Elevated proenkephalin-derived peptide levels in ACTH-producing adenomas: nucleus and cytoplasm localization.

The biosynthesis of met-enkephalin in human pituitary and human pituitary adenomas is still not well known. In this work, we studied the processing of proenkephalin-derived peptides in postmortem human pituitary (PMHP), ACTH-producing adenomas (ACTH-PA), nonfunctioning adenomas (NFA), and GH-producing adenomas (GH-PA). ACTH-PA contained at least 10 times more proenkephalin-derived peptides than PMHP, NFA,and GH-PA. Proenkephalin processing was different in the four tested tissues. In ACTH-PA, proenkephalin was processed to high-, intermediate-, and low-mol-wt products. The highest met-enkephalin-containing peptides levels corresponded to intermediate and low-mol-wt materials, although met-enkephalinArg-Phe and synenkephalin immunoreactivity appeared only in high-mol-wt peptides. In PMHP and NFA, met-enkephalin-Arg-Phe immunoreactivity was detected in intermediate- and low-mol-wt materials, and it was absent in GH-PA. Immunoblotting of ACTH-PA showed that met-enkephalin-Arg-Phe immunoreactivity corresponded to peptides of 44, 32-30, 27, and 17 kDa. The 32-30 and 17-kDa molecules were localized in the nuclear fraction where they were extracted after enzymatic digestion with DNase I. Plasmatic met-enkephalin levels did not increase in patients with Cushing's disease, suggesting that the pentapeptide stored in ACTH-PA was not released to the general circulation. In conclusion, we demonstrated that only ACTH-PA contained high levels of proenkephalin peptides, which were stored in cytoplasm organelles and in the nucleus, probably bound to chromatin. These results suggest an adenoma-specific physiological role of proenkephalin products.

Differential response to a stress stimulus of proenkephalin peptide content in immune cells of naive and chronically stressed rats.

Proenkephalin peptides produced by endocrine and nervous tissues are involved in stress-induced immunosuppression. However, the role of peptides produced by immune cells remains unknown. The present study examines the effect of acute and chronic foot-shock stress on proenkephalin peptide content in bone marrow (BMMC), thymus (TMC), and spleen (SMC) rat mononuclear cells. Proenkephalin was not processed to met-enkephalin in BMMC, while in TMC and SMC met-enkephalin represented 10% and 26% of total met-enkephalin-containing peptides, respectively. Naive rats receiving a stress stimulus showed a significant decrease of proenkephalin derived peptides in BMMC, TMC and SMC. However, in chronically stressed rats that already showed basal low peptide levels, a new stress stimulus produced a differential response in each immune tissue. That is, in BMMC peptide levels reached control rats values; in TMC remained unmodified; and in SMC, although precursors content increased, met-enkephalin levels were even lower than those observed in acutely stressed rats. Free synenkephalin content paralleled met-enkephalin changes in SMC of acutely and chronically stressed rats. The in vitro release of met-enkephalin and free synenkephalin increased in SMC of stressed rats. Met-enkephalin produced in SMC and partially processed proenkephalin peptides detected in BMMC, were only found in macrophages. However, met-enkephalin only appeared in bone marrow macrophages after at least 4 h of cell culture. Altogether, these results suggest that a stress stimulus induced proenkephalin peptide release from immune tissue macrophages. The differential response observed in chronically stressed rats suggest an alternative activation of heterogeneous proenkephalin-storing macrophage subpopulations.

Antibodies against the amino-terminal portion of pro-enkephalin inhibit DNA synthesis in human peripheral mononuclear cells.

