Search

An immunohistochemical study of opioid peptides in the hypophysis of the axolotl, Ambystoma mexicanum, was carried out with antisera against leu-enkephalin, beta-endorphin, met-enkephalin, and dynorphin A (1-8). We found leu-enkephalin immunoreactivity in some fibers of the neural lobe and the median eminence. In contrast to previous reports on mammals and other vertebrates, we found leu-enkephalin immunoreactivity in many cells scattered throughout the anterior lobe. As in other vertebrates, the beta-endorphin immunoreactivity was present in all the cells of the intermediate lobe and in a few cells of the anterior lobe. Met-enkephalin and dynorphin A (1-8) immunoreactivities were only present in the neural lobe and the median eminence. The presence of leu-enkephalin and beta-endorphin in the anterior lobe suggests that these peptides could be acting as hormones released from the hypophysis of the unmetamorphosed amphibian.

Bilateral pontine tegmental lesions produce in cats the phenomenon known as rapid eye movement (REM) sleep without atonia. During episodes of REM sleep without atonia cats are capable of exhibiting such complex behaviors as head-raising, body-righting, standing, and in some cases, walking and attacking. Since release of such behaviors implies disinhibition of specific motor systems, the purpose of this study was to determine whether the administration of chloramphenicol (CAP), which is known to attenuate the firing frequencies of cells that become activated during REM and motor activity, could reverse this phenomenon. Cats with dorsolateral tegmental pontine (DLTP) lesions producing REM without atonia were thoroughly studied in terms of the muscular and behavioral activity they displayed during REM before and after systemic CAP administration. Thiamphenicol (TAP), a CAP analogue that does not reduce neuronal firing frequency during REM sleep, was used as a control drug. The results of these experiments showed that CAP but not TAP induced a return of the atonia during REM sleep. It is suggested that the return of atonia induced by CAP in DLPT lesioned cats is caused by attenuation in the activity of medial reticular neurons which have somatotopical representation. Such cells, which have high levels of activity during REM sleep and motor activation in wakefulness, are normally overwhelmed by the inhibitory mechanism of the atonia of REM. The return of atonia and consequent reduction in complex behavior following CAP administration may be due to withdrawal of the excitatory influence of these neurons.

The in vivo spontaneous release of enkephalin in the globus pallidus of the rat increases from noon to evening by 100%; during this period the local release of exogenous gamma-aminobutyric acid (GABA) decreases by 60%. These diurnal rhythms are more marked in the K+-stimulated release: enkephalin-induced output increases 6-fold while GABA decreases 10-fold during the afternoon and evening hours. Since pallidal enkephalin and GABA are involved in the control of locomotor activity we suggest that these rhythms may be linked to the circadian changes of activity in the rat.

The brain levels of β-endorphin, α-endorphin and enkephalin were measured by radioimmunoassay after different methods of sacrifice. Microwave irradiation proved not to be better than decapitation followed by boiling of the intact tissue, the latter procedure giving values of β-endorphin 10 fold higher than decapitation alone. Concurrently when decapitation was followed by boiling, α-endorphin was no longer detected. Evaluation in brain tissue of several extraction media--phosphate buffered saline, 5% TCA, HCl methanol, and 1N HOAc--showed the last to be the most satisfactory for both β-endorphin and enkephalin. Since β-endorphin was found to be readily hydrolized by brain homogenates with consequent appearance of α-endorphin, these results indicate that disruption of tissue modifies the content of opioid peptides in brain.

A nonapeptide Thr-Ile-Ile-Asn-Val-Lys-Cys-Thr-Ser (NTX1-9) and a decapeptide Met-Asn-Gly-Lys-Cys-Lys-Cys-Tyr-Asn-Asn (NTX30-39) corresponding to the N-terminal and C-terminal sequences respectively of Noxiustoxin (NTX) were synthesized by the solid phase method of Merrifield (1963). The first synthetic peptide (NTX1-9) was shown to be toxic to mice independently of the route of administration: intraperitoneally, subcutaneously or intraventricularly (100-200 micrograms/20 g mouse weight). The second (NTX30-39) was not toxic even at higher dose (400 micrograms/20 g mouse). When the effects of the peptide NTX1-9 and of the authentic toxin (Noxiustoxin) were studied on the liberation of [3H] 4-aminobutyric acid (3H-GABA) from mouse synaptosomes, both gave essentially the same results, except that peptide NTX1-9 was needed at higher concentration. Synthetic peptide NTX30-39 had no effect in the same preparation at even higher doses. The GABA release produced by toxic peptide NTX1-9 was not affected by tetrodotoxin but was completely abolished by the presence of the K+ ionophore valinomycin, mimicking the effect of native NTX in the same system (Sitges et al., 1986). These results indicate that the toxic active site of Noxiustoxin is possibly located in or near the N-terminal amino acid portion of the molecule.

