Fragile X mental retardation protein (FMRP) interacting proteins exhibit different expression patterns during development.

Fragile X syndrome is caused by the lack of expression of fragile X mental retardation protein (FMRP), an RNA-binding protein involved in mRNA transport and translation. FMRP is a component of mRNA ribonucleoprotein complexes and it can interact with a range of proteins either directly or indirectly, as demonstrated by two-hybrid selection and co-immunoprecipitation, respectively. Most of FMRP-interacting proteins are RNA-binding proteins such as FXR1P, FXR2P and 82-FIP. Interestingly, FMRP can also interact directly with the cytoplasmic proteins CYFIP1 and CYFIP2, which do not bind RNA and link FMRP to the RhoGTPase pathway. The interaction with these different proteins may modulate the functions of FMRP by influencing its affinity to RNA and by affecting the FMRP ability of cytoskeleton remodeling through Rho/Rac GTPases. To better define the relationship of FMRP with its interacting proteins during brain development, we have analyzed the expression pattern of FMRP and its interacting proteins in the cortex, striatum, hippocampus and cerebellum at different ages in wild type (WT) mice. FMRP and FXR2P were strongly expressed during the first week and gradually decreased thereafter, more rapidly in the cerebellum than in the cortex. FXR1P was also expressed early and showed a reduction at later stages of development with a similar developmental pattern in these two regions. CYFIP1 was expressed at all ages and peaked in the third post-natal week. In contrast, CYFIP2 and 82-FIP (only in forebrain regions) were moderately expressed at P3 and gradually increased after P7. In general, the expression pattern of each protein was similar in the regions examined, except for 82-FIP, which exhibited a strong expression at P3 and low levels at later developmental stages in the cerebellum. Our data indicate that FMRP and its interacting proteins have distinct developmental patterns of expression and suggest that FMRP may be preferentially associated to certain proteins in early and late developmental periods. In particular, the RNA-binding and cytoskeleton remodeling functions of FMRP may be differently modulated during development.

Monoclonal antiidiotypic antibodies against delta opioid receptors as an electron microscopy probe.

Four different rat monoclonal antibodies were produced against delta opioid receptor using an antiidiotypic approach in which antibodies directed against the opioid agonist DADLE were used as immunogen. In the first step, seven hybridomas were selected on the basis of their ability to inhibit the DADLE-anti-DADLE antibody interaction. After purification from ascitic fluids, these monoclonal antibodies were characterized. Four antiidiotypic antibodies, named 5, 11, 16, and 51, directed toward different epitopes, recognized the delta opioid receptor: (i) they bound directly to the NG108-15 cells, (ii) they inhibited the [3H]DADLE binding on the NG108-15 cells, (iii) they immunoprecipitated a 52,500 dalton protein present on the surface of the NG108-15 cells. The four monoclonal antiidiotypic anti-opioid receptor antibodies were used to immunocytologically detect the opioid receptors under light and electron microscopy in the rat spinal cord. The regional distribution of the immunoreactivity corresponded to layers known to be rich delta opioid receptor subtype. Moreover, at the ultrastructural level, the labeling was located mainly on plasma membranes, especially on non-synaptic zones. Our results show that monoclonal antiidiotypic antibodies constitute a valuable tool for visualizing cell surface receptors.

Proteolytic degradation of hemoglobin by endogenous lysosomal proteases gives rise to bioactive peptides: hemorphins.

Hemorphin generation by mice peritoneal macrophages has been recently reported, nevertheless no conclusive data exist to localize clearly the macrophage proteolytic activity implicated in their generation. Because lysosomes are believed to be the main site of degradation in the endocytic pathway, we have studied their potential implication in the generation of hemorphins from hemoglobin. When this protein is submitted to purified rat liver lysosomes, an early generation of hemorphin-7-related peptides, detected by a radioimmunoassay, was observed. These peptides seemed to be relatively stable during the first hours of hydrolysis.

