The influence of experimental inflammation and axotomy on leucine enkephalin (leuENK) distribution in intramural nervous structures of the porcine descending colon.

BACKGROUND: The enteric nervous system (ENS), located in the intestinal wall and characterized by considerable independence from the central nervous system, consists of millions of cells. Enteric neurons control the majority of functions of the gastrointestinal tract using a wide range of substances, which are neuromediators and/or neuromodulators. One of them is leucine-enkephalin (leuENK), which belongs to the endogenous opioid family. It is known that opioids in the gastrointestinal tract have various functions, including visceral pain conduction, intestinal motility and secretion and immune processes, but many aspects of distribution and function of leuENK in the ENS, especially during pathological states, remain unknown. RESULTS: During this experiment, the distribution of leuENK - like immunoreactive (leuENK-LI) nervous structures using the immunofluorescence technique were studied in the porcine colon in physiological conditions, during chemically-induced inflammation and after axotomy. The study included the circular muscle layer, myenteric (MP), outer submucous (OSP) and inner submucous plexus (ISP) and the mucosal layer. In control animals, the number of leuENK-LI neurons amounted to 4.86 ± 0.17%, 2.86 ± 0.28% and 1.07 ± 0.08% in the MP, OSP and ISP, respectively. Generally, both pathological stimuli caused an increase in the number of detected leuENK-LI cells, but the intensity of the observed changes depended on the factor studied and part of the ENS. The percentage of leuENK-LI perikarya amounted to 11.48 ± 0.96%, 8.71 ± 0.13% and 9.40 ± 0.76% during colitis, and 6.90 ± 0.52% 8.46 ± 12% and 4.48 ± 0.44% after axotomy in MP, OSP and ISP, respectively. Both processes also resulted in an increase in the number of leuENK-LI nerves in the circular muscle layer, whereas changes were less visible in the mucosa during inflammation and axotomy did not change the number of leuENK-LI mucosal fibers. CONCLUSIONS: LeuENK in the ENS takes part in intestinal regulatory processes not only in physiological conditions, but also under pathological factors. The observed changes are probably connected with the participation of leuENK in sensory and motor innervation and the neuroprotective effects of this substance. Differences in the number of leuENK-LI neurons during inflammation and after axotomy may suggest that the exact functions of leuENK probably depend on the type of pathological factor acting on the intestine.

Oxidative and nitrative modifications of enkephalins by human neutrophils: effect of nitroenkephalin on leukocyte functional responses.

Neutrophils play a major role in acute inflammation by generating reactive oxygen/nitrogen species. Opioid peptides, including enkephalins, are present at inflammation sites. Neutrophils contribute to protect against inflammatory pain by releasing opioid peptides. In this investigation, the ability of human polymorphonuclear cells to induce oxidative and nitrative modifications of Leu-enkephalin has been investigated in vitro. Activated human neutrophils mediate the oxidation of Leu-enkephalin resulting in the production of dienkephalin. In the presence of nitrite at concentrations observed during inflammatory and infectious process (10-50 µM), nitroenkephalin, a nitrated derivative of Leu-enkephalin, is additionally formed. The yield of nitroenkephalin increases with nitrite concentration and is significantly inhibited by the addition of catalase or 4-aminobenzoic acid hydrazide (ABAH), a specific inhibitor of peroxidases. These results suggest that neutrophils induce nitration of Leu-enkephalin by a mechanism that is dependent on myeloperoxidase activity and hydrogen peroxide. Oxidative/nitrative modifications of Leu-enkephalin have been also evidenced when cells were treated with the NO-donor molecule, DEANO. The nitrated enkephalin has been examined for its effect on leukocyte functional responses. The data reveal that nitroenkephalin at micromolar concentrations inhibits superoxide anion generation and degranulation of azurophilic granules of human polymorphonuclear cells. Moreover, nitroenkephalin inhibits spontaneous apoptosis of neutrophils, as evaluated by measuring caspase-3 activity. Collectively, our data indicate that the nitrated enkephalin attenuates neutrophil activation and promotes the short-term survival of these cells, suggesting a possible role of the nitrocompound in the efficiency and resolution of inflammatory processes.