Push-pull cannulae were acutely positioned through previously implanted guides in the globus pallidus of unanesthetized freely moving cats and rats. During slow-flow perfusions, enkephalin release was detected in resting conditions and increased more than 3-fold when both 50 mM K+ and 1.8 mM Ca2+ were present in the perfusing medium. Local perfusion with veratrine also enhanced enkephalin release. Furthermore, in vivo, electrical stimulation of the rat caudo-putamen enhanced enkephalin release in the pallidum. This latter finding is consistent with a functional strio-pallidal enkephalin-containing pathway previously postulated by immunohistochemical or lesion experiments.

Noxiustoxin (NTX), a 39 amino acid peptide purified from the venom of the Mexican scorpion Centruroides noxius, has been shown to block voltage-dependent K+ currents in the squid giant axon (Possani et al., 1982; Carbone et al., 1982). Although several other drugs known as K+ channel blockers in the squid axon also act on isolated nerve terminals to produce an increase in transmitter release, these releasing effects have not been shown to be related to a decrease of K+ permeability in synaptosomes (Vizi et al., 1977; Tapia and Sitges, 1982). In this work we show that NTX increases 3H-GABA release from perfused mouse brain synaptosomes. This effect was not blocked by TTX. Ca2+ channel blockers (verapamil or Co2+) or the absence of external Ca2+ prevents the releasing effect of this toxin. NTX does not seem to induce transmitter release by directly increasing Ca2+ permeability: The K+ ionophore valinomycin completely inhibits the release induced by NTX, as well as that evoked by the K+ channel blocker 4-aminopyridine; in contrast, the release evoked by a Ca2+ ionophore is not blocked by valinomycin. These findings strongly suggest that the releasing effect of NTX is mediated by a decrease in K+ permeability. External Ca2+ is needed only in order to couple this stimulus with the release process. Consistent with this hypothesis, we present evidence that NTX blocks the efflux of 86Rb+ from synaptosomes. An extended comparison of the effect of 4- aminopyridine with that of NTX is also reported.

Toxic peptides II-9.2.2 and II-10, purified from Centruroides noxius venom, bear highly homologous N-ter-minal amino acid sequences, and both toxins are lethal to mice. However, only toxin II-10 is active on the voltage-clamped squid axon, selectively decreasing the voltage-dependent Na+ current. Here, we have tested toxins II-9 and II-10 on synaptosomes from mouse brain: both toxins increased the release of γ-[3H]aminobutyric acid ([3H]GABA). Their effect was completely blocked by tetro-dotoxin or by the absence of external Na+. Also, both toxins increased Na+ permeability in isolated nerve terminals. Besides the observation that toxin II-9 is active on synaptosomes, the effect of toxin II-10 in this preparation is opposite to that observed in the squid axon. Thus, our results reflect functional differences between the populations of Na+ channels in mouse brain synaptosomes and in the squid axon. The release of GABA evoked by these toxins from synaptosomes required external Ca2+ and was blocked by Ca2+ channel blockers (verapamil and Co2+). This latter observation is in sharp contrast to the releasing action of veratrine, which evoked release even in the absence of external Ca2+. Furthermore, the action of both C. noxius toxins was potentiated by veratrine, a result suggesting they have different mechanisms of action. Among drugs that release neuro-transmitters by increasing Na+ permeability, it is noteworthy that scorpion toxins are the only ones yet reported to have a strict requirement for external Ca2+.

