Characterization of sodium-independent β-alanine binding to cerebral cortical membranes from 7-day-old and adult mice

The sodium-independent binding of β-alanine to cerebral cortical membranes from adult (3- and 12-month-old) and developing (7-day-old) mice was characterized for the first time. The binding was saturable in each age group, consisting of only one component. The affinity for β-alanine was highest and the number of available binding sites greatest in young animals. The binding was not affected by strychnine, but inhibited by β-alanine itself, glycine, l-alanine and l-serine, the IC₅₀ values being lower in immature mice. Glycine was shown to be a competitive inhibitor. The binding was also inhibited, albeit only in adults, by N-methyl-d-aspartate receptor antagonists acting at the glycine modulatory site and by some GABAergic substances. It is concluded that even though β-alanine may possess binding sites of its own, particularly in the immature cerebral cortex, β-alanine could at least partly bind to strychnine-insensitive glycine sites in the N-methyl-d-aspartate receptor complex.     

Binding sites for the glutamate-analogue [H]AMPA in cultured rat brainstem and spinal cord

By means of light microscopic autoradiography, binding sites for the glutamate-analogue [ [³H]AMPA [(RS)-α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid] were observed on the soma and processes of many brainstem and spinal neurons but not on glial cells. Unlabeled AMPA, glutamate or the glutamate antagonist glutamic acid diethyl ester at high concentrations inhibited or reduced binding of [³H]AMPA. Our data indicate that binding sites for [³H]AMPA exhibit characteristics expected for glutamate receptors.     

Opiate antagonism of glycine-evoked membrane polarizations in cultured mouse spinal cord neurons

The effect of several opiate receptor agonists on the responses of spinal cord neurons to putative inhibitory and excitatory amino acids was studied using an in vitro model system, cultured fetal mouse spinal cord neurons, and bath application of opiates. Intracellular recordings were made from the cultured neurons with conventional voltage recording techniques or under voltage clamp conditions. The putative amino acid neurotransmitters were applied by iontophoresis or micropressure ejection.Our main finding is that the opiate agonists, morphine and levorphanol (5–100 μM), consistently depressed the responses evoked by the putative inhibitory amino acid neurotransmitters glycine and β-alanine but not GABA. Dextrorphan, the inactive isomer of levorphanol, also depressed the glycine and β-alanine responses, but higher concentrations were required. The excitatory glutamate response was unaltered by these opiates. Leucine enkephalin, an opioid peptide, had no effect on the amino acid responses in the neurons where it was also tested. The opiate antagonist naloxone (10–100 μM) did not reverse the morphine or levorphanol depression of the amino acid responses making it unlikely that opiate receptors mediate this effect.Strychnine was considerably more effective than morphine as a glycine antagonist, producing depressions at nM concentrations compared to the μM concentrations required for morphine. Preliminary studies indicate that both morphine and strychnine act in a non-competitive manner. However, additional studies will be required before the sites of action for these agents can be identified.The possible pharmacological or toxicological significance of the present work remains to be determined. Considering the high doses of opiates (μM concentrations) required to depress the glycine and β-alanine responses, it is unlikely that this action is relevant to normal therapeutic situations. However, such concentrations of opiates are often utilized in pharmacological studies and may be achieved when opiates are applied by iontophoresis. Our data indicate that consideration of the present opiate action should be made when μM concentrations or iontophoretic application of opiates are used for pharmacological studies of CNS tissue.     

Immunocytochemical demonstration of β-endorphin and β-lipotropin in cultured human spinal ganglion neurons

Human fetal spinal ganglion neurons isolated and cultured in vitro were found to contain immunoreactive β-endorphin and β-lipotropin as demonstrated by immunoperoxidase and immunofluorescence techniques. The specificity of the immunoreactions was confirmed by the negative staining by prior absorption of the specific antisera with added peptides. The culture system described may provide a valuable model system in which cellular mechanisms underlying the functions of opioid peptides can be investigated.     

