RESUMEN
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.
Asunto(s)
Adenoma/metabolismo , Hormona Adrenocorticotrópica/metabolismo , Encefalinas/metabolismo , Neoplasias Hipofisarias/metabolismo , Precursores de Proteínas/metabolismo , Adulto , Anciano , Carboxipeptidasa B , Carboxipeptidasas/metabolismo , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Desoxirribonucleasa I/metabolismo , Encefalina Metionina/metabolismo , Femenino , Hormona de Crecimiento Humana/metabolismo , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Peso Molecular , Hipófisis/metabolismo , Procesamiento Proteico-Postraduccional , Tripsina/metabolismoRESUMEN
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.
Asunto(s)
Células de la Médula Ósea/metabolismo , Encefalinas/metabolismo , Leucocitos Mononucleares/metabolismo , Precursores de Proteínas/metabolismo , Bazo/metabolismo , Estrés Fisiológico/metabolismo , Timo/metabolismo , Animales , Electrochoque , Encefalina Metionina/metabolismo , Macrófagos/metabolismo , Masculino , Ratas , Ratas Wistar , Bazo/citología , Timo/citologíaRESUMEN
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.
Asunto(s)
Encefalinas/inmunología , Leucocitos Mononucleares/citología , Precursores de Proteínas/inmunología , Secuencia de Aminoácidos , División Celular , Encefalinas/química , Humanos , Técnicas Inmunológicas , Técnicas In Vitro , Activación de Linfocitos , Datos de Secuencia Molecular , Neuroinmunomodulación , Precursores de Proteínas/químicaRESUMEN
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)
Asunto(s)
Química Encefálica/fisiología , Encéfalo/embriología , Encefalinas/metabolismo , Precursores de Proteínas/metabolismo , Envejecimiento/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Encéfalo/citología , División Celular , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Encefalinas/inmunología , Femenino , Neostriado/metabolismo , Embarazo , Precursores de Proteínas/inmunología , Radioinmunoensayo , Ratas , Ratas WistarRESUMEN
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.
Asunto(s)
Médula Ósea/metabolismo , Encefalinas/metabolismo , Monocitos/metabolismo , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Bazo/metabolismo , Animales , Células de la Médula Ósea , Cromatografía/métodos , Encefalinas/química , Peso Molecular , Precursores de Proteínas/química , Ratas , Bazo/citologíaRESUMEN
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.
Asunto(s)
Glándulas Suprarrenales/efectos de los fármacos , Animales Recién Nacidos/metabolismo , Dexametasona/farmacología , Encefalina Metionina/biosíntesis , Encefalinas/metabolismo , Precursores de Proteínas/metabolismo , Glándulas Suprarrenales/crecimiento & desarrollo , Glándulas Suprarrenales/metabolismo , Animales , Carbacol/farmacología , Cromatografía , Encefalina Metionina/metabolismo , Humanos , Recién Nacido , Nicotina/farmacología , Pilocarpina/farmacología , Ratas , Reserpina/farmacologíaRESUMEN
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.
Asunto(s)
Cefalalgia Histamínica/sangre , Encefalina Metionina/sangre , Neutrófilos/química , Péptidos/sangre , Adulto , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
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)
Asunto(s)
Médula Suprarrenal/metabolismo , Cuerpo Estriado/metabolismo , Encefalina Metionina/metabolismo , Encefalinas/metabolismo , Ventrículos Cardíacos/metabolismo , Microsomas/metabolismo , Precursores de Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Membranas Intracelulares/metabolismo , Datos de Secuencia Molecular , Especificidad de Órganos , Procesamiento Proteico-Postraduccional , Ratas , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Vertebrados/genética , Xenopus/genéticaRESUMEN
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.