Opioid peptides derived from prodynorphin were localized immunocytochemically to dentate granule cells and mossy fibers of the rat hippocampus with antisera against dynorphin A(1–17) and dynorphin B. Extracts of microdissected hippocampal regions were resolved by reverse phase and molecular exclusion chromatography to identify the molecular forms of the dynorphin A immunoreactivity and to quantify regional contents. Results demonstrated that the relative concentration of dynorphin A within each dissected region of hippocampus agreed well with the distribution of dynorphin A detected by immunocytochemical methods. Immunostaining of proenkephalin-derived opioid peptides, [Leu5]enkephalin and bovine adrenal medullary peptide-22P, was concentrated in cell bodies of the entorhinal cortex, nerve fibers in the perforant pathway, and terminals in the outer molecular layer of the dentate gyrus. Light immunostaining of granule cells and mossy fibers with these antisera was also found. The relative concentration of [Leu5]enkephalin immunoreactivity in each microdissected region of the hippocampus also agreed well with the distribution of [Leu5]enkephalin immunostaining. Chromatography of hippocampal regional extracts demonstrated that the immunoreactivity measured was due to the presence of authentic [Leu5]enkephalin. The probable neurotransmitter function of both [Leu5]enkephalin and dynorphin A was shown by their calcium-dependent release after in vitro depolarization of hippocampal tissue. The reported presence of beta-endorphin in hippocampus was not verified. Comparison of the hippocampal distribution and content of prodynorphin and proenkephalin-derived opioids suggests that separate populations of neurons containing these two peptide families form distinct neurotransmitter systems of roughly equal concentration.

— Seven N-(5′-phosphopyridoxyl) amino acids, reduced analogs of the glutamate-pyridoxal phosphate Schiff base, were synthesized and purified. All of them inhibited mouse brain glutamate decarboxylase activity. The four most potent inhibitors were the aminooxyacetate, GABA, cysteinesul-finate and glutamate derivatives, and the effect of these compounds was studied kinetically. The inhibition produced was in all cases mixed function with respect to glutamate and competitive with respect to pyridoxal phosphate. The inhibition kinetics were non-linear. These results are interpreted in terms of an ordered binding of pyridoxal phosphate and glutamate to the enzyme. Furthermore, they are consistent with previous findings suggesting the existence of two kinds of glutamate decarboxylase activity differing in their dependence on free pyridoxal phosphate.

The effects of intracerebroventricular injection of colchicine or cytochalasin B were compared on radioimmunoassayable endorphin and enkephalin in several rat brain regions and pituitary gland. In addition, colchicine effects on brain immunoreactivity were also assessed by immunocytochemistry. Colchicine treatment increased endorphin levels in the hypothalamus without changing pituitary levels. Cytochalasin B did not change brain endorphin levels but increased pituitary content. These contrasting drug effects probably reflect the neuronal nature of the brain endorphin system and the endocrine nature of the pituitary system. Colchicine treatment also increased enkephalin radioimmunoreactivity in the hypothalamus with a corresponding decrease in the pituitary. This colchicine effect is compatible with reduced axonal transport of enkephalin in a hypothalamo-neurohypophysial pathway: immunocytochemistry after colchicine reveals stained hypothalamic neurons not seen normally. However, colchicine treatment did not alter enkephalin radioimmunoassay values in the other brain regions, even when these regions also revealed groups of cell bodies not seen in normal brain. This inconsistency between unchanged assay values and increased cell reactivity might be explained in part if most enkephalin neurons have short axons; thus, colchicine arrest of axonal transport might cause a local redistribution of enkephalin immunoreactive material that would not be detected by radioimmunoassay due to the limitations of tissue dissection. These results demonstrate the enhanced interpretive value of data obtained from both radioimmunoassay and immunocytochemistry.

