Proenkephalin system in human polymorphonuclear cells. Production and release of a novel 1.0-kD peptide derived from synenkephalin.

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.

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)

Adrenal proenkephalin-derived peptides during postnatal development in spontaneously hypertensive rats.

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.

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.

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)

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.

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)

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.

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.

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.

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.

Diurnal variations of IR-Met-enkephalin in the brain of pentylenetetrazol-kindled rats.

Pentylenetetrazol (PTZ) kindling was induced in male Wistar rats by daily i.p. injections of 40 mg/kg of the convulsant agent. Immunoreactive (IR)-Met-enkephalin was quantified in the amygdala, hippocampus and hypothalamus 17 days after the last stimulus, in groups of 6-7 rats, every 4 h, beginning at 08.00 h. IR-Met-enkephalin level displayed diurnal variations in brain regions of control animals. In the amygdala and the hippocampus the peptide peaked at 24.00 h and in the hypothalamus at 20.00 h; the troughs were at 08.00, 16.00 and 08.00 h, respectively. Diurnal variations were abolished in the amygdala and hypothalamus of kindled rats. In the amygdala the effect was characterized by an IR-Met-enkephalin increase at 04.00, 08.00 and 12.00 h; in the hypothalamus the peptide was enhanced at 08.00 and 12.00 h; in the hippocampus IR-Met-enkephalin increased at 12.00 h and showed a displacement of the peak during the dark phase. The results suggest that PTZ kindling in rats produces a long-lasting alteration on diurnal variations of IR-Met-enkephalin levels in limbic structures.

Pentylenetetrazol kindling produces a long-lasting elevation of IR-Met-enkephalin but not IR-Leu-enkephalin in rat brain.

Pentylenetetrazol (PTZ) kindling was induced in male Wistar rats by daily i.p. injections of 40 mg/kg of the convulsant agent. IR-Met-enkephalin and IR-Leu-enkephalin were quantified in several brain regions 16 days after the last stimulus. In addition other rats received another PTZ dose on the sixteenth day and the samples were analyzed 1 and 24 h later. The results showed a long-lasting elevation in amygdala, septum, hypothalamus and hippocampus of IR-Met-enkephalin only. These brain structures also showed a decrease of IR-Met-enkephalin 1 h after the PTZ-induced seizure, but reached newly elevated levels 24 h later. IR-Leu-enkephalin levels only showed a decrease in striatum 1 h after PTZ-induced convulsions. The data suggest that Met-enkephalin is related to permanent changes in brain function induced by PTZ kindling, while Leu-enkephalin may depend on the occurrence of epileptic seizures.

Pentylenetetrazol-induced seizures produce an increased release of IR-Met-enkephalin from rat striatum in vitro.

In this work we analyzed the immunoreactive-methionine-enkephalin (IR-Met-enkephalin) levels in several brain regions of rats sacrificed during the tonic extension, induced by acute treatment with pentylenetetrazol (PTZ). The results show an increased of IR-Met-enkephalin content in striatum but not in amygdala, hypothalamus, septum, hippocampus and cortex. To characterize whether this elevation of enkephalin levels in striatum corresponded to the releasable pool, we studied the in vitro efflux of this peptide in striatal slices of rats sacrificed during the seizures, in acute PTZ and in PTZ-kindled rats (kindling group I). In addition, PTZ-kindled rats were analyzed 24 h after the last stimulus (kindling group II). The striatal slices of acute group and kindling group I displayed a significant increase in the evoked release of IR-Met-enkephalin. However, no significant changes occurred from striatal slices of kindling group II animals. In vitro superfusion of GABA (100 microM) produced a approximately equal to 63% decrease in IR-Met-enkephalin released from striatal slices in both saline and acute PTZ-treated rats. Several studies suggest that opioid peptides may be released in the ictal phase of seizure in order to mediate some transient postictal behavior. Our results suggest that of several brain regions tested, only the striatal IR-Met-enkephalin may be released during the ictus to mediate postictal behavior in the acute PTZ treated and in PTZ-kindled rats. This effect may be regulated by the GABA system.

Brain content of immunoreactive [Leu5]enkephalin and [Met5]enkephalin after pentylenetetrazol-induced convulsions.

Wistar rats were injected daily with convulsant doses of PTZ (40 mg/kg). The brain content of immunoreactive (IR) enkephalins was measured after repetitive clonic convulsions and after the 'kindling' state was reached. In both instances we found a significant increase of IR [Leu5]enkephalin and IR [Met5]enkephalin in septum, striatum and amygdala 24 h after PTZ administration. These results support the suggestion that the peptide increase occurs with repetitive convulsions and not only after the 'kindling' state is reached.

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)

Amygdaloid kindling enhances the enkephalin content in the rat brain.

Established amygdaloid kindling causes an increase in the immunoassayable content of both Leuenkephalin and Met-enkephalin in the rat brain. Control and sham-operated (electrode implanted but not stimulated) rats do not show statistically significant differences in brain enkephalin content, while kindled rats show a 40% enkephalin increase in both hemispheres. The present finding is in agreement with several lines of evidence and suggest that enkephalins may play a role in epileptic seizures.

Brain content of leu5- and met5-enkephalin changes independently during the development of kindling in the rat.

Brain content of leucine-enkephalin and methionine-enkephalin changes independently during kindling of the amygdala. Both peptides were measured after 4, 8, 15 and 21 days of stimulation. Leu-enkephalin showed a progressive increase during the kindling: the results on the 4th day did not differ significantly from the controls, whereas those on the 8th day displayed an increase only in the stimulated left hemisphere and those on the 15th day showed an increase in both hemispheres. This increase was also seen after 21 days of stimulation. In contrast, Met-enkephalin was not modified during the early stages of kindling, but was found to be increased after the animals had presented five or more generalized seizures. These results show that Leu-enkephalin is related to the development of kindling, whereas Met-enkephalin levels change only as a consequence of the generalized seizures.

Mitogenic activation of the human lymphocytes induce the release of proenkephalin derived peptides.

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.

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.