Dietary β-alanine results in taurine depletion and cerebellar damage in adult cats

We have used the taurine analogue, β-alanine, to perturb the taurine concentrations in taurine-supplemented and taurine-deprived adult cats. By using 5 % β-alanine in the drinking water for 20 weeks, both groups of cats had greatly reduced brain taurine concentrations. Taurine-supplemented cat brain accumulated relatively small amounts of β-alanine whereas taurine-deprived cats accumulated large amounts of β-alanine. The cerebellum of cats treated with β-alanine had a number of pathological changes compared with similar cats drinking water alone. The changes were more severe in the taurine-deprived cats, and included reduced numbers of granule and Purkin je cells, with many of those remaining appearing pyknotic and dying. Long swollen fibers were seen in the white matter, resembling Rosenthal fibers described in some human cerebellar diseases. There was also prominent gliosis. Using antibodies to β-alanine and taurine, β-alanine was localized in Purkinje cell soma and dendrites, in Golgi II cells, and in some granule cells, especially in taurine-deprived cats treated with β-alanine. Taurine appears to have been virtually eliminated from Purkinje and granule cells, and concentrated in Golgi II cells and glia. We conclude that β-alanine is responsible for these neuro toxic pathological changes. © 1996 Wiley-Liss, Inc.     

Electrophysiological investigation of β, β′-iminodipropionitrile neuropathy: Intracellular recordings in spinal cord

β, β′-Iminodipropionitrile (IDPN) was given to cats (50 mg/kg/week for 5 weeks) to induce giant axonal swellings in the proximal internodes of motor axons. Conventional intracellular recording techniques were used to investigate the influence of the axon swellings on axonal impulse conduction and generation of action potentials in unidentified lumbosacral motoneurons (MN).Action potentials recorded from axon swellings, verified by lack of orthodromically or antidromically elicited EPSPs or IPSPs, afterhyperpolarization potentials or initial segment-somaldendritic (IS-SD) inflections, were variable in shape. Some were indistinguishable from recordings in normal axons. Component or extra potentials occurred in 45% of recordings from axon swellings; their position on the action potential depended on the direction of impulse invasion into the swelling. Many action potentials were broad, with amplitudes less than 30 mV. Impulse conduction was estimated to be blocked in 19% of motor axons tested.Action potentials recorded in MN of IDPN treated cats resembled in many aspects those recorded in chromatolytic MN, with increased latencies upon antidromic stimulation and decreased IS conduction times and SD thresholds; other parameters did not differ significantly. The minimal interval between two stimuli which each evoked action potentials increased from3.3 ± 0.1to5.8 ± 0.5ms. IS-SD portions of the action potentials could not be fractionated in 49% of cells regardless of interpulse interval. Many MN failed to follow frequencies as low as 10 Hz. Delayed depolarizations were observed in 14% of MN recordings. Repetitive action potentials were elicited by single stimuli in 14% of MN and more frequently by orthodromic than antidromic stimulation. Action potentials could often be elicited in the same MN by stimulation of more than one ventral root filament. The incidence of this ephaptic transmission or crosstalk was estimated to be 12%. The findings are discussed in terms of the influence of proximal axon swellings on action potential generation in MN, propagation along non-homogeneous regions of axons and functional chromatolysis.     

Pro-opiocortin fragments in human and rat brain: β-endorphin and α-MSH are the predominant peptides

The ‘pro-opiocortin’ fragments, β-lipotropin, β-endorphin, ACTH and α-MSH, were estimated in discrete areas of rat and human brain and pituitaries by means of radioimmunoassay in combination with gelfiltration. These peptides exihibited parallel patterns of distribution, but with β-endorphin and α-MSH predominant in the brain of rat and man, and, in contrast, their respective precursors, β-LPH and ACTH predominant in the adenohypophysis of rat and man. These data may be indicative of important differences in post-translational processing of ‘pro-opiocortin’ between these contrasting tissues.     

