Opioid Peptide Precursor Proenkephalin Research Articles

Proenkephalin A gene products activate a new family of sensory neuron--specific GPCRs.

Several peptide fragments are produced by proteolytic cleavage of the opioid peptide precursor proenkephalin A, and among these are a number of enkephalin fragments, in particular bovine adrenal medulla peptide 22 (BAM22). These peptide products have been implicated in diverse biological functions, including analgesia. We have cloned a newly identified family of 'orphan' G protein--coupled receptors (GPCRs) and demonstrate that BAM22 and a number of its fragments bind to and activate these receptors with nanomolar affinities. This family of GPCRs is uniquely localized in the human and rat small sensory neuron, and we called this family the sensory neuron--specific G protein--coupled receptors (SNSRs). Receptors of the SNSR family are distinct from the traditional opioid receptors in their insensitivity to the classical opioid antagonist naloxone and poor activation by opioid ligands. The unique localization of SNSRs and their activation by proenkephalin A peptide fragments indicate a possible function for SNSRs in sensory neuron regulation and in the modulation of nociception.

Expression of opioid receptors and ligands in pregnant mouse uterus and placenta.

The endogenous opioid system has been implicated in the regulation of hormonal secretion, pain perception, and uterine contractility during pregnancy, but there is only limited information about the cellular location of opioid receptor and opioid peptide gene expression in the pregnant rodent uterus and placenta. In this study, we have used in situ hybridization to identify expression sites of mRNAs encoding the delta (delta), kappa (kappa), and mu ( micro) opioid receptors as well as the endogenous opioid peptide precursors proenkephalin (PENK), prodynorphin (PDYN), and proopiomelanocortin (POMC) in pregnant mouse uterus and placenta. Soon after implantation, all three opioid receptor genes as well as POMC and PENK, but not PDYN, were detected in the uterine environment. Each expressed gene exhibited a distinct expression pattern that was generally retained until late gestation. The delta receptor and POMC were coexpressed in the trophoblast giant cells, which remained the only cells of the placenta/uterus to express these two genes throughout gestation. Cells expressing kappa receptors were absent from the placenta but instead were found in the basal part of the decidualized uterine endometrium. While kappa and micro receptors were transiently expressed in the uterine myometrium (until embryonic day 8.5), substantial levels of PENK were continuously detected in this region until at least embryonic day 18. In addition, complementary expression of the micro receptor and PENK genes in the uterus was detected. Taken together, these results suggest multiple roles for the opioid receptors and opioid peptides in maternal adaptation to pregnancy and in supporting embryo growth.

Regulation of striatal proenkephalin and prodynorphin gene expression by transcription factor CREB

There has been controversy over the precise mechanisms by which neural stimuli acting via cAMP and Ca 2+ pathways regulate expression of the genes encoding the opioid peptide precursors proenkephalin and prodynorphin. Evidence has been adduced for both AP-1 proteins, including Fos, and CREB-like proteins as the key factors transducing signals to the DNA. Here we present evidence from in vivo systems that CREB, rather than Fos, is likely the key protein regulating cAMP responsiveness of these two opioid genes. In addition, we show that CREB is phosphorylated in vivo in response to stimuli known to regulate these opioid genes.

Proenkephalin products are stored in the sperm acrosome and may function in fertilization.

Previous studies have shown that spermatogenic cells are a major source of testicular RNA encoding the opioid peptide precursor proenkephalin, suggesting that proenkephalin-derived peptides may function as intratesticular paracrine factors produced by male germ cells. However, direct evidence for the production of proenkephalin by spermatogenic cells has been lacking. In this report, we have used polysome profile analysis, peptide quantitation, and immunocytochemistry to show that proenkephalin products are synthesized during spermatogenesis and are retained within spermatozoa of humans, hamsters, rats, and sheep. We further show that these peptides are stored in the sperm acrosome and are depleted from sperm following the acrosome reaction, an exocytotic event required for fertilization. Proenkephalin products thus may serve a dual function as sperm acrosomal factors released during the fertilization process as well as intratesticular regulators secreted by spermatogenic cells.