Peripheral stimulation of CCK-B receptors by BC264 induces a hyperexploration, dependent on the delta opioid system in the nucleus accumbens of rat.

This study analyses the influence of the CCKergic system on the enkephalinergic system in the exploratory behavior of rats, using both behavioral and biochemical approaches. The results show that the increase of the spontaneous alternation behavior induced by the selective CCKB agonist, BC264 (3 microg/kg) was not suppressed by the opioid antagonists, naloxone (100 microg/kg), or naltrindole (300 microg/kg). In contrast, BC264 injected at the same dose induced a hyperlocomotor activity measured in the open-field test, which was antagonized by the selective delta opioid antagonist, naltrindole. BC264 (3 microg/kg) significantly increased the extracellular levels of Met-LI in the anterior part of the nucleus accumbens. Furthermore, local injection of naltrindole (0.25 microg/0.5 microl) in the anterior nucleus accumbens completely suppressed the hyperlocomotion induced by BC264. The behavioral effects induced by BC264 cannot be explained by its interaction with gastrinic receptors mediating gastric acid release, since BC264 produced a long-lasting increase of gastric acid output from conscious gastric fistula rats only at doses 100 times higher than those inducing behavioral modifications. The hyperlocomotion obtained after stimulation by BC264 of probably peripheral CCKB receptors, indicates that this receptor type could participate in the transmission of information between the peripheral system and some regions of the CNS involved in motivations and emotions.

Anti-idiotypic antibodies: a useful alternative for studying the biochemical expression of mu/delta opioid binding sites in mammalian brain.

A polyclonal antiserum was produced against opioid binding sites using an anti-idiotypic approach whereby antibodies directed against the opioid agonist DSLET were used as immunogen. The anti-idiotypic antiserum recognized specific brain proteins with molecular masses of 76 +/- 4, 73 +/- 4 and 59 +/- 3 kDa, respectively. The immunolabeling of these proteins was mainly inhibited by mu, delta opioid agonists and a general antagonist, naloxone. The inhibition of immunoprecipitation by opioid agonists and antagonist and the developmental expression of these immunoreactive proteins found to occur during brain ontogeny strongly suggest that these three proteins were mu, delta but not kappa opioid binding sites. The anti-idiotypic antiserum both inhibits 3H-DADLE binding and mimics the inhibitory agonist effects on the stimulated cAMP level of NG 108-15 cells which expressed delta opiate receptors. Numerous mammalian brain opioid binding sites were labeled, due to the fact that the binding site was the epitope recognized by the anti-idiotypic antibodies. From the numerous studies performed with a view to characterizing the specificity of the anti-idiotypic antibodies, it was strongly suggested that the anti-idiotypic antibodies specifically recognize mu/delta opioid binding sites and they can therefore be powerful tools for studying the biochemical expression of these opioid binding sites in mammalian brains.

Detection of methionine-enkephalin at the 10(-16) mole level.

A high performance radioimmunoassay for Met5-enkephalin has been developed, using two ways to circumvent the difficulties of obtaining such an assay for the opiate peptides. First, hemocyanin was used as the carrier for the antigen and secondly, a new iodination method for Met5-enkephalin was established, resulting in a [125I]probe with an intact methionyl residue. The Met5-enkephalin antibodies exhibited a very high affinity (Kd = 5 X 10(-12) M) and Met5-enkephalin could be detected at the 10(-16) mole level. The Met5-enkephalin content of individual cervical ganglia and individual cochlea was measured. We also measured the Met5-enkephalin content of brain from different areas of two rat strains: no significant differences were found.

The rat dermorphin-like immunoreactivity is supported by an aminopeptidase resistant peptide.