Regional distribution of β-lipotropin converting enzymes in rat pituitary and brain

Among the brain areas studied only pars distalis and pars intermedia are found to contain β-lipotropin activating enzyme indicating that these may be the exclusive organs for a physiologically significant conversion of β-lipotropin into β-endorphin. β-Endorphin inactivating enzyme is found to be rather uniformly distributed in all the pituitary and brain regions, α- and γ-endorphins are presumably formed by the action of this enzyme on β-endorphin.     

Specific [H]strychnine binding associated with glycine receptors in bovine retina

Saturable, specific [³H]strychnine binding can be demonstrated in homogenates of bovine retina. Scatchard plots revealed only one set of binding sites with a dissociation constant (K_d) of about 60 nM and a maximal number of binding sites of about 1.5 pmol/mg protein. The structural specificity of [³H]strychnine binding sites in bovine retina parallels the properties found for [³H]strychnine binding sites in the spinal cord of several vertebrates. Thus, the data do not give any evidence that specific [³H]strychnine binding in bovine retina labels taurine rather than glycine receptors and favors glycine rather than taurine as inhibitory neurotransmitter in bovine retina. The subcellular distribution of specific [³H]strychnine binding in bovine retina parallels that of sodium-dependent, high-affinity uptake of glycine and taurine. All 3 parameters are mainly found in the P₂ fractions of bovine retina homogenates, containing conventional synaptosomes, most abundant in the inner plexiform layer, but can also be found in the P₁ fractions, containing large synaptosomes from the photoreceptor cell layer.     

Characterization of β-adrenergic receptors in rat brain and pituitary using a new high-affinity ligand, [I]iodocyanopindolol

The binding of [¹²⁵I]iodocyanopindolol (ICYP) to membrane preparations from rat cerebral cortex, hypothalamus and anterior pituitary gland was characterized in regard to specificity, density, and the proportion of β-adrenergic receptor subtypes. By employing a mixture of ligands specific for α-adrenergic, serotoninergic and dopaminergic receptors, it was possible to eliminate most of the less-specific contributions to ICYP binding profiles, which resulted in narrowing the range of measured dissociation constants to 35–50 pM for all neural tissues studied. These values corresponded well with constant for the ‘slow’ component discernible in ICYP association with cerebral cortical membranes at 37° C. The maximum binding values were 63, 29 and 5.6 fM/mg membrane protein in cortical, hypothalamic and anterior pituitary membrane fractions, respectively.Evaluation of the β-adrenergic receptor subtypes using 4 selective competitors indicated an average 19% content of the β₂-subtype in cortical membranes, while in hypothalamic membranes 47% of the receptors could be assigned to that subtype. In the anterior pituitary as well as in the cerebellum, the receptors were predominantly ofβ₂-subtype. These findings are discussed in terms of possible physiological functions of β-receptors in these tissues, including the regulation of the release of pituitary hormones.     

Regional distribution of α- and γ-type endorphins in rat brain

The regional distribution of Met-enkephalin, beta-endorphin and alpha- and gamma-type endorphins in rat brain was investigated. To that end, brains were dissected into anatomically defined areas. Acetic acid tissue extracts were fractionated using an HPLC system suitable for separation of endorphins and peptide concentrations were subsequently measured by specific radioimmunoassay systems. The distribution of Met-enkephalin and beta-endorphin through the brain was fairly uneven and in accordance with results obtained by others. The peptides alpha-endorphin, gamma-endorphin, des-Tyr-alpha-endorphin (DT alpha E) and des-Tyr-gamma-endorphin (DT gamma E) were detectable in almost all brain areas. Their distribution, however, appeared to be uneven. Hypothalamus and septum showed the highest levels of alpha- and gamma-type endorphins. These regions also contained high amounts of beta-endorphin, underscoring a precursor function of this peptide in the formation of alpha- and gamma-type fragments. In general, levels of alpha-endorphin were higher than those of des-Try-alpha-endorphin, whereas the opposite was found for gamma- and des-Tyr-gamma-endorphin.     