Translational Status of Proenkephalin mRNA in the Rat Reproductive System

The mRNA for the opioid peptide precursor proenkephalin is widely distributed throughout the male and female reproductive systems of rodents. In the present studies, the concentrations of proenkephalin-derived peptides in selected reproductive tissues of the rat have been determined. When compared with previously characterized tissues such as brain, the peptide contents in reproductive tissues were unexpectedly low relative to the abundance of proenkephalin mRNA. This suggested that either translation of proenkephalin mRNA is relatively inefficient in reproductive tissues or that the turnover of proenkephalin products occurs at a higher rate, or both. To distinguish between these possible mechanisms, the polysomal distributions of proenkephalin mRNA in different rat reproductive tissues and in rat brain were determined. In adult rat testis, in which the predominant proenkephalin RNA is the 1700-nucleotide form present in spermatogenic cells, the transcript was found to be mainly associated with translationally inactive ribonucleoprotein fractions. In contrast, the 1450-nucleotide form of proenkephalin mRNA appeared to be translated to a similar extent in rat brain, epididymis, ovary, and somatic cells of the immature rat testis. It therefore appears that inefficient translation of proenkephalin mRNA in spermatogenic cells is a major determinant of the low ratio of proenkephalin peptides to RNA in the adult rat testis, while posttranslational mechanisms (most likely peptide turnover) are involved in the rat epididymis, ovary, and presumably other reproductive tissues. These findings also indicate that mRNA and/or translation product concentrations within a given tissue are not always accurate indicators of the level of peptide or protein production.

N-linked glycosylation of a proenkephalin A-derived peptide. Evidence for the glycosylation of an NH2-terminally extended Met-enkephalin Arg6-Gly7-Leu8 variant.

To investigate the possibility that the opioid peptide precursor proenkephalin A was glycosylated, we utilized an antiserum raised against the COOH terminus of Met-enkephalin Arg6-Gly7-Leu8 (MERGL) to identify and characterize enkephalin-containing peptides from extracts of bovine adrenal medulla. Sephadex G-50 gel filtration separated two immunoreactive peaks which had apparent masses of 9 and 6 kDa. Anion-exchange chromatography and reverse-phase high pressure liquid chromatography (HPLC) revealed that the 9-kDa material was a heterogenous mixture of immunoreactive peptides, of which one (9K-MERGL Ia) was purified to homogeneity. The 6-kDa material separated into two major immunoreactive peaks (6K-MERGL I and 6K-MERGL II) on anion-exchange chromatography, and these were obtained in an homogenous form after reverse-phase HPLC. Amino acid sequencing, together with immunological characterization, indicated that the three peptides were identical in chain length, and corresponded to proenkephalin A 116-165. They contained the sequence Asn-Ser-Ser which is a potential N-glycosylation site. In 9K-MERGL Ia, but not the others, automated Edman amino acid sequencing was unable to detect the relevant asparagine residue, suggesting that this residue has been chemically modified. Further investigation of the 9K-MERGL material using lectin affinity chromatography provided direct evidence of glycosylation. Verification of this result was obtained using the specific enzyme glycopeptidase F (glycopeptide-N-glycosidase) which demonstrated that 9K-MERGL contained, in part, N-linked oligosaccharide chains. These results show that an NH2 terminally extended Met-enkephalin Arg6-Gly7-Leu8 variant was N-glycosylated, and hence indicate that the precursor polypeptide proenkephalin A can be glycosylated during translation in the rough endoplasmic reticulum.

Proenkephalin A gene expression in bovine adrenal chromaffin cells is regulated by changes in electrical activity.

Concentrations of mRNA coding for the opioid peptide precursor proenkephalin A (mRNAENK) were measured in primary cultures of bovine adrenal chromaffin cells maintained in serum-free medium. Using a sensitive solution hybridization assay, an increase in mRNAENK levels from 45 to 300% above control with K+ (10-20 mM), Ba2+ (1 mM) and veratridine (5 microM) was found. The highest increase (300% above control) was obtained with the Na+ channel agonist veratridine. This effect was nearly abolished in the presence of the Na+ channel antagonist tetrodotoxin (TTX) (1 microM). Moreover, TTX partially inhibited the increase in mRNAENK levels caused by K+ (20 mM) depolarization (from 185 to 130% of control), but had no effect on the stimulation by Ba2+ (1 mM). The Ca2+ channel antagonists D600 (50 microM) verapamil (50 microM) and Co2+ (1 mM) inhibited the responses to either K+, Ba2+ or veratridine, whereas the Ca2+ channel agonist Bay K 8644 (0.1 microM) potentiated the effect of 20 mM K+ from 185 to 230% of control. The K+-induced increase in the mRNAENK levels was associated with an increase of immunoreactive proenkephalin A-derived peptides in both tissue and medium, indicating an enhanced production of opioid peptides. These results suggest that membrane depolarization may play an important role in the regulation of proenkephalin A gene expression in bovine adrenal chromaffin cells. It may represent a mode by which substances acting directly on Na+ or Ca2+ channels may modulate the regulation of proenkephalin A mRNA biosynthesis and opioid peptide production.