Pro-enkephalin (PENK) mRNA and PENK-derived peptides have been reported in lymphocytes, monocytes, and macrophages. Met-enkephalin (ME) and/or synenkephalin (SYN)-containing peptides are produced and released by human peripheral blood lymphocytes (HPBL) activated with phytohemagglutinin (PHA). Furthermore, SYN (PENK 1-70) was cleaved to low-molecular-mass peptides in HPBL. In this work we studied the effect of a mouse monoclonal antibody (mAb) and a rabbit antiserum (pAb) against the C-terminal portion of SYN on DNA synthesis in PHA-activated HPBL. [3H]Thymidine incorporation into HPBL incubated with 0.1 microgram/ml of PHA was tested in the presence of different concentrations of mAb immunoglobulin (Ig) G or different dilutions of pAb. mAb induced a concentration-dependent decrease of [3H]thymidine incorporation into HPBL: 7%, 19%, 28%, and 35% of inhibition was observed with 0.1, 1, 1.5, and 2 micrograms IgG, respectively, reaching values of 65% with 10 micrograms IgG. Similarly, pAb dilutions of 1/500, 1/1000, 1/2000 and 1/4000 inhibited DNA synthesis by 63%, 61%, 43%, and 30%, respectively. The inhibitory effect of mAb and pAb was specific since it was not produced by non-immune mouse IgG or several non-immune rabbit sera and was completely reversed by 1 microM of the synthetic peptide [Tyr63](syn 63-70) synenkephalin. These results suggest that low-molecular-mass SYN-derived peptides released by PHA-activated HPBL may participate in the proliferative response of these cells. This is further evidence that the non-opioid portion of PENK--that is, SYN-derived peptides--may be involved in tissue development.

Prohormone convertases PC2 and PC3 in rat neutrophils and macrophages. Parallel changes with proenkephalin-derived peptides induced by LPS in vivo.

Prohormone- or proneuropeptide-converting enzymes PC2 and PC3 have been observed exclusively in nervous and endocrine tissues. In this work the presence of these enzymes in cells of the immune system was demonstrated. PC2 was detected in peripheral and liver-infiltrating polymorphonuclear leukocytes (PMN) but not in alveolar macrophages (AM) or spleen mononuclear cells (SMC). PC2 proteins corresponded to 75, 71 and 56 kDa forms. PC3 appeared in AM and SMC but not in PMN, and a 66 kDa protein was the only PC3 form detected. Proenkephalin-derived peptides (PENKp) were observed in PMN and AM, showing peptides of 35, 28, 21, 18 and 14 kDa in the former cells and a doublet of 35 and 32 kDa in the latter. PC2 proteins and PENKp decreased in liver PMN and peripheral PMN 90 min after intravenous (i.v.) infusion of LPS, suggesting an increased release. However, in vitro assays showed that the chemotactic peptide FMLP but not LPS increased the basal secretion of PC2 proteins and PENKp in PMN. These results indicate that PC2 proteins are released from PMN, together with PENKp, and suggest that LPS in vivo may act through an indirect mechanism. Low levels of PC3 and PENK were detected in the AM of rats treated for 90 min with SAL or LPS. However, a significant increase of PC3 and PENKp appeared 30 h after LPS infusion. These results show for the first time that PC2 and PC3 are differentially expressed in PMN and AM, respectively, which were paralleled by the presence of different post-translational products of PENK. In addition, the in vivo effect of LPS on PC2, PC3 and PENKp levels in PMN and AM resembles the effect of LPS on prohormone levels in endocrine tissues, suggesting that similar mechanisms may control the turnover of PENK in endocrine and in these immune cells.

Valproic acid-induced rapid changes of met-enkephalin levels in rat brain. Probable association with abstinence behavior and anticonvulsant activity.