Site-directed antibodies against synthetic related dermorphin peptides were previously produced and characterized. One of them, which specifically recognizes the crucial 'opioid message' (the N-terminal part of the dermorphin molecule (i.e. Tyr-D-Ala-Phe-Gly) was selected in order to detect and locate endogenous dermorphin-like molecules in rat, mouse and guinea pig tissues. Dermorphin-like peptides were found to be present in tissues known to contain peptides such as neurons in the central nervous system, nerve fibers in the gut and B and T immune cells. With all the tissues assayed, the HPLC profile obtained on the immunoreactive material showed the same main peak eluted at a retention time of 32 +/- 1 min. The results of biochemical experiments in which enzymatic treatments were performed on the dermorphin-like immunoreactivity indicate the immunoreactivity is a peptide resistant to aminopeptidase hydrolysis. This finding suggests the presence of a residue conferring resistance to proteolytic processes of this kind, which is likely to be a D-amino acid residue.

Monoclonal antibodies against Met-enkephalin as probe in the central nervous system.

Peptides with transmitter-like properties have been found in many brain areas. Immunochemical techniques have contributed most to clarification of the function and the pathway of these substances in neuronal systems. In this paper we report the production of 4 monoclonal antibodies against Met-enkephalin and their use in studying this peptide in rat cervical cord. Two of the antibodies recognize the COOH-terminus part of the Met-enkephalin, and do not cross-react with other known peptides. The other two antibodies are mainly directed against the NH2-terminus part of the peptide. Specific interactions of these monoclonal antibodies with regions of rat cervical cord were shown by immunochemistry techniques.

Characterization of methionine-enkephalin release in the rat striatum by in vivo dialysis: effects of gamma-hydroxybutyrate on cellular and extracellular methionine-enkephalin levels.

The opioïd system is implicated in mediating the effects produced upon administration of gamma-hydroxybutyrate. Gamma-hydroxybutyrate occurs endogenously in the mammalian brain, and is most probably involved in the regulation of some basic brain functions, particularly those concerning the dopaminergic nigrostriatal pathway, which is closely linked to the expression of enkephalins in the striatum. In the present study, in vivo microdialysis was used to examine the basic characteristics of methionine-enkephalin (met-enkephalin) release in the striatum of Wistar rats, using a high performance radioimmunoassay. Administration of gamma-hydroxybutyrate to the rats induced a dose-dependent decrease in the extracellular release of met-enkephalin. In parallel, a dose- and time-dependent gamma-hydroxybutyrate-induced accumulation of met-enkephalin in striatum was observed. These two phenomena (tissue accumulation and inhibition of release) were blocked by NCS-382, a gamma-hydroxybutyrate receptor antagonist. The striatal met-enkephalin accumulation does not seem to be exclusively due to the inhibition of its release. Thus, a gamma-hydroxybutyrate mediating effect on met-enkephalin synthesis is suggested, most probably occurring via functional modulation of striatal dopamine synthesis and release. To understand the role of this dopaminergic mechanism, unilateral lesions of the nigrostriatal dopaminergic pathway were carried out. In gamma-hydroxybutyrate-treated rats, striata exhibited a similar increase in met-enkephalin content. In untreated rats, only the lesioned striatum showed an identical increase in met-enkephalin levels. Thus, striatal met-enkephalin accumulation could be attributed to the suppression of the dopaminergic impulse flow, due to gamma-hydroxybutyrate or to the action of 6-hydroxydopamine. In the extracellular spaces (microdialysis experiments), gamma-hydroxybutyrate administration induced identical modifications of met-enkephalin release in lesioned or non-lesioned striata. These modifications could be reproduced by peripheral or striatal administration of sulpiride, a D2/D3 antagonist. From a functional point of view, the dopaminergic D2 receptor blockade or the gamma-hydroxybutyrate-induced inhibition of dopamine release could be considered to induce similar results, with identical consequences on striatal met-enkephalin accumulation and release. These results suggest that gamma-hydroxybutyrate-induced modifications in met-enkephalin release, presumably potentiated by 6-hydroxydopamine treatment, act via a functional modification of the nigrostriatal dopaminergic pathway.

Release of proenkephalin-derived opioid peptides from rat striatum in vitro and their rapid degradation.

