Alejandro Bayón

Hypothalamic enkephalin neurones may regulate the neurohypophysis.

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.

Perinatal development of the endorphin- and enkephalin- containing systems in the rat brain

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.

Radioimmunoassay of brain peptides: Evaluation of a methodology for the assay of β-endorphin and enkephalin

Abstract 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.

β-Endorphin: cellular localization, electrophysiological and behavioral effects

Tremendous excitement has been generated by the isolation, purification, and subsequent synthesis of the opioid peptides. Our collaborative efforts have been directed at the questions of where β-endorphin is stored in brain and pituitary, how such structures are related to those storing the enkephalins and possibly other biogenic substances, and the functions of these peptides expressed through their electrophysiological properties. Although none of these questions is yet completely answered, sufficient data have been accumulated (Bloom et al., 1976, 1977a, b; Guillemin et al., 1977a, b, c; Rossier et al., 1977a, b, c; Nicoll et al., 1977; Henriksen et al., 1977; French et al., 1977) to enable us to give this overview and progress report.

Redistribution of endorphin and enkephalin immunoreactivity in the rat brain and pituitary after in vivo treatment with colchicine or cytochalasin B.

Abstract 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.

Regional distribution of endorphin, met5-enkephalin and leu5-enkephalin in the pigeon brain

The distribution of beta-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 Met5-enkephalin and Leu5-enkephalin regional distribution. These data support the view that enkephalin neurons and endorphin neurons are independent central neuronal systems.

Kinetics of brain glutamate decarboxylase. Interactions with glutamate, pyridoxal 5'-phosphate and glutamate-pyridoxal 5'-phosphate Schiff base.

Abstract— The kinetic behavior of glutamate decarboxylase from mouse brain was analyzed in a wide range of glutamate and pyridoxal 5′‐phosphate concentrations, approaching three limit conditions: (I) in the absence of glutamate‐pyridoxal phosphate Schiff base; (II) when all glutamate is trapped in the form of Schiff base; (III) when all pyridoxal phosphate is trapped in the form of Schiff base. The experimental results in limit condition (I) are consistent with the existence of two different enzyme activities, one dependent and the other independent of free pyridoxal phosphate. The results obtained in limit conditions (II) and (III) give further support to this postulation. These data show that the free pyridoxal phosphate‐dependent activity can be abolished when either all substrate or all cofactor are in the form of Schiff base. The free pyridoxal phosphate‐independent activity is also abolished when all substrate is trapped as Schiff base, but it is not affected by the conversion of free pyridoxal phosphate into the Schiff base. A kinetic and mechanistic model for brain glutamate decarboxylase activity, which accounts for these observations as well as for the results of previous dead end‐inhibition studies, is postulated. Computer simulations of this model, using the experimentally obtained kinetic constants, reproduced all the observed features of the enzyme behavior. The possible implications of the kinetic model for the regulation of the enzyme activity are discussed.

In vivo release of enkephalin from the globus pallidus

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.

Synthetic peptides corresponding to the sequence of noxiustoxin indicate that the active site of this K+ channel blocker is located on its amino-terminal portion

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 μg/20 g mouse weight). The second (NTX30–39) was not toxic even at higher dose (400 μg/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.

Kinetics of brain glutamate decarboxylase. Inhibition studies with N-(5'-phosphopyridoxyl) amino acids.

Abstract— 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.