fragment (16 K 1–74). Only about 50% of 16 K 1–74 was found to be processed to give rise to the extreme amino-terminal 1 to 49 sequence (16 K 1–49) and the carboxyl-terminal 50 to 74 sequence (Lys 1 γ 3 melanotropin). Sufficient 16 K 1–77 and 16 K 1–49 was purified from bovine posterior pituitaries in order to determine if there are any structural features controlling the limited degree of processing of 16 K within the intermediate lobe. Both bovine 16 1–77 and 16K 1–49 were found to have cystine bridges linking cystine residues 2 and 24 and linking cystine residues 8 and 20. While 16K 1–77 was found to be O-glycosylated at threonine 45 and N-glycosylated as asparagine 65, 16 K 1–49 was found to have no carbohydrate content. Thus the presence of O-glycosylation at threonine 45 apparently inhibits cleavage at -Arg 49-Lys 50-. Lys 1γ 3 MSH 16 K 1–74 and 16 K 1–49 purified from rat neurointermediate pituitaries were tested for their ability to potentiate the action of corticotropin (ACTH) in an isolated rat adrenal cell bioassay. None of the 16 K-related peptides showed any intrinsic steroidogenic activity. Experiments were performed in which dispersed adrenal cells were incubated with serial dilutions of ACTH. Constant amounts of test peptides were added in concentrations ranging from 10 pM to 5 nM. Lys 1 γ 3 MSH potentiated the steroidogenic activity of ACTH by up to 2-fold with an ED 50 of approx 0.5 nM. 16 K 1–49 showed no ability to potentiate the action of ACTH. In contrast the most highly glycosylated form of 16 K 1–74 potentiated the action of ACTH by up to 6-fold.

Biosynthetic fate of the amino-terminal fragment of pro-opiomelanocortin within the intermediate lobe of the mouse pituitary

All the biosynthetic derivatives of pro-opiomelanocortin (POMC) were purified from an extract of 300 mouse neurointermediate pituitaries. Inspection of the amino acid composition of these peptides indicated that cleavage at all available dibasic processing sites within POMC was essentially complete except for -Arg 49-Lys 50- within the 1 to 74 amino-terminal sequence. Only about 50% of the 1 to 74 fragment was processed to the 1 to 49 sequence and Lys 1 γ 3MSH (i.e., the 50 to 74 sequence). The existence of these derivatives of the 1 to 74 fragment was confirmed by pulse-labelling explant cultures of mouse neurointermediate pituitaries with tritiated amino acids. Pulse/chase biosynthetic experiments indicated that the cleavage of the 1 to 74 sequence takes place 3 to 6 hours post-translation. This time course of biosynthesis suggests that the cleavage of the 1 to 74 sequence is a secretory granule event. Time course studies revealed that the minimum time required for newly synthesized derivatives of POMC to emerge from the intermediate lobe tissue was approximately 3 hours.

Changes in the immunoreactivities of an opioid peptide leumorphin in the hypothalamus and anterior pituitary during the estrous cycle of the rat and their relation to sexual behavior

Leumorphin, an opioid peptide whose functions are unknown, is found in mammalian brain and pituitary and stimulates lordosis behavior in estrogen-treated female rats. To elucidate the role of leumorphin in the physiological control of female sexual behavior, the levels of immunoreactive (ir) leumorphin as well as ir dynorphin (dynorphin A) were measured in the rat brain and pituitary during the estrous cycle. There was a clear variation of ir leumorphin in the hypothalamus and anterior pituitary during the estrous cycle. The levels of ir leumorphin in the hypothalamus and anterior pituitary on the afternoon of proestrus were significantly higher ( P < 0.01) than those on the afternoons of estrus and metestrus. The rise in the hypothalamic levels of ir leumorphin on the afternoon of proestrus was correlated with the receptivity of lordosis during the estrous cycle. Furthermore, there was a close correlation with ir dynorphin levels. These findings are in agreement with studies demonstrating a common precursor for leumorphin and dynorphin. Ir leumorphin in the hippocampus and neurointermediate pituitary did not change significantly during the estrous cycle. Because the leumorphin antiserum used recognizes rimorphin (dynorphin B) 1.78 times more than porcine leumorphin on a molar basis, high performance-gel permeation chromatography was done on pooled extracts of hypothalamus taken at proestrus and estrus. The peak in the leumorphin elution position showed a clear variation similar to that of total ir leumorphin. The physiological significance of this leumorphin-like substance in the activation of sexual behavior is discussed.