Extra-synaptic localization of α-bungarotoxin receptors in cultured chick ciliary ganglion neurons

Surface [¹²⁵I]α-bungarotoxin receptors were localized on dissociated cultures of embryonic chick ciliary ganglion neurons by electron microscopic autoradiography. Grain density was 4–5-fold higher on cell body membrane versus neuritic process membrane, and there was no apparent concentration of grains at morphologically identifieble inter-neuronal synapses.     

Release of β-lipotropin and β-endorphin from rat hypothalami in vitro

Hypothalamic tissue extracts of rats were chromatographed and β-endorphin immunoreactivity (β-End_i) was measured. The two major peaks of β-End_i co-eluted with β-lipotropin (β-LPH) and β-End respectively. Hypophysectomy caused a local decrease of β-LPH and β-End concentrations in the mediobasal hypothalamus. During superfusion of hypothalamic tissue blocks in vitro, membrane depolarization by electric stimulation or 45 mM K⁺ induced a Ca²⁺-dependent release of both β-LPH and β-End.     

Modulation of rat brain α- and β-adrenergic receptor populations by lesions of the dorsal noradrenergic bundle

Bilateral lesion of the ascending noradrenergic fibers in the dorsal bundle of adult Wistar rats with 4 μg 6-hydroxydopamine caused extensive depletion of norepinephrine in all forebrain areas, but led to a 54% increase in norepinephrine levels in the cerebellum. β-Adrenergic receptor binding of [³Hdihydroalprenolol was significantly increased in all forebrain areas depleted of norepinephrine except hypothalamus. The increase in [³Hdihydroalprenolol binding was due to 62% and 34% increases in the number of β-receptor sites in the frontal cerebral cortex and hippocampus respectively. Binding of [³HWB-4101 toα₁-adrenergic receptors after dorsal bundle lesion was augmented generally to a lesser extent than β-receptor binding, with significantly increased numbers of sites only in the frontal cortex (74%), thalamus (20%) and septum. Bothα₁-andβ-receptor binding sites were reduced in number by 25–28% in the cerebellum of dorsal bundle-lesioned rats, whereas intraventricular administration of 6-hydroxydopamine to adult rats, which depletes norepinephrine in the cerebellum by 96%, increased cerebellarα₁-andβ-receptor binding by 33–40%. Binding of [³Hclonidine to forebrainα₂-adrenergic receptors was significantly elevated in the frontal cortex, but reduced in the amygdala and septum, after dorsal bundle lesion.     

Effects of α-endorphin, β-endorphin and (des-tyr)-γ-endorphin on α-MPT-induced catecholamine disappearance in discrete regions of the rat brain

Following the intracerebroventricular administration of α-endorphin, β-endorphin and (des-tyrosine¹)-γ-endorphin in a dose of 100 ng, the α-MPT-induced catecholamine disappearance was found to be altered in discrete regions of the rat brain. In the regions in which α-endorphin exerted an effect, it without exception caused a decrease in catecholamine disappearance. Thus, in rats treated with α-endorphin the disappearance of noradrenaline was decreased in the medial septal nucleus, dorsomedial nucleus, central amygdaloid nucleus, subiculum, the ventral part of the nucleus reticularis medullae oblongatae and the A1 region, and that of dopamine in the caudate nucleus, globus pallidus, medial septal nucleus, nucleus interstitialis striae terminalis, paraventricular nucleus, zona incerta and central amygdaloids nucleus. β-endorphin was found to decrease noradrenaline disappearance in the ventral part of the nucleus reticularis medullae oblongatae, dopamine disappearance in the lateral septal nucleus and the disappearance of both amines in the rostral part of the nucleus tractus solitarii. Dopamine disappearance was increased in the medial septal nucleus and the zona incerta following β-endorphin treatment. Following treatment with (des-tyrosine¹)-γ-endorphin, noradrenaline disappearance was enhanced in the anterior hypothalamic nucleus, whereas dopamine disappearance was increased in the paraventricular nucleus, the zona incerta and the rostral part of the nucleus tractus solitarii. In addition to this the latter peptide also caused a decreased noradrenaline disappearance in the periventricular thalamus and the A7 region. The results fit well with the suggestion that endorphins act as modulators of catecholamine neurotransmission in particular brain regions. The pattern of effects of the endorphins differ from that previously observed following intracerebroventricular administration of methionine-enkephalin. This is in keeping with the notion that the enkephalin containing network in the brain and that containing β-LPH represent two independent systems with distinct differences in their projections to various brain regions.     