In a previous paper we demonstrated that the heptapeptide [Met]enkephalyl-Arg6-Phe7 was released from rat striatal slices by high K+ concentration and rapidly degraded by peptidases, even in the presence of the neutral endopeptidase 24.11 ("enkephalinase")-inhibitor, thiorphan (0.1 microM), the angiotensin-converting enzyme inhibitor, captopril (1 microM), and the aminopeptidase inhibitor, bestatin (20 microM). In this study the pattern of degradation of exogenous [3H]heptapeptide by rat striatal slices has been studied. The angiotensin-converting enzyme and aminopeptidase(s) were partly responsible for this degradation. In addition an enzymatic activity that cleaved the Phe4-Met5 bond was involved in the degradation of the heptapeptide by striatal slices. This activity was inhibited by the dipeptide Leu-Arg (1 mM) and the tripeptide Leu-Arg-Leu (1 mM). The simultaneous presence of thiorphan (0.1 microM), captopril (1 microM), bestatin (20 microM) and Leu-Arg (1 mM) almost completely inhibited the degradation of [3H]heptapeptide by striatal slices. In the presence of these peptidase inhibitors a concomitant release of [Met]enkephalin, the heptapeptide [Met]enkephalyl-Arg6-Phe7 and the octapeptide [Met]enkephalyl-Arg6-Gly7-Leu8 was evoked by KCl or veratridine. The K+-evoked release was by a Ca2+-dependent mechanism and the release evoked by veratridine was blocked by tetrodotoxin. In both cases the ratio of [Met]enkephalin to heptapeptide amounts released was close to that found in their common precursor, proenkephalin. Thus the enkephalinergic neuron appears to be capable of synthesizing, from a unique precursor, four different putative opioid neurotransmitters, namely [Met]enkephalin, [Leu]enkephalin, the heptapeptide [Met]enkephalyl-Arg6-Phe7 and the octapeptide [Met]enkephalyl-Arg6-Gly7-Leu8, to store these peptides and to release them upon depolarization.

Quantification and characterization of enkephalins in the upper part of the cat digestive tract and the coeliac ganglia.

The [Met]enkephalin, [Leu]enkephalin and [Met]enkephalin-arg-gly-leu contents of the upper part of the digestive tract (lower oesophageal sphincter, fundus, antrum, pylorus, duodenum, ileum) and coeliac ganglia of the cat were determined and identified. The enkephalin content of all the structures studied, expressed in femtomole/mg of wet tissue, was found to range from 83 to 446 with [Met]enkephalin; 19 to 63 with [Leu]enkephalin; 2.5 to 13 with [Met]enkephalin-arg-gly-leu. In the muscular and plexus layers the [Met]- and [Leu]enkephalin contents increase gradually from the lower oesophageal sphincter to the pylorus and then decrease from the duodenum to the ileum. The [Met]enkephalin versus [Leu]enkephalin ratio is 2.7 in the coeliac ganglia and ranges from 4.3 to 8.1 in the areas of the digestive tract investigated. In addition, the presence of authentic [Met]- and [Leu]enkephalin was confirmed in all the structures assayed by high pressure liquid chromatography. Owing to the low amounts of [Met]enkephalin-arg-gly-leu detected in individual samples of the coeliac ganglia and in the areas of the digestive tract investigated, it was not possible to characterize this peptide using high pressure liquid chromatography and therefore to confirm the presence of authentic [Met]enkephalin-arg-gly-leu in these structures. The differences in the enkephalin concentrations observed among these various areas of the digestive tract suggest that these peptides may act differently from one area to another, thus playing a complex integrative role in the nervous control of gastrointestinal tract motility.

Ultrastructural study of delta-opioid receptors in the dorsal horn of the rat spinal cord using monoclonal anti-idiotypic antibodies.