Asunto(s)
Cefalalgia Histamínica/sangre , Encefalina Metionina/sangre , Adulto , Cefalalgia Histamínica/fisiopatología , Epinefrina/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Norepinefrina/sangre , Dolor/sangre , Dolor/fisiopatología , Factores de TiempoRESUMEN
In the hematopoietic system a pluripotent stem cell generates precursors for lymphoid and myeloid lineages. Proenkephalin-derived peptides were previously detected in differentiated lymphoid cells. We have studied whether the proenkephalin system is expressed in a typical differentiated cell of the myeloid lineage, the neutrophil. Human peripheral polymorphonuclear cells contain and release proenkephalin-derived peptides. The opioid portion of proenkephalin (met-enkephalin-containing peptides) was incompletely processed, resulting in the absence of low molecular weight products. The nonopioid synenkephalin (proenkephalin 1-70) molecule was completely processed to a 1.0-kD peptide derived from the COOH-terminal. This molecule was characterized in neutrophils by biochemical and immunocytochemical methods. The chemotactic peptide FMLP and the calcium ionophore A23187 induced the release of the proenkephalin-derived peptides, and this effect was potentiated by cytochalasin B. The materials secreted were similar to those present in the cell, although in the supernatant a higher proportion corresponded to more processed products. The 1.0-kD peptide was detected in human, bovine, and rat neutrophils, but the chromatographic pattern of synenkephalin-derived peptides suggests a differential posttranslational processing among species. These findings demonstrate the existence of the proenkephalin system in human neutrophils and the production and release of a novel 1.0-kD peptide derived from the synenkephalin molecule. The presence of opioid peptides in neutrophils suggests their participation in the inflammatory process, including a local analgesic effect.
Asunto(s)
Encefalinas/metabolismo , Neutrófilos/metabolismo , Precursores de Proteínas/metabolismo , Animales , Calcimicina/farmacología , Bovinos , Cromatografía en Gel , Humanos , Técnicas para Inmunoenzimas , Técnicas In Vitro , Peso Molecular , N-Formilmetionina Leucil-Fenilalanina/farmacología , RatasRESUMEN
The association of endogenous synenkephalin and met-enkephalin containing peptides with the membrane of bovine chromaffin granules and physicochemical characteristics of this association were studied. The associated materials were only released at a non physiological pH range and this effect was enhanced with growing salt concentrations (0.5, 1.0 and 2.0 M KSCN). A higher peptide dissociation occurred with membrane solubilizing agents (SDS greater than Triton X-100 greater than digitonin). In microsomes the materials dissociated with 2 M KSCN (pH 7.4) corresponded to peptides larger than 12.0 kDa, while in granules corresponded to molecules smaller than 8.5 kDa, displaying synenkephalin and met-enkephalin immunoreactivities. These data suggest that some sequence of the C-terminal portion of synenkephalin may be responsible for the association of proenkephalin derived peptides with microsome and granule membranes.
Asunto(s)
Médula Suprarrenal/ultraestructura , Encefalina Metionina/metabolismo , Encefalinas/metabolismo , Membranas Intracelulares/metabolismo , Precursores de Proteínas/metabolismo , Animales , Bovinos , Fenómenos Químicos , Química Física , Gránulos Cromafines/ultraestructura , Cromatografía en Gel , Digitonina/farmacología , Concentración de Iones de Hidrógeno , Membranas Intracelulares/efectos de los fármacos , Microsomas/ultraestructura , Peso Molecular , Octoxinol , Concentración Osmolar , Polietilenglicoles/farmacología , Dodecil Sulfato de Sodio/farmacología , Tiocianatos/farmacologíaRESUMEN
Several reports indicate that enkephalins participate in lymphocyte proliferation and several events of the immune response. It has been proposed that peptides involved in these processes may originate in the nervous system or endocrine glands. We have found that human peripheral blood lymphocytes (PBL) activated with a mitogenic agent contain and release proenkephalin derived peptides. The kinetics of met-enkephalin and cryptic products of proenkephalin in PBL activated with phytohemaglutinin (PHA) were studied. Peptides were released to the supernatant of stimulated PBL, reaching the highest values after 18 to 24 hours. The material secreted corresponds to high, intermediate and low molecular weight peptides derived from proenkephalin, displaying met-enkephalin and synenkephalin (proenkephalin 1-70) immunoreactivity. Therefore, an intrinsic lymphocytic proenkephalin system is induced by PHA and may play an important role in the regulation of the immune response.