Plasma endogenous enkephalin levels in early systemic sclerosis: clinical and laboratory associations.

OBJECTIVE: Met- and leu-enkephalins are endogenous opioid neuropeptides with potent analgesic, vasoactive, immunomodulatory and anti-apoptotic properties. We hypothesised that clinical or immunological variables of early systemic sclerosis (SSc) might be correlated to plasma enkephalin levels. METHODS: Plasma samples were collected at study entry of the Genetics versus Environment in Scleroderma Outcomes Study (GENISOS) cohort (early SSc, n=116). Plasma met-enkephalin and leu-enkephalin levels (microg/ml) were measured by high performance liquid chromatography (HPLC) and correlated to clinical and laboratory parameters in the GENISOS database. Statistical analyses were performed by nonparametric Wilcoxon rank sum tests and Pearson correlation coefficients. RESULTS: Significantly lower plasma met-enkephalin levels were associated with anti-topoisomerase-I seropositivity (6+8.3 vs. 14.9+22.8 microg/ml, p=0.02). Plasma leu-enkephalin levels were significantly higher in SSc patients with digital pulp loss (95.6+130 vs. 64.9+101 microg/ml, p=0.02). Lower mean plasma met-enkephalin levels and inversely higher leu-enkephalin levels were noted in SSc patients with Raynaud's phenomena (p=NS). CONCLUSION: The associations of plasma enkephalin levels to immunologic or clinical pathologies may underscore their vasogenic or fibrogenic significance and potential as therapeutic targets in early SSc.

Distribution of enkephalin immunoreactivity in germinative cells of developing rat cerebellum.

The cellular distribution of enkephalin, an endogenous opioid, in the developing rat cerebellum was determined by immunocytochemistry. Methionine and leucine enkephalin were concentrated in the external germinal layer, a matrix of proliferative cells; staining was confined to the cortical cytoplasm. Enkephalin was not detected by immunocytochemistry in differentiated neural cells. These results indicate that endogenous opioids are involved specifically in early phases of nervous system development, particularly cell proliferation and differentiation.

Impulse activity differentially regulates [Leu]enkephalin and catecholamine characters in the adrenal medulla.

Regulation of the putative peptide neurohumour [Leu]enkephalin and the catecholaminergic enzymes tyrosine hydroxylase and phenylethanolamine-N-methyl-transferase was examined in the rat adrenal medulla in vivo and in vitro. Surgical denervation of the adrenal gland or pharmacologic blockade of synaptic transmission, treatments known to decrease catecholamine traits, increased [Leu]enkephalin content. Medullas explanted to culture exhibited a 50-fold rise in [Leu]enkephalin in 4 days, whereas tyrosine hydroxylase remained constant, and phenylethanolamine-N-methyltransferase decreased to a new baseline level. Veratridine-induced depolarization prevented the accumulation of [Leu]enkephalin, an effect that was blocked by tetrodotoxin, which antagonizes transmembrane Na+ influx. These studies suggest that enkephalinergic and catecholamine characters are differentially regulated by impulse activity and depolarization in the adrenal medulla.

Leucine-enkephalin promotes wound repair through the regulation of hemidesmosome dynamics and matrix metalloprotease.

The skin responds to environmental stressors by coordinated actions of neuropeptides and their receptors. An endogenous peptide for δ-opioid receptor (DOPr), Leu-enkephalin (L-ENK), is expressed in the skin and its expression is altered in pathological conditions. Although the importance of DOPr is rapidly gaining recognition, the molecular mechanisms underlying its effects on wound healing are largely undefined. We show here that L-ENK induced activation of Erk, P90(RSK), and Elk-1 and promoted the disruption of hemidesmosomes and the expression of matrix metalloprotease (MMP)-2 and MMP-9, important processes for wound healing. Treatment with Erk inhibitor blocked activation of P90(RSK) and Elk-1 and significantly blunted wound repair. Therefore, our results suggest that activation of Erk and its downstream effectors, P90(RSK) and Elk-1, are critical for DOPr-mediated skin homeostasis.

Topography and associations of leu-enkephalin and luteinizing hormone-releasing hormone neuronal systems in the human diencephalon.