Valproic acid (VPA) induces abstinence behavior and analgesia and displays an anticonvulsant effect, but its exact mechanism of action is not yet clear. In order to view whether proenkephalin derived-peptides are involved in the mechanism of VPA-induced behavior, we analyzed immunoreactive-met-enkephalin (IR-ME) in rat striatum, midbrain, and amygdala 10, 20, and 45 min after i.p. injection of 200 mg/kg of VPA. VPA induced body shakes that peaked within 5 to 10 min. IR-ME increased in the striatum and decreased in the midbrain at 10, 20, and 45 min, reaching the highest and lowest levels at 10 and 20 min, respectively. No changes occurred in the amygdala. Gel filtration chromatography followed by HPLC of striatum extracts showed that the increased IR-ME levels corresponded to low molecular weight peptides, including ME. These results indicate that VPA produced rapid changes of IR-ME levels in rat brain and suggest peptide participation in the mechanisms of VPA-induced behavior. The anticonvulsant effect of VPA was tested in rats treated with pentylenetetrazol (70 mg/kg) 30 min after VPA (400 mg/kg) administration, and IR-ME was analyzed in striatum 15 min later. No changes in striatal IR-ME levels occurred in protected rats (no behavioral convulsions), compared with those treated only with VPA, but a significant decrease appeared in unprotected animals (clonic convulsions). These results suggest that striatal ME may participate in the mechanism of VPA-induced abstinence behavior and in the anticonvulsant effect. Otherwise, midbrain ME might be involved in other VPA behaviors such as analgesia.

Rapid cleavage of the endogenous PC3 prosegment and slow conversion to 74 kDa and 66 kDa proteins in AtT-20 cells.

AtT-20 cells synthesize 87 kDa and 66 kDa forms of the prohormone convertase PC3 (also known as PC1). In the present work, using biosynthetic labeling experiments (performed both at 20 degrees C and at 37 degrees C), followed by immunoprecipitation with aminoterminally and carboxyterminally-directed antisera, we have found that the first PC3 translational product was a 94 kDa protein that was then converted to an 84 kDa form. This processing was extremely rapid, occurring with a half-life of less than 2 min at 20 degrees C. The 84 kDa form was endoglycosidase H-sensitive, indicating a lack of acquisition of sugar transferred in the medial golgi. Dithiothreitol, a reducing agent that prevents the disulfide bond formation of newly synthesized proteins in the endoplasmic reticulum (ER), inhibited the processing of the 94 kDa to the 84 kDa form. However, brefeldin A (BFA), an inhibitor of ER/golgi transport, and monensin, an inhibitor of the medial/trans-golgi transport, did not affect the cleavage of the 94 kDa to the 84 kDa protein. The 84 kDa protein was converted to an endoglycosidase H-resistant form of 87 kDa that was sequentially processed to 74 kDa and 66 kDa proteins. The 87 kDa protein was immunoprecipitated by the PC3 aminoterminally and carboxyterminally-directed antisera, while the 74 kDa and 66 kDa protein were only detected with the aminoterminally-directed antibody. Radiosequencing of the 87 kDa and 66 kDa proteins indicated that the biosynthesis of the 87 kDa proteins involves the removal of the 83 amino acid prosegment, and that the processing of the 87 kDa to 66 kDa form occurred by cleavage at the carboxyterminal portion. BFA and monensin effectively interrupted the processing of the 84-87 kDa protein to the 74 and 66 kDa species. In addition, while the 84-87 kDa protein produced in monensin-treated cells was still sensitive to endoglycosidase H, the 66 kDa protein was resistant to this enzyme. These results indicate that the post-translational processing of PC3 occurs in three steps: (1) rapid conversion, probably in the ER, of the 94 kDa precursor to the 84 kDa protein by removal of the aminoterminus prosegment; (2) cleavage of the 87 kDa protein to an intermediate product of 74 kDa; and (3) production of the 66 kDa protein. The second and third steps occur in late cellular compartments such as the trans-golgi network or secretory granules and involve sequential cleavages at the carboxyterminus.(ABSTRACT TRUNCATED AT 400 WORDS)

[Prolactin, a link between the neuroendocrine and immune systems. Role in the pathogenesis of rheumatic diseases]. / La prolactine, un lien entre le système neuro-endocrinien et le système immunitaire. Rôle dans la pathogénie des maladies rhumatismales.