The presence of the opioid peptides [5-methionine] -enkephalin (Met-enkephalin) and [5-leucine] -enkephalin (Leu-enkephalin) (7) in several regions of the central nervous system (17) and the immunohistochemical evidence for their location in fibers and cell bodies (6) first supported their consideration as putative central neurotransmitters. The search for a model system to study the central neurobiology of the enkephalins has led to the striatum and especially the globus pallidus because this region has the highest content of immuno-reactive enkephalin and enkephalin-containing fibers. The early studies in one of our laboratories (8) showed that during in vitro perfusion of slices from rat globus pallidus, enkephalin-like immuno-reactive material was released by 50 mM K+ in a Cat2+dependent manner. In subsequent work (1) we have analyzed the composition of these enkephalin-like substances, both in fresh and perfused tissue and in the released material, and also some aspects of the metabolic changes of enkephalins during in vitro release experiments. Based on these studies, we have explored the basic characteristics of enkephalin release from pallidal tissue in unanesthetized-freely moving animals using a push-pull cannula perfusion technique. Our results show that enkephalins can be released from the pallidum, not only by chemical stimulation of the local fibers but also, in a more physiological manner, by electrical stimulation of the caudate nucleus.

Radioimmunoassay and microdissection procedures were used to study the perinatal development of the endorphin- and enkephalin-containing systems in the rat brain. In contrast to values reported on adult rat, endorphin levels are much higher than enkephalin levels on embryonic day 16. The highest endorphin values are found in the diencephalon, midline telencephalon and medulla-midbrain regions. Perinatally, enkephalin content increases at a faster rate than endorphin in all brain regions, producing a marked drop of the endorphin/enkephalin ratios. Between postnatal days 6 and 25, both endorphin and enkephalin levels increase, approaching their adult distribution pattern. No correlation was found between regional distributions or rates of increase of endorphin and enkephalin in any of these developmental stages, suggesting that the two peptide systems develop independently from each other.

WE have previously reported that significant amounts of immunoreactive (ir)-Leu5-enkephalin were present in extracts of the neurointermediate lobe of the rat pituitary1. Negligible amounts of the pentapeptide were detected in the anterior lobe. In these assays, the concentration of Leu5-enkephalin in the neurointermediate lobe was higher than in the globus pallidus, the brain region reported to contain the densest enkephalinergic innervation2. The high content of (ir)-Leu-enkephalin in the neurointermediate lobe of the pituitary led us to further investigation of its distribution and possible function. We report here that (ir)-enkephalins in the pituitary are concentrated in nerve fibres projecting from the hypothalamus to the pars nervosa and that this pathway may be involved in the regulation of neurohypophysial neurosecretion.

Endogenous [5-methionine]enkephalin (Metenkephalin) and [5-leucine]enkephalin (Leu-enkephalin) are released from perfused slices of rat globus pallidus by increased K + in a Ca 2+ -dependent manner. Tissue perfused for 40 min contained only 26% of the Met-enkephalin and 44% of the Leu-enkephalin found in the freshly dissected tissue. After perfusion, the mean (±SEM) ratio (wt/wt) of Met-enkephalin to Leu-enkephalin was 3.4 ± 0.2 compared with 5.8 ± 0.2 in the fresh tissue. The degradation of trace amounts of synthetic [ 3 H]enkephalins in the perfusing medium during stimulated release seems to reflect the accelerated degradation of enkephalin released from the tissue: 63% of the Met-enkephalin and 23% of the Leu-enkephalin were degraded in a medium containing bacitracin (30 μg/ml). The mean ratio (wt/wt) of the Met-enkephalin to the Leu-enkephalin recovered after release by exposure of slices to 50 mM K + was 2.7 ± 0.3. When perfusates were corrected for degradation, this ratio increased to about 5.5 which is higher than that found in the perfused tissue. The differences in release, tissue loss, and catabolism of the two enkephalins may be reflecting differences in the metabolic systems operating on the pentapeptides, but this interpretation will have to be validated by in vivo release experiments. In any event these observations strongly suggest that both enkephalins can be considered candidate neurotransmitters in the rat globus pallidus.

The distribution of β-endorphin and enkephalin in the pigeon forebrain by immunohistochemistry and radioimmunoassay is essentially analogous to mammals. Both endorphin- and enkephalin-reactive fibers have a similar periventricular distribution, but the enkephalin fibers are more extensive and are also found in the paleostriatum, limbic regions and brain stem, pituitary stalk and notably, penetrating the organum vasculosum hypothalami. There was poor correlation between endorphin and enkephalin regional contents by radioimmunoassay. In contrast, a highly significant correlation was observed between Met 5 -enkephalin and Leu 5 -enkephalin regional distribution. These data support the view that enkephalin neurons and endorphin neurons are independent central neuronal systems.