Although several studies indicated that leu-enkephalin controls gonadal function, the morphological substrate of this modulation is unknown. To reveal potential interaction sites between leu-enkephalin and LH-releasing hormone (LHRH) in the hypothalamus, the distribution and connections of leu-enkephalin-immunoreactive (IR) and LHRH-IR systems were examined in the human diencephalon using double-label immunohistochemistry. First the leu-enkephalin-IR and LHRH-IR neural elements were mapped, then the maps of the two different neurotransmitter systems were superimposed unveiling the overlapping areas. The putative juxtapositions between leu-enkephalin-IR and LHRH-IR structures were revealed with double label immunocytochemistry. Close contacts were detected in the medial preoptic area and in the infundibulum/median eminence. In these areas, diaminobenzidine-silver-intensified, black leu-enkephalin-IR fibers abutted fusiform, brown, diaminobenzidine-labeled LHRH neurons often forming multiple contacts. Examination of semithin sections of these close associations with the aid of oil immersion revealed no cleft between the contacting LHRH-IR and leu-enkephalin-IR elements. Our findings indicate that the juxtapositions between LHRH-IR and leu enkephalin-IR neurons may be functional synapses forming the morphological substrate of the leu-enkephalin-modulated LHRH secretion in the human diencephalon. Moreover, the wide distribution of leu-enkephalin-IR elements suggests leu-enkephalin control of other diencephalic functions as well.

Synaptic organization of enkephalinlike-immunoreactive amacrine cells in the goldfish retina.

Immunoelectron microscopy was used to examine the synaptic organization of enkephalinlike-immunoreactive amacrine cells in the goldfish retina. Enkephalin-immunostained processes sometimes contained dense-cored vesicles (115-145 nm) in addition to a generally homogeneous population of small, round, clear synaptic vesicles. A total of 194 synaptic relationships were observed that involved the immunostained processes of enkephalin-amacrine cells. The large majority of these were observed in sublayer 5 of the inner plexiform layer. In greater than 95% of the synaptic relationships, the enkephalin-immunostained profile served as the presynaptic element. In 58.8% of these relationships, enkephalin processes synapsed onto amacrine cell processes, while 30.4% of their synapses were onto processes that lacked synaptic vesicles. They also occasionally formed synaptic contacts (6.7%) onto the somas of cells located either in the inner nuclear or in the ganglion cell layers. Enkephalin profiles received synaptic input only from amacrine cells (4.1%), while no direct synaptic interaction was observed between enkephalin processes and bipolar cells. However, in sublayer 1, enkephalin profiles were found to synapse onto amacrine cell processes that were presynaptic to bipolar cell terminals. In the proximal inner plexiform layer, enkephalin processes were presynaptic to amacrine cell processes that as a group surrounded and sometimes provided synaptic input to extremely large and round bipolar cell endings.

Serotoninergic, noradrenergic, and peptidergic innervation of Onuf's nucleus of normal and transected spinal cords of baboons (Papio papio).

We have investigated with light and electron microscope immunocytochemistry the aminergic and peptidergic innervation of Onuf's nucleus in adult baboons. This nucleus, located in the ventrolateral part of the sacral spinal cord (S2 and S3), is considered to control urethral and anal sphincters and penile muscles. By comparison of intact and transected spinal cords, we have found that serotoninergic innervation has two origins: first, supraspinal, innervating the whole nucleus, with a possible predominance in the dorsal half; and second, intraspinal, corresponding to the ventral half of the nucleus. Thyrotropin-releasing hormone innervation appears largely coincident with serotonin, both in intact and transected spinal cords. Noradrenaline is exclusively of supraspinal origin, as attested by its disappearance below the level of the section. Substance P, calcitonin gene-related peptide, and Leu- and Met-enkephalin, which profusely innervate Onuf's nucleus, are on the contrary not affected by the transection. They most likely originate from the cord itself or the dorsal root ganglia. Thus, Onuf's nucleus innervation in the baboon arises both from supraspinal and intraspinal sources. The present study provides an anatomical basis for both voluntary and reflex controls of excretory and sexual functions in a primate. The same neurotransmitter (serotonin) according to its cell origin and discrete topography could exert different influences upon the same effector system.