The ultrastructural localization of delta-opioid receptors was studied using monoclonal anti-idiotypic antibody prepared with an anti-D-Ala2-D-Leu5-enkephalin. Immunocytochemical techniques were used on vibratome sections from rats perfused with paraformaldehyde. A high density of immunoreactivity was observed in the dorsal horn of the spinal cord, particularly the two superficial layers, the dorsolateral funiculus and the area surrounding the central canal. The labelling was absent when the antibody was preincubated with the immunogen. Competition between the anti-idiotypic antibody and different ligands, delta or mu, was controlled by preincubation of tissue sections with the ligand in the presence of peptidase inhibitors for 3-4 h before addition of the anti-idiotypic antibody. Enkephalin, dermenkephalin and naltrindole induced disappearance of the labelling at 10(-9) M while dermorphin or dermorphin Lys7 were ineffective at the same concentration. Lamina II of the dorsal horn was studied by electron microscopy. The immunolabelling was mainly localized on cell membranes at appositions between the two neurons. About one third were localized between an axon terminal and a dendrite, the same proportion of labellings were between two axon terminals. Labelling was occasionally observed at appositions between a glomerular terminal and a dendrite or a terminal or at axoglial appositions. Axosomatic localizations were rare. The presynaptic localization of the labelling is in favor of a presynaptic mechanism of action for delta-opioids in the spinal cord, providing that these receptors are functional. delta-Opioid peptides probably act non-synaptically since receptors were never localized on synaptic differentiations.

Chromatographic analysis of the enkephalin immunoreactivity in the nucleus locus coeruleus of the cat after local colchicine administration.

Cell bodies immunoreactive for methionine- and leucine-enkephalin are found in the area of the locus coeruleus (dorsolateral pons) of the cat after injection of colchicine in the ascending projections of the nucleus. Using radioimmunoassay procedures, it is shown that colchicine induces a significant increase in methionine- and leucine-enkephalin-immunoreactive material in this area of the brain. High pressure liquid chromatography analysis demonstrated that the immunoreactive materials were authentic methionine- and leucine-enkephalin. The methionine- and leucine-enkephalin patterns were identical in the colchicine injected and non-injected sides of the dorsolateral pons. It is suggested that, in this area of the brain, colchicine (i) does not significantly modify the processing of proenkephalin to form the pentapeptides methionine- and leucine-enkephalin, and (ii) does not induce the appearance of new substances reactive to the enkephalin antisera employed.

Immunohistochemical and biochemical evidence for the presence of the pentapeptide met-enkephalin and the heptapeptide met-enkephalin-Arg(6)-Phe(7) but not the octapeptide met-enkephalin-Arg(6)-Gly(7)-Leu(8) in amphibian chromaffin cells.

Using specific antibodies to met-enkephalin, met-enkephalin-Arg(6)-Phe(7) and met-enkephalin-Arg(6)-Gly(7)-Leu(8), we have studied the distribution of these opioid peptides in the frog adrenal gland by means of the indirect immunofluorescence technique. Bright staining of all chromaffin cells was observed by application of met-enkephalin and met-enkephalin-Arg(6)-Phe(7) antisera. No nerve endings could be detected. A few chromaffin cells were weakly stained by met-enkephalin-Arg(6)-Gly(7)-Leu(8) antiserum. Using a specific radioimmunoassay for met-enkephalin, the dilution curve of frog adrenal extracts was parallel to that of synthetic met-enkephalin. The concentration of met-enkephalin-like material measured in crude acetic extracts of frog adrenals (2.31 +/- 0.16 pmol/mg w. wt) was high when compared to those reported for most mammalian species. No leu-enkephalin and virtually no met-enkephalin-Arg(6)-Gly(7)-Leu(8) were detected by the corresponding radioimmunoassays. Reverse phase HPLC analysis revealed that oxidized met-enkephalin was the main form of met-enkephalin detected in acetic extracts of frog adrenals. HPLC separation showed the presence of a peptide co-eluting with synthetic met-enkephalin-Arg(6)-Phe(7). Higher molecular weight forms were also separated by HPLC. These results show the presence of both met-enkephalin and the heptapeptide met-enkephalin-Arg(6)-Phe(7) and the lack of met-enkephalin-Arg(6)-Gly(7)-Leu(8) in frog chromaffin cells.