Increasing evidence suggest that prolactin (PRL) has important immunoregulatory properties and may play a role in the pathogenesis and disease expression of certain autoimmune diseases. Prolactin is co-mitogenic on murine and human lymphocytes, induces the formation of IL-2 cell surface receptor and modulates the expression of various growth factor related genes. Prolactin also stimulates antibody production both in vivo and in vitro. Specific high affinity prolactin-receptors have been described on T and B lymphocytes as well as on monocytes. They are distributed on heterogeneous lymphocyte subsets and they showed imbalance in autoimmune situations. Lymphocytes may produce PRL-like proteins biologically active that function as autocrine growth factors for lymphoproliferation. Hyperprolactinemia has been found in male patients with systemic lupus erythematosus (SLE) and also during pregnancy in SLE patients. Hyperprolactinemia is correlated with clinical and serological activity in a subset of SLE patients. High levels of PRL aggravates disease activity and accelerates mortality in the B/W mouse model of SLE. In rheumatoid arthritis an excessive and upregulated secretion of PRL has been shown. Hyperprolactinemia has also been shown in a subset of patients with primary Sjögren's syndrome. High PRL levels have been found in Reiter's syndrome patients and bromocriptine treatment has been reported effective in these patients and psoriatic arthritis patients. These data support a potential role of this immunoregulatory hormone in the pathogenesis of some rheumatic diseases.

Synenkephalin processing in embryonic rat brain.

Synenkephalin (proenkephalin 1-70) is produced and secreted as an intact molecule or as a part of precursors in the adult brain and adrenal medulla, respectively. However, it is cleaved to low molecular weight peptides in proliferating immune cells. Considering that the pre-proenkephalin gene is expressed in the embryonic rat brain during the cell proliferation stage, we studied the processing of synenkephalin in embryonic rat brains (E18) and compared it with the processing in adult rat brains. IR-synenkephalin was measured by RIA using a C-terminally directed antiserum. Adult rat brains contained higher concentrations of immunoreactive (IR)-synenkephalin (2,612 + 264) than embryonic rat brain (1,361 + 100) (results in fmol/mg proteins, n = 5). Gel filtration chromatography (Sephadex G-50) showed that in the extracts of adult rat brain, 50% of the IR-synenkephalin eluted in the position of the authentic peptide (8 kDa) and the rest of the immunoreactivity corresponded to partially processed peptides of 4.0 and 2.5 kDa. In embryonic rat brains synenkephalin was processed to intermediate peptides of 2.5, 1.7 and mainly to a low molecular weight peptide of 1.0 kDa. The concentration of this last peptide, which was further characterized by affinity column and HPLC, represented 45% of the total immunoreactivity. IR-met-enkephalin in embryonic rat brains (analyzed before and after enzymatic digestion with trypsin and carboxypeptidase B) corresponded principally to non-processed or partially processed products. However, these were cleaved to free met-enkephalin in adult rat brains.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of response of proenkephalin A and sympathetic nervous system in chronic pain associated with lung cancer.

Since the discovery of the link between peripheral endogenous opioid peptides and pain regulation, these substances have been studied in relation to certain pain conditions. In order to elucidate the effect of chronic pain on both peripheral opioid system and sympathetic nervous activity, we assayed plasma met-enkephalin (ME), neutrophil met-enkephalin containing peptides (NMECP) and plasma free and conjugated catecholamines (CA) in lung cancer patients with chronic pain related to bone metastases and without pain. No significant difference was found in ME levels when the pain cancer group (0.36 +/- 0.06 pmol/ml) was compared to the pain-free group (0.37 +/- 0.04 pmol/ml); results were similar for NMECP levels (14.1 +/- 1.66 pmol/mg prot and 18.41 +/- 1.93 pmol/mg prot, respectively). CA levels in both groups were also similar. These results differ from those we have reported previously for acute pain, suggesting that a non-permanent painful stimulus may be necessary for peripheral opioid system stimulation.

Immunocytochemical localization of the neuropeptide-synthesizing enzyme PC1 in AtT-20 cells.