Distribution of opioid peptides in the preoptic region: immunohistochemical evidence for a steroid-sensitive enkephalin sexual dimorphism.

The distribution of cells and fibers that contain opioid peptides within the preoptic region of the rat was examined immunohistochemically. Cells and/or fibers that contain peptides derived from each of the three major opioid peptide families were differentially stained by using antisera that recognize unique derivatives of each precursor molecule and do not cross-react with members of the other opioid peptide families. A beta-endorphin (beta E) antiserum was used to stain fibers that contain peptides derived from the proopiomelanocortin molecule, and dynorphin-containing cells were identified by using an antiserum directed toward dynorphin B (Dyn B) that does not show detectable cross-reactivity with enkephalin-related peptides. An antiserum raised against peptide E (PE), which does not appear to cross-react significantly with dynorphin peptides, was used to localize enkephalin cells and fibers. Each family of opioid peptides showed a unique distribution in the preoptic region. beta E-immunoreactive fibers were primarily localized to the preoptic part of the periventricular nucleus, with moderate densities of fibers contained in the anteroventral periventricular nucleus (AVPv) and medial preoptic nucleus (MPN). Dyn B-immunoreactive fibers showed a somewhat more uniform distribution throughout the region, and only a few Dyn B-stained cells bodies were found within the medial preoptic area. In contrast, the preoptic region contained hundreds of PE-immunoreactive cells, which were particularly numerous within the AVPv, MPN, and anterodorsal preoptic nucleus. The AVPv and MPN also contained discretely localized plexuses of PE-stained fibers. Although the overall distributions of opioid peptide-containing fibers within the preoptic region were quite similar in male and female rats, differential distributions of fibers were found in certain nuclei such as the AVPv and MPN, and they were correlated with previously identified cytoarchitectonic sexual dimorphisms. Such differential distributions were particularly distinct for enkephalin-containing fibers. Although the AVPv is larger in female rats, it contained more PE-immunoreactive cell bodies in male rats, and we have shown here that this sexual dimorphism appears to be at least partially dependent on perinatal levels of gonadal steroids. In contrast, no difference in the number of PE-stained cells was found within the anterodorsal preoptic nucleus of male and female animals, indicating that sexual differences are not a general characteristic of enkephalinergic cells in the preoptic region of the rat.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of some peptides (substance P, [Leu]enkephalin, [Met]enkephalin) in the brain stem and spinal cord of a lizard, Varanus exanthematicus.

The distribution of substance P-like and [Leu]- and [Met]enkephalin-immunoreactive cell bodies, fibers and terminal structures in the brain stem and spinal cord of a lizard, Varanus exanthematicus, was studied with the indirect immunofluorescence technique, using antibodies to these peptides. Substance P-like immunoreactive cell bodies were found in the hypothalamus, in a periventricular cell group in the rostral mesencephalon, in the interpeduncular nucleus, in and ventral to the descending nucleus of the trigeminal nerve, in and directly ventral to the nucleus of the solitary tract, scattered in the brain stem reticular formation and in the trigeminal and spinal ganglia. A rather widespread distribution of substance P-like immunoreactivity was found in the brain stem and spinal cord, mainly concentrated in striatotegmental projections related to visceral and/or taste information (nucleus of the solitary tract, parabrachial region), in the descending nucleus of the trigeminal nerve and in the dorsal horn of the spinal cord (areas I and II). In the spinal cord also around the central canal (area X and adjacent parts of area V-VI) a distinct substance P innervation was found. The ventral horn receives only a very sparse substance P innervation. The distribution of [Leu]- and [Met]enkephalin in the brain stem and spinal cord of Varanus exanthematicus is less impressive than that of substance P. Enkephalinergic cell bodies were found particularly in the caudal hypothalamus. Small populations of enkephalinergic cell bodies were found in the vestibular nuclear complex, in the nucleus of the solitary tract, in and around the descending nucleus of the trigeminal nerve and throughout the rhombencephalic reticular formation. Enkephalins are likely to be present in efferent projections of the striatum, in projections related to taste and/or visceral information (nucleus of the solitary tract, parabrachial region) and in descending pathways to the spinal cord. Enkephalinergic fibers are present in the lateral funiculus and enkephalin-immunoreactive cell bodies are found in the reticular formation, particularly the inferior reticular nucleus which is known to project to the spinal cord. In the spinal cord enkephalinergic terminal structures were found especially in the superficial layer of the dorsal horn (areas I and II) and around the central canal. The ventral horn including the motoneuron area receives only a relatively sparse enkephalinergic innervation.