Effects of serotonin depleters on the contents of β-endorphin, α-melanotropin and adrenocorticotropin in rat brain and pituitary

Serotonin depleters, 5,7-dihydroxytryptamine (5,7-DHT) andp-chlorophenylalanine (PCPA), were injected into adult male rats and β-endorphin (β-EP), α-melanotropin (α-MSH) and adrenocorticotropin (ACTH) levels in rat brain and pituitary were each estimated by radioimmunoassay combined with a gel column chromatography. (1) 5,7-DHT, injected intracerebroventricularly combined with pargyline, decreased the levels of immunoreactive-β-EP, -α-MSH and -ACTH significantly and concomitantly in hypothalamus, thalamus, and brainstem. (2) PCPA, repeatedly injected intraperitoneally 8 times every 3 days, decreased the levels of these peptides in some of these brain regions. (3) There was no significant change of IR-β-EP, -α-MSH, -ACTH in the anterior and the inter-mediate-posterior pituitaries after the treatment of 5,7-DHT, or PCPA. (4) A single injection of the same dose of PCPA induced no significant effects on these peptide levels in both brain and pituitary. These data suggest that central serotoninergic neurons might affect β-EP-α-MSH-ACTH containing neurons in rat brain.     

Binding of {H}-dihydroalprenolol and {H}-acetobutolol to human blood platelets is not related to occupancy of β-adrenoceptors

Binding of {³H}-dihydroalprenolol to human platelet lysates is inhibited by (±)-propranolol and (±)-butoxamine, but less effectively by (±) practolol. (−)-Isoprenaline causes no significant inhibition of binding where stimulation of adenylate cyclase can be shown. Binding of {³H}-acetobutolol is also inhibited by (±)-propranolol. “Specific” binding of {³H}-dihydroalprenolol and {³H}-acetobutolol defined by (±) propranolol shows a non-classical saturation curve. 50% maximal binding is observed in the range 15 – 25 mM. The extent of “specific” binding is 2-fold greater for {³H}-dihydroalprenolol. Similar and rapid rates of binding of {³H}-dihydroalprenolol are observed at 4°C and 20°C. No stereoselectivity is observed for inhibition of {³H}-dihydroalprenolol binding by (+) and (−)-propranolol. Binding of {³H}-dihydroalprenolol and {³H}-acetobutolol may relate to the lipophilic character of these radioligands and does not represent interaction with β-adrenoceptors.     

Comparison of taurine, hypotaurine and β-alanine uptake in brain synaptosomal preparations from developing and adult mouse

The properties of taurine, hypotaurine and β-alanine uptakes were compared in brain synaptosomal preparations from 6-day-old and adult mice. The uptakes of these structurally related amino acids resembled each other, being concentrative, sodium-dependent and inhibited by the same analogues. The absolute sodium requirement of uptake was already evident in developing brain. The affinity of the lowaffinity uptake for taurine was higher in immature than in adult brain. Both affinity and maximal velocity increased in hypotaurine uptake during development, whereas in β-alanine uptake only the maximal velocity did so. The efficient synaptosomal taurine and hypotaurine transport systems in immature brain could contribute to the high taurine level in developing brain.