Isolation and structure of a C-terminally amidated nonopioid peptide, amidorphin-(8-26), from bovine striatum: a major product of proenkephalin in brain but not in adrenal medulla.

We have isolated and sequenced a C-terminally amidated peptide from bovine striatum. The peptide was purified to homogeneity by adsorption to XAD-2 resins and four different HPLC steps. Amino acid composition analysis and gas-phase sequence analysis revealed identity of this peptide with residues 8-26 of the proenkephalin-derived opioid peptide amidorphin, which we have recently isolated from bovine adrenal medulla. C-terminal amidation of amidorphin-(8-26) from bovine striatum was demonstrated by its stability to carboxypeptidase A digestion and full crossreactivity in a radioimmunoassay that required the C-terminal amide group as part of the recognition site. The nonopioid peptide amidorphin-(8-26), which lacks the N-terminal [Met]enkephalin sequence of amidorphin, is a major product of the opioid peptide precursor proenkephalin in the brain. In the adrenal medulla, however, where amidorphin occurs in remarkably high concentrations, amidorphin-(8-26) could not be detected. This is indicative of differential post-translational processing of proenkephalin in different tissues. In the brain, as opposed to the adrenal medulla, amidorphin is further processed at the typical cleavage signals of two basic residues, giving rise to the nonopioid peptide amidorphin-(8-26) and, possibly, to the opioid peptide [Met]enkephalin. Thus, proenkephalin in the brain might be considered as a precursor in which an opioid peptide is linked with a nonopioid peptide of possibly different biological function.

Reptilian enkephalins: Implications for the evolution of proenkephalin

To shed light on the evolution of the opioid peptide precursor proenkephalin, we have investigated the profile of immunoreactive enkephalins in reptilian brain using six antisera directed toward different portions of the proenkephalin molecule. Three different orders of reptiles—turtles, alligators, and lizards—were studied; these orders represent lineages diverging more than 250 million years ago. Reptilian brain was found to contain large quantities of Met 5-enkephalin (Met 5-enk), Leu 5-enkephalin (Leu 5-enk), and Met 5-enk-Arg 6-Phe 7 (MERF); gel filtration and reverse-phase chromatography of acid extracts prepared from turtle brain indicated that nearly all of the immunoreactivity corresponding to these peptides could be ascribed to the authentic penta- to heptapeptides. Immunoreactive metorphamide, but not amidorphin, was present in all three species. Unlike mammalian brain, reptilian brain does not contain immunoreactive Met 5-enk-Arg 6-Gly 7-Leu 8 (MERGL). These results indicate that reptilian proenkephalin, while similar to mammalian enkephalin in containing Met 5-enk, Leu 5-enk, MERF, and metorphamide, nevertheless differs from mammalian proenkephalin. Reptilian proenkephalin either may contain the MERGL sequence in a form not recognized by the MERGL antibody, or may lack the MERGL sequence entirely.

Quantitation of proenkephalin A messenger RNA in bovine brain, pituitary and adrenal medulla: correlation between mRNA and peptide levels.

Concentrations of mRNA coding for the opioid peptide precursor proenkephalin A were measured in bovine brain areas, pituitary and adrenal medulla. In all tissues, a single hybridizable species of 1400 bases in size was found by Northern blot analysis using as a probe a single-stranded (ss) cDNA complementary to bovine proenkephalin A mRNA. In solution hybridization experiments the distribution of the mRNA was quantified. Considerable differences were found for the abundance of proenkephalin A mRNA in the various tissues: from 0.023% in the adrenal medulla to 0.00002% in the adenohypophysis. Relative abundance in the various brain areas varied greater than 20-fold, being highest in the caudate nucleus (0.0025%) and lowest in the thalamus and substantia nigra (0.0001%). Comparison with immunoreactive peptide concentrations in these tissues showed a close correlation between the levels of proenkephalin A mRNA and the immunoreactive peptides.

Isolation and structure of a novel C-terminally amidated opioid peptide, amidorphin, from bovine adrenal medulla.