Asunto(s)
Encefalinas/sangre , Activación de Linfocitos/fisiología , Linfocitos/metabolismo , Fitohemaglutininas/farmacología , Precursores de Proteínas/sangre , Adulto , Células Cultivadas , Cromatografía en Gel , Encefalina Metionina/sangre , Humanos , Cinética , Linfocitos/inmunología , Peso MolecularRESUMEN
Adrenal enkephalin and enkephalin-containing peptides were studied during postnatal development in normotensive (WKY) and spontaneously hypertensive rats (SHR). The effect of chronic treatment with the ganglionic blocker chlorisondamine (5 mg/kg) was also assessed. Free enkephalin immunoreactivity and total enkephalin immunoreactivity, as determined by enzymatic digestion of large enkephalin containing fragments, were quantitated in the adrenal glands at 11 days and 7, 16, and 24 weeks of age. Both total and free metenkephalin were significantly diminished in the adrenal of SHR when compared to WKY at all ages tested. The analysis of the chromatographic profile showed that SHR displayed reduced levels of high and low molecular weight materials at 11 days and 16 weeks of age; however intermediate compounds were high in the glands of these animals. Similar increased values for free met-enkephalin were found in adrenals of WKY and SHR after ganglionic blocker treatment, which means that the relative increase was larger in SHR than WKY; while for total enkephalin the relative increase and the concentration reached in SHR was about half of those presented in WKY. These and other results presented suggest that the basic alteration of the adrenal proenkephalin system of SHR may be due to a genetic reduction of proenkephalin levels. Otherwise, the free enkephalin decrease could be related to changes in nervous input to the adrenal gland.
Asunto(s)
Glándulas Suprarrenales/metabolismo , Envejecimiento/metabolismo , Encefalinas/metabolismo , Hipertensión/metabolismo , Precursores de Proteínas/metabolismo , Glándulas Suprarrenales/efectos de los fármacos , Animales , Clorisondamina/farmacología , Cromatografía en Gel , Encefalina Leucina/metabolismo , Encefalina Metionina/metabolismo , Masculino , Peso Molecular , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKYRESUMEN
We have compared the enkephalin-like material derived from proenkephalin released from perfused cat adrenal glands stimulated with pilocarpine (5 X 10(-4)M) and nicotine (5 X 10(-6) M). In addition, two doses of acetylcholine (10(-5) and 10(-4) M) and 50 mM K+ were tested. Free Met-enkephalin immunoreactivity and total Met-enkephalin immunoreactivity, as determined by enzymatic digestion of large enkephalin-containing fragments, were coreleased with catecholamines. Free Met-enkephalin immunoreactivity represented 13% of total immunoreactivity for nicotinic stimulation, 46% for pilocarpine, 33% for 10(-5) M acetylcholine, 22% for 10(-4) M acetylcholine, and 16% for 50 mM K+. Analysis of the perfusate by gel filtration showed that 80% of the total Met-enkephalin immunoreactivity whose release was induced by pilocarpine was eluted in fractions corresponding to fragments of low molecular weight, whereas these fractions accounted only for 10% of the total Met-enkephalin immunoreactivity whose release was induced by nicotine. HPLC analysis of low-molecular-weight peptide fractions revealed that Met-enkephalin, Met-enkephalin-Arg-Gly-Leu, and Met-enkephalin-Arg-Phe represented 69% of total Met-enkephalin immunoreactivity whose release was induced by pilocarpine. These results indicate that selective activation of muscarinic receptors is followed by release of low-molecular-weight material, whereas nicotine application also yielded high-molecular-weight peptides. Furthermore, increasing the acetylcholine concentration from 10(-5) to 10(-4) M and using 50 mM K+ increased proportionally the high-molecular-weight peptide secretion. Results are discussed in relation to the existence of a heterogeneous population of granules either in the same cell or in different cells, containing proenkephalin-derived peptides. (ABSTRACT TRUNCATED AT 250 WORDS)