The subtilisin-like enzyme PC1 (also known as PC3) cleaves the neuropeptide precursor proopiomelanocortin at paired basic residues in transfection experiments, thus providing evidence for a critical role in precursor processing. While mRNA for this enzyme is highly enriched in neuroendocrine tissues, little is known about the tissue and subcellular distribution of the PC1 protein. This study used immunocytochemical techniques to investigate the anatomical distribution of PC1, both alone and compared to met-enkephalin (MET-enk), in AtT-20 pituicytes transfected with proenkephalin cDNA. A high density of PC1 immunostaining was observed in a small region adjacent to the nucleus and in the tips of the processes of these cells. Dual-staining immunocytochemistry of whole cells illustrated that both PC1 and MET-enk immunoreactivity were present in the tips, but PC1 was concentrated in a region adjacent to the nucleus while MET-enk punctate staining was dispersed throughout the soma. This codistribution was confirmed in semithin sections of dual-stained cells cut at 1-1.5 microns through the thickness of the cells. PC1 staining resembled that of TGN38, a marker for the trans-Golgi network. When PC1 immunocytochemistry was performed in cells that were pretreated with brefeldin A, a drug that redistributes the proximal Golgi compartments to the endoplasmic reticulum, there was a complete disruption of the defined locus of PC1 immunoreactivity. Taken together, our data indicate that (1) PC1 is concentrated in a region of the cell body resembling the trans-Golgi network and (2) both the enzyme and the processed peptide are transported to the tips of the processes.(ABSTRACT TRUNCATED AT 250 WORDS)

Release of the prohormone convertase PC1 from AtT-20 cells.

AtT-20 cells are known to synthesize two molecular weight forms of the prohormone converting enzyme PC1 with molecular masses of 87 and 66 kDa. In this study we have analyzed basal and stimulated secretion of these proteins. Western blot results show that basal secretion medium of cultured AtT-20 cells contained low concentrations of both the 87 and 66 kDa forms of PC1 with the former protein predominant. During the stimulation period with CRF, cAMP and cAMP + BaCl2, increased release of both proteins was observed, but the 66 kDa protein predominated. Secretion medium obtained from stimulated and unstimulated cells was enzymatically active against the Cbz-Arg-Ser-Lys-Arg-AMC fluorogenic substrate as well as against 35S-proenkephalin. This activity was Ca+2 dependent and was inhibited by the chelating agent EDTA. The activity was insensitive to acid and thiol proteinase inhibitors as well as to N-alpha-p-tosyl-L-Lys-chloromethyl ketone; it was slightly sensitive to phenylmethyl sulfonyl fluoride and was strongly inhibited by D-Tyr-Ala-Lys-Arg-chloromethyl ketone. This inhibitor profile exhibits strong similarities to furin and kexin. After partial purification of medium by gel filtration chromatography, a portion of the enzymatic activity and immunoreactivity for both 87 kDa and 66 kDa proteins eluted with an apparent molecular weight of 400 kDa (suggesting aggregation); however the highest activity appeared in the elution position of the 66 kDa monomer. When the 87 kDa protein was removed from the medium by means of an affinity column containing an antibody against the carboxyl terminal portion of PC1, the column flow-through, which included the 66 kDa protein, still remained enzymatically active. These data support the notion that the 66 kDa protein, which is the most concentrated PC1 product stored in AtT-20 cells and is released during stimulation, is enzymatically active.

Differential posttranslational processing of proenkephalin in rat bone marrow and spleen mononuclear cells: evidence for synenkephalin cleavage.