Opioid neurons and pain modulation: an ultrastructural analysis of enkephalin in cat superficial dorsal horn.

To clarify the circuitry through which opioid compounds modulate spinal and trigeminal nociceptive transmission, we have examined the synaptic associations formed by leucine-enkephalin-containing (enkephalin) neurons in the superficial dorsal horn of the cat. As described previously, punctate enkephalin immunoreactivity is concentrated in the marginal layer (lamina I) and in both the outer and inner layers of the substantia gelatinosa (lamina IIo and IIi). In colchicine treated cats, enkephalin perikarya are most numerous in lamina I and at the border between laminae I and II. Ultrastructural analysis reveals that enkephalin cells receive a diverse afferent input. The majority of afferent inputs are presynaptic to the enkephalin dendrites; few axosomatic synapses are seen. Among these presynaptic axonal profiles are unlabeled axons which resemble primary afferent terminals, including the characteristic central axonal varicosity. Enkephalin dendrites are also postsynaptic to enkephalin immunoreactive axons. Two types of enkephalin axonal profiles appear in the superficial dorsal horn. Class I profiles are only found in lamina I. These are large profiles which form few synapses; those synapses made are axodendritic. Class II enkephalin axons are smaller and are distributed in both layers I and II. While Class II axons most commonly form axo-dendritic synapses, they also form axo-axonic synapses with flat vesicle-containing profiles; the latter are generally presynaptic to the enkephalin terminals. Serial analysis further revealed that both the enkephalin and the flat vesicle-containing profile synapse onto a common dendrite. Although enkephalin axons frequently lie adjacent to round vesicle-containing profiles, anatomical evidence that opioid axons form synapses with this type of ending was not found. An additional type of enkephalin vesicle containing-profile is found in layer IIi; its morphological features do not clearly distinguish its axonal or dendritic origin. These endings are typically postsynaptic to unlabelled central endings, and provide minimal presynaptic input to other elements in the neuropil. Like some class II axons, these labelled profiles contain vesicles which cluster at the membrane immediately adjacent to unlabelled central axons. These results indicate that spinal enkephalin neurons receive a variety of synaptic inputs. These include inputs which may derive from primary afferent axons. Enkephalin neurons, in turn, influence nociceptive transmission predominantly through postsynaptic mechanisms. Finally, while we did not observe enkephalin terminals presynaptic in an axoaxonic relationship, the possibility that enkephalin neurons modulate the excitability of fine fiber nociceptive and nonnociceptive afferents via "nonsynaptic interactions" is discussed.

Evidence for localized and discrete roles for enkephalins during memory formation in the chick.

This study examined effects on memory formation produced by [Leu]enkephalin and [Met]enkephalin administration in 2 regions of the 2-day-old chick brain involved in memory formation: the intermediate medial hyperstriatum ventrale (IMHV) and the lobus parolfactorius (LPO). Basal concentrations of endogenous [Leu]enkephalin and [Met]enkephalin were determined for 5 brain regions, and effects of 1-trial peck-avoidance training on enkephalin concentrations were examined in the IMHV and LPO. [Leu]enkephalin was amnestic when administered in the IMHV but not in the LPO. In contrast, [Met]enkephalin may be amnestic when administered in the LPO but not in the IMHV. Training decreased [Met]enkephalin concentration in the LPO but not in the IMHV. Training had no effect on [Leu]enkephalin concentration in either the IMHV or the LPO. Thus, amnestic effects of [Leu]- or [Met]enkephalin administration are brain-region specific. Regional activity of endogenous [Met]enkephalin during memory formation is consistent with localized amnestic effects produced by [Met]enkephalin administration.

Stimulation and antagonism of opioid delta-receptors produce opposite effects on active avoidance conditioning in mice.