Binding of a pure 125I-monoiodoleptin analog to mouse tissues: a developmental study.

The preparation of a pure 125I-labeled monoiododerivative of mouse leptin is described. This radiolabeled analog has been used to characterize and localize central and peripheral leptin binding sites (Ob-R) of the mouse at different stages of its development. The affinity values found in membrane homogenates of various mouse tissues are similar and range between 0.1 and 0.3 nM, indicating that all the Ob-R isoforms have a similar affinity. Leptin binding sites are highly expressed at the membrane level in lung, intestine, kidney, liver, and skin and to a lesser degree in stomach, heart, and spleen. Brain, thymus, and pancreas homogenates are devoid of any specific binding. The distribution of mouse Ob-R has also been explored by autoradiography and dipping techniques on whole mouse sections. In lung, leptin binding sites are located at the pulmonary parenchyma and at the bronchiolar epithelial level. Binding sites are expressed all along the digestive tract from the tongue to the rectum (esophagus, stomach, intestine, colon, and rectum). In muscular visceral structures (stomach, intestine, and bladder) the binding is mainly present in the lamina propria. During development, leptin receptors are early expressed in the liver, kidney, and bone. In the lung, the Ob-R level increased gradually from birth to adulthood where the expression is maximal. By contrast, leptin receptors located in the medulla of the kidney remain remarkably constant all along the development. A broad signal is present in cartilage and bone particularly in vertebrae, limb, and ribs. Interestingly, leptin receptors are barely detectable in the mouse brain except in the choroid plexus and leptomeninges, whereas in the rat brain leptin binding sites are located in the thalamus, the piriform cortex, the cerebellum (at the granular and molecular cell layer), and the pineal gland.

Purification and characterization of enkephalin-related peptides released by in vitro peptic digestion of bovine plasma proteins.

In vitro pepsin treatment of plasma proteins generates biologically active peptides such as enkephalin-related peptides. These peptides were characterized using chromatographic techniques along with a radioimmunoassay procedure involving the use of Leu-enkephalin and Met-enkephalin antisera. Serum albumin is the only existing source of Met-enkephalin-immunoreactive peptides. One of these peptides consists of nine residues with the sequence NH2-Glu-Lys-Leu-Gly-Glu-Tyr-Gly-Phe-Gln; a second immunoreactive peptide might be the hexapeptide NH2-Gly-Glu-Tyr-Gly-Phe-Gln, which has been already identified in a rat serum albumin hydrolysate. Our results indicate that immunoglobulins constitute the main source of Leu-enkephalin-immunoreactive peptides. Immunoreactive NH2-Tyr-Phe-Leu was isolated from pepsin-treated bovine immunoglobulins. Binding experiments and cyclic nucleotide measurements suggested that this peptide was an enkephalin-related peptide. Similar experiments could be carried out to identify the proteins that contain enkephalin-like peptide sequences with the view to investigating the various biological processes occurring in enzymatically treated proteins.

Production and characterization of site-directed antibodies against dermorphin and dermorphin-related peptides.

To detect and purify endogenous dermorphin-like molecules in mammalian tissues, an immunological approach was developed. Site-directed antibodies against synthetic dermorphin and related dermorphin peptides were produced. The immunogenic forms of dermorphin were selected to obtain antibodies recognizing different epitopes overlapping the whole dermorphin molecule. One of them specifically recognized the crucial "opioid message" (the N-terminal part of the molecule), which is required for a ligand to exert its full opioid activity. The validity of our immunological approach was analyzed by studying the dermorphin-related peptide distribution in Phyllomedusa sauvagei skin. The finding that tetrapeptide Y-A-G-F-OH was present in Phyllomedusa sauvagei extracts suggested that either the Tyr3-Pro6 peptidic bond may be relatively unstable or endogenous proteolytic enzymes present in Phyllomedusa skin may inactivate this peptidic bond.