Proenkephalin (PENK) messenger RNA was reported to be present in bone marrow mononuclear cells (BMMC) and spleen mononuclear cells (SMC). Nevertheless, the pattern of PENK products in normal cells of the rat immune system, which is important for defining the physiological role of PENK gene expression, has not been well established. In this work we have characterized the processing of the opioid portion (met-enkephalin-containing peptides) and nonopioid portion (synenkephalin-derived peptides) of PENK in rat BMMC and SMC. Met-enkephalin-containing peptides were detected in mononuclear cells of both hematopoietic tissues. In BMMC, free immunoreactive (IR)-met-enkephalin corresponded only to the 15% of total met-enkephalin-IR, whereas in SMC it represented the 66.5%. Gel filtration chromatography showed that BMMC contained partially processed PENK-derived peptides of high and intermediate molecular weight, whereas SMC displayed fully processed products containing met-enkephalin and/or the carboxyterminal portion of synenkephalin. HPLC purification of low molecular weight products showed that free IR-met-enkephalin in SMC mainly corresponded to met-enkephalin and oxidized met-enkephalin. In addition we have characterized in SMC three peptides lower than 3.0 kilodalton containing the C-terminal sequence of synenkephalin. These peptides were purified by gel filtration, affinity chromatography, ion exchange chromatography, and HPLC. These results show that PENK was processed in mononuclear cells of the primary (bone marrow) and secondary (spleen) organs of the rat hematopoietic system, as occurs in neural and endocrine tissues. Nevertheless, the precursor was cleaved only in the latter tissue to low molecular weight peptides. Furthermore we demonstrated that synenkephalin (proenkephalin 1-70) in SMC was processed to low molecular weight peptides containing the C-terminus free. This last result suggests that a dibasic Lys-Lys and monobasic (Lys) sites were cleaved.

Early complete maturation of proenkephalin processing induced by dexamethasone in the adrenal gland of neonatal rats.

We have previously found that proenkephalin processing is incomplete in the neonatal rat adrenal medulla and have postulated that immaturity of either the nervous input to the gland or the endocrine hypothalamus-pituitary-adrenal axis might be involved in the failure of the gland to yield free met-enkephalin. Therefore, we investigated whether cholinergic and glucocorticoid agonists may act in vivo on neonatal proenkephalin processing; reserpine, a strong activator of precursor cleavage, was also tested. Acute administration of nicotine, pilocarpine and reserpine to 24-hour-old rats increased the content of enkephalin-containing peptides (ECP) after 72 h (4-day-old rats) and activated the posttranslational processing of proenkephalin to high, intermediate and low molecular weight peptides respectively, although free met-enkephalin was not produced. Chronic treatment with nicotine and pilocarpine neither modified the concentration of ECP nor were able to induce free metenkephalin production. Chronic administration of dexamethasone increased ECP levels in the adrenal of 4-day-old rats and caused proenkephalin processing to intermediate- and low-molecular-weight products including the production of free met-enkephalin. These results indicate that only dexamethasone was able to induce the production of met-enkephalin in the adrenal of neonatal rats, suggesting an involvement of the hypothalamus-pituitary-adrenal axis in the proteolytic maturation of proenkephalin during the ontogeny of rat adrenal medulla.

Biosynthesis of the prohormone convertase mPC1 in AtT-20 cells.

A new family of mammalian subtilisin-like enzymes, probably involved in the processing of proproteins in regulated and constitutive cells at paired basic residues, has recently been discovered. Little information exists as yet concerning the biosynthesis of these endogenous subtilisin-like enzymes. In the present work the biosynthesis and release of the endogenous prohormone convertase PC1 in AtT-20 cells were studied. As predicted from mRNA studies, AtT-20 cells contain high levels of PC1 protein. Through immunoblotting, 87-kilodalton (kDa) and 66-kDa bands were detected with an amino terminally directed antiserum; however, only the 87-kDa product was detected with carboxyl terminally directed antiserum, indicating carboxyl terminal truncation. Pulse-chase experiments, using [35S]methionine/cysteine, showed that after 20 min pulse the main product in the cells was the 87-kDa protein. Cells chased for varying amounts of time exhibited a progressive increase in the intensity of a 66-kDa band, along with a corresponding decrease of the 87-kDa band. The 87-66 kDa conversion was nearly complete after 4 h of chase. This posttranslational processing was inhibited by the ionophore monensin, a Golgi disruptor, with a corresponding accumulation of the 87-kDa protein within the cell. Both the 87 kDa- and 66 kDa-labeled proteins were detected as membrane-bound rather than soluble proteins. The 87-kDa protein was the main product secreted by nonstimulated AtT-20 cells, while the 66-kDa product was only released when the cells were stimulated with CRF or BaCl2 and Bromo-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in neutrophil met-enkephalin containing peptides in episodic cluster headache.