The effects of opioid delta-receptor activation on conditioning in a one-way active avoidance paradigm were investigated in mice. Peripheral administration of 30 and 100 micrograms/kg of [Leu5]enkephalin (LE) and 11.6 micrograms/kg of [D-Pen2,D-Pen5]enkephalin (DPDPE), a synthetic enkephalin analog with high delta-selectivity, impaired acquisition of avoidance responding. The dose response functions for both peptides were U-shaped. Deficits in responding were also present 24 hours after training, which suggests that the impaired performance observed during training was not an impairment in the ability of the mice to perform. Also, neither LE nor DPDPE decreased footshock sensitivity or open-field activity, which rules out analgesia and decreased activity levels as likely explanations for the deficits in conditioning. Finally, antagonizing endogenous ligands with the delta-selective antagonist ICI 154,129 enhanced acquisition, an effect opposite that produced by LE and DPDPE. The results suggest that activation of delta-receptors is normally involved in the modulation of active avoidance learning.

Basal forebrain neurons and memory: a biochemical, histological, and behavioral study of differential vulnerability to ibotenate and quisqualate.

The differential vulnerability of basal forebrain cells to ibotenate (IBO) or quisqualate (QUIS) was investigated in rats. IBO was also coinjected with cystine (CYS) or zinc (Zn). Cortical choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) activity, neurotensin receptors, and high-affinity choline uptake sites were quantified in conjunction with radioimmunoassays for neurotensin, substance P, and somatostatin; immunocytochemistry for neurotensin-, somatostatin-, Leu-enkephalin-, and ChAT-positive cells; and in situ hybridization histochemistry of somatostatin, substance P, and enkephalin mRNAs. Compared with the performance of controls, continuous alternation performance in a T maze of IBO+Zn or IBO+CYS rats was better than that of IBO rats, whereas the performance of QUIS rats was unimpaired. Of those neurotransmitter systems examined, only ChAT-immunoreactive cells were vulnerable to IBO or QUIS. However, cholinergic cell loss did not correlate with impaired performance.

Enkephalinergic nerve terminals target inhibitory interneurons in the rat hippocampus.

Using correlated light and electron microscopic preembedding enkephalin immunocytochemistry combined with post-embedding GABA immunogold staining, we found morphological evidence of a direct connection between the enkephalinergic and GABAergic systems in the rat hippocampus. Enkephalin-immunoreactive boutons were found to be presynaptic to GABA-immunoreactive postsynaptic profiles, establishing type 2 symmetrical synapses on GABA-positive cell bodies and dendritic shafts in strata radiatum and lacunosum moleculare of the CA1 region. Thirty-six percent of all studied postsynaptic targets (n = 40) were non-pyramidal, including all somatic (n = 7) and 47% of the dendritic (n = 13) postsynaptic targets. The remaining 64% consisted of pyramidal dendritic shafts and spines. These results support previous physiological experiments suggesting that the opioidergic system takes part in disinhibitory processes in the hippocampal formation.

Antisense oligodeoxynucleotide to the CCKB receptor produces naltrindole- and [Leu5]enkephalin antiserum-sensitive enhancement of morphine antinociception.

Cholecystokinin (CCK) has been shown to attenuate, while CCK antagonists enhance, the antinociceptive activity of morphine, suggesting that this peptide may act as an endogenous modulator of the opioid system. Here, we have investigated the effects of administration of a synthetic oligodeoxynucleotide (oligo) complementary to the 5' coding region of the cloned mouse CCKB receptor (antisense), or a mismatch oligo, on the antinociceptive effects of morphine. Intracerebroventricular (i.c.v.) treatment of mice with CCKB antisense, but not mismatch, oligo for 3 days resulted in an enhancement of the antinociceptive potency of i.c.v. morphine, as indicated by an approximately 6-fold leftward shift of the dose-effect curve. The antinociceptive effects of morphine in control and CCKB antisense-treated animals were investigated in the presence or absence of naltrindole, an opioid delta receptor antagonist, as well as in the presence or absence of antisera directed against either [Leu5]- or [Met5]enkephalin. The enhanced potency of morphine in mice pretreated with CCKB antisense oligo was blocked by a delta-selective dose of naltrindole and antisera to [Leu5]enkephalin, but not [Met5]enkephalin; naltrindole, or antisera towards [Leu5]enkephalin or [Met5]enkephalin did not produce antinociceptive effects when given alone and did not alter the antinociceptive actions of morphine in control mice. These data suggest that CCK may act via CCKB receptors to tonically inhibit the release of [Leu5]enkephalin, or a [Leu5]enkephalin-like peptide. The enhancement of morphine antinociception seen in the presence of blockade of the CCKB receptor may be the result of the well-known enhancement of morphine antinociception by opioid delta agonists.