Coordinate regulation of peptide acetyltransferase activity and proopiomelanocortin gene expression in the intermediate lobe of the rat pituitary.

Proopiomelanocortin (POMC) is posttranslationally processed in the intermediate lobe of the pituitary to both N-terminally acetylated and nonacetylated forms of alpha MSH and beta-endorphin (beta END). N-Acetylation substantially modifies the physiological responses produced by both peptides, suggesting that the activity of the peptide acetyltransferase, which posttranslationally acetylates beta END and des-acetyl-alpha MSH, may play an important role in defining the biological activity of the secretory products of the intermediate pituitary lobe. The present results demonstrate that peptide acetyltransferase activity is induced by treating rats chronically with the dopamine receptor antagonist haloperidol. Haloperidol administration produced parallel and essentially equivalent increases in acetyltransferase activity, POMC mRNA levels, and the content and secretion of POMC-derived peptides in the neurointermediate pituitary. Time-course and dose-response studies further demonstrated that acetyltransferase activity covaried with POMC mRNA and peptide levels. Chronic treatment with the dopamine receptor agonist bromocriptine had the opposite effects; it lowered acetyltransferase activity, POMC mRNA levels, and alpha MSH and beta END immunoreactivities. Subcellular fractionation showed that acetyltransferase activity was localized in three subcellular compartments corresponding in density to secretory vesicles, rough endoplasmic reticulum and Golgi apparatus, and cytosol. Haloperidol treatment significantly increased the specific activity of the secretory vesicle-associated acetyltransferase without affecting the specific activity of the enzymes present in the endoplasmic reticulum or cytosol. Together, these data indicate that peptide acetyltransferase activity and POMC biosynthesis are coregulated.

Met-enkephalin-like immunoreactivity in neurointermediate pituitary is decreased by DA receptor stimulation

The effect of dopamine receptor stimulation by administration of the dopamine analogue bromocriptine on Met-enkephalin-LI was examined in rat hypothalamus, and neurointermediate and anterior lobes of pituitary. Bromocriptine treatment resulted in a dramatic decline of Met-enkephalin-LI in neurointermediate pituitary which was significant by 3 days of treatment. Maximal reduction of Met-enkephalin-LI ranged between 60–70% of pretreatment values and was maintained as long as bromocriptine was administered (4 weeks), with no evidence of desensitization or “escape.” The effects of bromocriptine on neurointermediate lobe were of long duration and persisted for at least 4 days after discontinuation of treatment. No significant effects of bromocriptine were detected on Met-enkephalin-LI in hypothalamus or anterior pituitary. Whether these differences represent truly different regional regulation of Met-enkephalin-LI or whether the changes are more sensitively reflected in an area such as neurointermediate lobe that largely consists of nerve terminals, remains to be shown.

Purification and characterization of peptidylglycine alpha-amidating monooxygenase from bovine neurointermediate pituitary.

Extracts of bovine neurointermediate pituitary secretory granules and frozen bovine neurointermediate pituitary contain multiple forms of peptidylglycine alpha-amidating monooxygenase (PAM) activity differing in apparent molecular weight and in charge. Metal chelate affinity chromatography, substrate affinity chromatography, and gel filtration resulted in the purification of two forms of amidation activity from frozen bovine neurointermediate pituitary: PAM-A, apparent molecular weight 54,000, was purified 7,000-fold and PAM-B, apparent molecular weight 38,000, was purified 21,000-fold. Enzyme activity of similar molecular weights was observed in the starting material. Purified PAM-A and PAM-B correspond to two of the three charge forms present in crude extracts, and both exhibited optimal activity at alkaline pH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of PAM-B revealed the presence of two bands with apparent molecular weights of 42,000 and 37,000; autoradiography of 125I-labeled PAM-B revealed only the same two bands, and 125I-labeled PAM-B co-eluted with enzyme activity during gel filtration. PAM-A was still heterogeneous based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The properties of purified PAM-A and PAM-B were very similar to those of amidation activity in crude extracts: activity was reduced upon removal of molecular oxygen; activity was stimulated by the addition of CuSO4 and eliminated by the addition of diethyldithiocarbamate; activity was stimulated by the addition of ascorbate, with optimal levels of ascorbate increasing as the concentration of peptide substrate was increased. In the presence of 1.25 mM ascorbate, PAM-B exhibited a Km of 7.0 microM for D-Tyr-Val-Gly and a Vmax of 84 nmol/micrograms/h.