We have previously demonstrated an increase in plasma met-enkephalin levels during the pain attacks in episodic cluster headache. The present study was undertaken in order to clarify the source of the plasma met-enkephalin increase. Recent evidence has shown that peripheral blood polymorphonuclear cells contain peptides derived from the proenkephalin A system, which can be released by specific stimuli. We studied neutrophil met-enkephalin containing peptides (NMECP) in 27 episodic cluster headache patients: 24 in a cluster period (6 of them during a pain attack), and 3 in the remission period. Neutrophil met-enkephalin containing peptide levels (after sequential enzymatic digestion with trypsin and carboxypeptidase B) were determined by radioimmunoassay with specific antiserum. Neutrophil peptide concentration (pmol/mg prot) was lower (p less than 0.01) in patients during the pain attack (14.4 +/- 0.36) than after their pain had subsided (36.7 +/- 0.31) and lower than in the remission period patients (35.8 +/- 0.4). We conclude that neutrophil met-enkephalin containing peptides decrease during pain in episodic cluster headache, and that they may be involved in the concomitant plasma met-enkephalin increase.

Differential association of endogenous proenkephalin-derived peptides with membranes of microsomes from rat striatum, adrenal medulla and heart ventricle.

Proenkephalin-derived peptides, in common with other prohormones, are associated with membranes of microsomes and secretory granules in the bovine adrenal medulla. Post-translational processing of the precursor molecule varies depending upon the tissue. The relationship between post-translational events in different tissues was examined by studying the membrane association of endogenous proenkephalin-derived peptides in the crude microsomal fraction of rat adrenal medulla, brain striatum and heart ventricle. [Met]-Enkephalin and synenkephalin (proenkephalin(1-70)) immunoreactivities were quantified by radioimmunoassay after sequential enzymatic digestion with trypsin and carboxypeptidase B. Between 60 and 75% of total immunoreactive peptides present in intact microsomes of the three tissues were associated with membranes and specifically released with 2 M KSCN (pH 7.4). Analysis of the chromatographic profile of materials present in the soluble and associated fractions produced the following results. In the three tissues the materials associated with microsomal membranes corresponded to peptides larger than 3-5 kDa and displayed synenkephalin and [Met]-enkephalin immunoreactivity. Adrenal and heart microsomes showed a continuous pattern of membrane-associated proenkephalin-derived peptides of high, intermediate and low molecular weights containing the synenkephalin and [Met]-enkephalin sequences. These tissues, however, presented quantitative differences, as the highest concentrations belonged to materials larger and smaller than 12.5 kDa in adrenal and heart microsomes respectively. On the other hand, brain striatal microsomes displayed a discontinuous pattern of associated materials, with the absence of some products of high and intermediate molecular weight. Only in the soluble fraction of striatal microsomes were peptides detected of high and intermediate molecular weight containing the [Met]-enkephalin but not the synenkephalin sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Increase in plasma methionine-enkephalin levels during the pain attack in episodic cluster headache.

Since high levels of endogenous opioids (endorphins, enkephalins) were found in brain areas classically related to nociception, their peripheral levels in humans were studied in different pain syndromes yielding contradictory results. This study was undertaken to assess changes in plasma methionine-enkephalin (met-enkephalin) levels in patients with episodic cluster headache associated with the pain period. Twenty-nine patients, 24 in the cluster period (6 of them during an attack) and 3 in the remission period were studied. Two other patients were subjected to a longitudinal follow-up. Plasma met-enkephalin levels were determined by radioimmunoassay (RIA) with specific antibody. Plasma peptide concentration (pmol/ml) was higher (p less than 0.001) in patients during the pain attack (3.97 +/- 1.18) than in controls (0.25 +/- 0.03). When measured 4 and 48 h after the pain attack lower levels were found (0.46 +/- 0.06) which decreased to control values after 24 h. These results may suggest involvement of peripheral enkephalins in pain modulation in patients with episodic cluster headache.