Colocalized peptides in gonadotrophs: LeuEnkephalin and ACTH interact differently on GnRH induced LH and FSH release.

Two peptides that have been previously colocalized in the gonadotrops (LeuEnkephalin with LH and ACTH with FSH) can stimulate the release of gonadotropins in primary culture of anterior pituitary cells. In presence of both substances, and in contrast to LHRH induced secretion, the augmentation of LH release is never dose dependent. It is always significantly higher than controls, for high (10(-6) M) or low concentrations (10(-12) M). Prolactin release is only modified in presence of Leu-Enkephalin. Met-Enkephalin does not increase the liberation of LH, FSH, or Prolactin in vitro. Furthermore, while Leu-Enkephalin can enhance the stimulating effect of LHRH, ACTH has a negative interaction with LHRH, when these two peptides are added together in incubation medium. These results demonstrate that Leu-Enkephalin and ACTH can in vitro modulate the release of gonadotrophins at the pituitary level suggesting a physiological role for such colocalization.

Regional brain IR-Met-, IR-Leu-enkephalin concentrations during progress and full electrical amygdaloid kindling.

Using amygdaloid kindling in chronic rats, we were able to observe behavioral, electrographic and IR-Met- and IR-Leu-enkephalin changes throughout the progress of different stages of convulsive activity. Rats presenting the initial stages of kindling, rats presenting the first generalized motor seizure, and rats with at least 10 generalized seizures were sacrificed 24 h after the last stimulus; also rats with at least 10 generalized seizures but sacrificed 21 days after the last seizure were compared with control and sham-operated groups of rats. The IR-Met and IR-Leu enkephalin concentrations in each group were measured in the striatum, amygdala, hypothalamus, medulla oblongata (including pons), hippocampus, mid-brain, spinal cord and cerebral cortex. A progressive increase in IR-Leu-enkephalin in amygdala and hippocampus was observed over the course of kindling. These increases remained until 21 days after rats were fully kindled (at least 10 generalized seizures). We observed increased and decreased concentration of each peptide in different regions. We discussed the regional and the differential effects of each peptide. The increased concentrations in limbic structures were associated with the amygdaloid increased excitability through the kindling process. We suggest that the decreases in concentrations are related with structures involved in the output behavior manifestations produced by kindling stimulation.

Inhibition of gastric acid secretion by immunoneutralization of endogenous brain thyrotropin-releasing hormone.

Previous studies have shown that intracisternal (i.c.), but not intravenous administration of thyrotropin-releasing hormone (TRH), an endogenous tripeptide (pGlu-His-Pro-NH2), produces a time-, dose-dependent and vagus-mediated stimulation of acid secretion in rats. This study was designed to test the hypothesis that endogenous brain TRH plays a role in regulation of acid secretion in the pylorus-ligation model. In confirmation of previous reports, i.c. TRH (1 microgram) significantly (P less than 0.01) stimulated gastric acid output, gastric secretory volume and decreased gastric intraluminal pH. Intracerebroventricular (i.c.v.) infusion of TRH antiserum (anti-TRH) 30 min prior to pyloric occlusion significantly reduced acid output, secretory volume and raised gastric pH. This inhibitory gastric acid secretory response to i.c.v. anti-TRH appears to be specific since i.c.v. infusion of normal rabbit serum or antisera raised against neurotensin (NT), Leu-enkephalin (L-enk), gonadotropin-releasing hormone (GnRH), somatostatin (SRIF) and alpha-melanocyte stimulating hormone (alpha-MSH) were without measurable effect. The findings of this study indicate that endogenous brain TRH, but not NT, L-enk, GnRH, SRIF or alpha-MSH plays a physiological role in regulation of acid secretion.