Biologically active peptide hormones and neurotransmitters have been shown to be enzymatically liberated from larger, inactive precursor molecules by tissue-specific post-translational processing, particularly at the typical cleavage signals of paired basic residues. Subsequent N-terminal or C-terminal modifications may be of importance in regulating the biological activities of these peptides. C-terminal alpha-amidation is considered to be essential for the biological function of several non-opioid peptides. Here we present the isolation and structure of a novel C-terminally amidated opioid peptide, amidorphin, from bovine adrenal medulla. Amidorphin and the recently isolated octapeptide metorphamide (adrenorphin) are the only endogenous opioid peptides in mammals known to possess a C-terminal amide group. The amino acid sequence of amidorphin corresponds to the sequence 104-129 of bovine proenkephalin A. Very high concentrations of amidorphin were detected in bovine adrenal medulla and in a further endocrinological system, the hypothalamic-neurohypophyseal axis. Amidorphin may therefore be considered to be a major gene product of the opioid peptide precursor proenkephalin A in these endocrine tissues.

Evidence for a differential postnatal development of proenkephalin B (= prodynorphin)-derived opioid peptides in the rat hypothalamus.

[he concentrations of immunoreactive (ir-) peptides derived from the opioid peptide precursors proenkephalin A (Met-enkephalin), proenkephalin B [dynorphin (DYN)-(1-17), dynorphin-(1-8), dynorphin B, alpha-neoendorphin (alpha-NEO-E), beta-NEO-E] and proopiomelanocortin [beta-endorphin (beta-END)], and of the neurosecretory hormones vasopressin and oxytocin increased between approximately 10-fold and 50-fold from birth to adulthood in the rate hypothalamus. Gel filtration and HPLC analysis of proenkephalin B-derived opioid peptides revealed that in 3-day-old rats the predominant portion of ir-dynorphin-(1-17) and a substantial part of ir-dynorphin B consisted of a high (6000) mol wt species, a common precursor peptide for DYN-(1-17) and DYN B. In adults rats, however, authentic DYN-(1-17) and DYN B were found to be the major ir-forms. The mol wt patterns of ir-DYN-(1-8), ir-alpha-NEO-E and ir-beta-NEO-E did not differ between 3-day-old and adult rats and reflected predominantly the respective authentic opioid peptides. Taking into consideration the developmental changes in the mol wt pattern of ir-DYN-(1-17), authentic DYN-(1-17) was 5 times lower in concentration than DYN-(1-8) in 3-day-old rats, whereas in adults these opioid peptides occurred in equimolar concentrations. These findings suggest that the posttranslational processing of the precursor proenkephalin B changes in the course of postnatal development. Ir-beta-END in the hypothalamus of newborn and adult rats consisted exclusively of beta-END-sized peptides which were not (unlike those in the intermediate pituitary lobe) alpha-N-acetylated. Thus, in the hypothalamus, the enzymatic processing of the opioid peptide precursor proopiomelanocortin to beta-END seems to be fully active at birth, in contrast to that of proenkephalin B.

Proenkephalin B (prodynorphin)-derived opioid peptides: evidence for a differential processing in lobes of the pituitary.

The distribution of peptides derived from the novel opioid peptide precursor proenkephalin B (prodynorphin) was studied in lobes of the pituitary with antibodies against alpha-neoendorphin (alpha-neo-E) beta-neoE, dynorphin (DYN)-(1-17), DYN-(1-8), and DYN B in combination with gel filtration and high pressure liquid chromatography. In the posterior pituitary, all five opioid peptides occurred in high and about equimolar concentrations, whereas putative precursor peptides were found in only minor quantities. In contrast, in the anterior pituitary immunoreactive (ir-) DYN-(1-17) and ir-DYN B consisted exclusively of a common precursor species with a mol wt of about 6000. Six thousand-dalton DYN may be comprised of the C-terminal portion of proenkephalin B, with the sequence of DYN-(1-17) at its N-terminus. Moreover, the major portions of ir-alpha-neo-E and ir-beta-neoE in the anterior pituitary were found to be of an apparent mol wt of 8000. These findings indicate a differential processing of the opioid peptide precursor proenkephalin B in the two lobes of the pituitary. The anterior pituitary seems to process proenkephalin B predominantly into high mol wt forms of neo-E and DYNs, whereas in the posterior pituitary proenkephalin B undergoes further proteolytic processing to the smaller opioid peptides alpha-neo-E, beta-neo-E, DYN-(1-17), DYN-(1-8), and DYN B. Thus, processing differences may enable the selective liberation of different (opioid) peptides with distinct biological properties from one precursor within different tissues.