Hemorphin peptides are released from hemoglobin by cathepsin D. radioimmunoassay against the C-part of V-V-hemorphin-7: an alternative assay for the cathepsin D activity.

In order to investigate the putative physiological role of the in vivo release of hemorphins from hemoglobin in tissues, an immunological approach was developed. Specific and sensitive antiserum were raised against the C-part of the V-V-hemorphin-7. The antisera recognized to the same extent the related hemorphins V-V-hemorphin-7 and L-V-V-hemorphin-7. The validity of our immunological approach was analyzed by studying the in vitro release of hemorphin from hemoglobin by cathepsin D and compared to the pepsin hydrolysis. These two enzymes led to the release of these same products suggesting that cathepsin D acted as an accurate pepsin-like enzyme. Moreover, considering the poor sensitivity of the available methods of detection for the in vitro Cathepsin D activity, our specific and sensitive V-V-hemorphin-7 radioimmunoassay seems to be a useful alternative assay for this enzymatic activity.

Changes in enkephalin immunoreactivity of sympathetic ganglia and digestive tract of the cat after splanchnic nerve ligation.

The aim of the present study was to analyze changes in the enkephalin immunoreactivity of sympathetic prevertebral ganglia coeliac plexus and inferior mesenteric ganglion) and intestinal tract (myenteric plexus and external muscle layers) in cats 2 days after left thoracic splanchnic nerve ligation, using radioimmunoassay and immunohistochemical techniques. Specific polyclonal antibodies directed against methionine- and leucine-enkephalin were used. The nerve ligation led to a considerable increase in the enkephalin immunoreactivity in the cranial part of the ligated nerves. This finding confirms the presence, in the cat, of an enkephalin output originating from thoracic spinal structures which are probably enkephalin-containing preganglionic neurons. In prevertebral ganglia the nerve ligation induced a marked decrease in the enkephalin immunoreactivity, which was probably due to the interruption of thoracic enkephalin efferents projecting towards both the coeliac plexus and the inferior mesenteric ganglion. In the digestive tract, the nerve ligation depressed the methionine-enkephalin immunoreactivity only in the gastro-duodenal region, and had no effect on the ileo-colonic region. The results of the present study add to the growing evidence that the sympathetic nervous system is involved in regulating the enteric enkephalinergic innervation, which is probably involved in controlling the intestinal motility.

Distribution of enkephalin immunoreactivity in sympathetic prevertebral ganglia and digestive tract of guinea-pigs and rats.

The aim of the present study was to determine the distribution of methionine-enkephalin (ME) and leucine-enkephalin (LE) immunoreactivity in the sympathetic prevertebral ganglia (coeliac plexus and inferior mesenteric ganglion) and in the myenteric plexus-muscular layer complex of the digestive tract in guinea-pigs and rats. This study was performed using the same immunological approaches including radioimmunoassays and HPLC characterization as those used previously on cats in order to be able to make inter-region and inter-species comparisons. In rat and guinea-pig prevertebral ganglia, the distributions of the enkephalin immunoreactivities were comparable and were characterized by a low ME/LE concentration ratio, of less than 1. In the digestive tract of rats, the enkephalin immunoreactivities were homogeneously distributed, whereas in guinea-pigs, they were found to be very low in the lower oesophageal sphincter and high in the duodenum. In both species, the ME/LE concentration ratio was around 2. The ME/LE concentration ratio determined in the present study in peripheral nervous structures was much lower than that determined previously in the rat brain. Radioimmunoassay and biochemical data might indicate that different mechanisms are responsible for the processing and/or degradation of enkephalins in the central and peripheral nervous systems. The present study provides further evidences that there are tissue- and species-dependent differences in the distribution of enkephalin immunoreactivities. These differences should be taken into consideration when dealing with the effects and the role of enkephalins in the nervous control of intestinal motility in mammals.