Modulation by Leu-enkephalin of peptide release from perifused neurointermediate pituitary. II. Inhibition of calcium-mediated secretion of alpha-MSH and beta-endorphin.

The present study examines the effect of opiates on alpha-MSH and beta-endorphin release from perifused neurointermediate rat pituitaries, as stimulated by various secretagogues for which Ca ions and/or cAMP serve as messengers. alpha-MSH release stimulated by high K+ concentrations (5-min pulses) and veratridine depolarization, which is closely dependent on Ca2+ fluxes, was abolished by both Leu-enkephalin and beta-endorphin. A dose-response relationship between inhibition of alpha-MSH secretion and the concentration of Leu-enkephalin, with ED50 approximately 10(-9) M, was observed. High K+-induced release of beta-endorphin was likewise blunted by Leu-enkephalin. The stimulatory effect of the Ca2+ ionophore A 23187 was inhibited in a similar way as was that of CRF, which requires both Ca2+ fluxes and cAMP formation. The antagonist naloxone not only reversed the action of opiates, but also enhanced spontaneous hormonal output. In contrast, the effects of l-isoproterenol and forskolin, for which cAMP serves as a primary messenger, were unaffected in the absence of extracellular Ca ions and, also, in the presence of Leu-enkephalin. We conclude that opioid peptides may exert a direct inhibitory influence on the release of both alpha-MSH and beta-endorphin and do so by interfering with the Ca2+ messenger system. In addition, these data also suggest the existence of an opiate-opiate negative feedback mechanism.

Posttranslational processing of proadrenocorticotropin/endorphin-derived peptides during postnatal development in the rat pituitary.

The anterior pituitary content of pro-ACTH/endorphin-related peptides increased 5-fold from birth to 4 weeks and increased another 3-fold by adulthood. In contrast, the neurointermediate lobe content of pro-ACTH/endorphin-related peptides increased 15-fold from birth to 4 weeks and another 10-fold by adulthood. Despite the dramatic increase in content, posttranslational processing of pro-ACTH/endorphin in the neurointermediate lobe of the neonate closely resembled intermediate lobe processing in the adult; alpha MSH- and beta-endorphin-sized molecules (rather than ACTH and beta-lipotropin) accounted for more than 90% of the immunoreactivity in both neonates and adults. In the neurointermediate pituitary of both the neonate and the adult, the alpha MSH-sized material was largely diacetylated, and the beta-endorphin was both alpha-N-acetylated and C-terminally shortened. However, the extent of C-terminal shortening of beta-endorphin in the neurointermediate lobe of the neonate was not as great as that observed by postnatal day 21 or that in the adult. In the anterior pituitary, distinct differences in processing occurred between birth and adulthood. Proteolytic processing of pro-ACTH/endorphin was not as extensive on day 1 as in the adult, and pro-ACTH/endorphin accounted for 40-50% of the total immunoreactive peptide. The extent of processing of precursor increased around day 21, and a higher percentage of ACTH-(1-39) and beta-endorphin-(1-31)-sized material was found. Neonatal anterior pituitary contained substantial amounts of alpha MSH-sized material, whereas in adult anterior pituitary, less than 1-2% of the ACTH-related material was alpha MSH-sized. Despite these differences in the extent of proteolytic processing, neonatal anterior pituitary corticotropes resembled those of adults, in that they did not alpha-N-acetylate beta-endorphin or alpha MSH. Immunocytochemical studies demonstrated that a subset of the neonatal anterior pituitary corticotropes produced alpha MSH-related molecules.

N α-acetyl-γ-endorphin is an endogenous non-opioid neuropeptide with biological activity

N α-acetyl-γ-endorphin (AcγE) was identified in the rat neurointermediate pituitary, based on its immunological properties, comigration with synthetic AcγE on HPLC and resistance to aminopeptidase-M degradation. The peptide appeared to be the main form of γ-endophin (γE) in this tissue and in brain areas remote from the hypothalamus (hippocampus, septum, amygdala). The anterior pituitary, the hypothalamus and the thalamus contained almost exclusively the non-acetylated form of γE. In contrast to γE, AcγE was completely devoid of specific affinity for brain opiate binding sites. Yet, the peptide mimicked γE in that if potently attenuated passive avoidance behaviour in rats, when injected topically into the nucleus accumbens. It is concluded that AcγE is an endogenous neuropeptide with non-opioid biological activity. N α-acetylation may not merely represent a mechanism for the inactivation of opioid activities of endorphins, but rather allow the organism to select specific sets of biological activities that reside in the endorphin structure.