Characteristics of [125I]ω-conotoxin labeling using bifunctional cross linker DSP in crude membranes from chick brain

Characteristic of [¹²⁵I]-conotoxin (-CgTX) labeling using bifunctional cross linker (dithio bis[succinimidyl propionate]: DSP) was systematically investigated in crude membranes from chick whole brain. [¹²⁵I]-CgTX specifically labeled 216 kDa as a main and 236 kDa as a minor bands in the crude membranes under non-reduced condition, but not labeled under reduced condition. We investigated the effect of various Ca channel antagonists on [¹²⁵I]-CgTX labeling with DSP in detail, and found that there is a strong correlation between the effects of Ca channel antagonists on [¹²⁵I]-CgTX labeling of the 216 kDa band and specific [¹²⁵I]-CgTX binding. These results suggest that labeling of the 216 kDa band under non-reduced condition with [¹²⁵I]-CgTX using DSP involves the specific binding sites of [¹²⁵I]-CgTX, perhaps including one of the neuronal N-type Ca channel subunits in the crude membranes.     

Low molecular weight non-peptide mimics of ω-conotoxin GVIA

We report the synthesis and biological activity of a low molecular weight non-peptidic mimic of the analgesic peptide ω-conotoxin GVIA. The molecular weight of this compound presents a reduction by 193 g/mol compared to a previously reported lead. This compound exhibits an EC₅₀ of 5.8 μM and is accessible in only six synthetic steps compared to the original lead (13 steps). We also report several improvements to the original synthetic route.     

β-Endorphin: Characteristics of binding sites in the rabbit cerebellar and brain membranes

Specific binding of human β-endorphin to rabbit cerebellar and brain membranes was measured using $\text{[}{}^3\text{H}{}_2\text{-Tyr}{}^{27}\text{]-β}{}_{\mathrm{h}}\text{-endorphin}$ as the primary ligand. In both tissues binding was time dependent and saturable, with apparent equilibrium dissociation constants of 0.275 nM and 0.449 nM in the cerebellum and brain, respectively. The binding capacity of cerebellum is greater than that of brain. Kinetic studies showed that the association rate constants were 2.7 × 10⁷ M^?1min^?1 for cerebellum and 2.4 × 10⁷ M^?1min^?1 for brain. Dissociation of tritiated β_h-endorphin from both cerebellum and brain is not consistent with a first order decay from a single site. In the cerebellum, these is a time-dependent increase in slowly dissociating complex. The potency of several opioid peptides and opiates to inhibit the binding of tritiated β_h-endorphin was determined. Ligands with preference for μ, δ, and κ opiate receptor (morphine, Metenkephalin and ethylketocyclazocine) all have similar affinities toward β_h-endorphin sites in both brain and cerebellar membranes.     

G protein dependent alterations in [125I]iodocyanopindolol and±cyanopindolol binding at 5-HT1B binding sites in rat brain membranes

Several manipulations that affect G protein/receptor coupling also alter the binding of [¹²⁵I]iodocyanopindolol ([¹²⁵I]ICYP)±cyanopindolol (±CYP) to rat brain 5-HT_1B binding sites in radiologand binding assays. Inclusion of 5 mM MgSO₄ in these assays results in a small but significant increase in the affinity of [¹²⁵I]ICYP (fromK _D=0.046 nM toK _D=0.037 nM). In contrast, 100 M Gpp(NH)p, GTP, or GDP reduce [¹²⁵I]ICYP affinity (K _D=0.056 nM with GTP) while ATP and GMP are less effective.±CYP affinity for 5-HT_1B sites labeled by [³H]dihydroergotamine ([³H]DE) also displays a small but significant reduction (from K_i=1.4 nM to K_i=3.5nM) by the inclusion of 100 M GTP. Pre-treatment of the brain membranes with N-ethylmaleimide (NEM) in concentrations known to inactivate many G proteins reduces 5-HT_1B specific binding of [¹²⁵I]ICYP. The NEM induced reduction in [¹²⁵I]ICYP binding can be reversed by reconstitution with purified exogenous G proteins (Go and Gi), demonstrating directly that high affinity binding of [¹²⁵I]ICYP to 5-HT_1B sites is dependent on G proteins. The effects of Mg²⁺ ion, guanine nucleotides, NEM and G protein reconstitution on [¹²⁵I]ICYP and ±CYP binding are all hallmarks of agonist binding to G protein linked receptors. The effect of GTP, however, is quantitatively much less for the binding of these pindolol derivatives than for the binding of 5-HT, a presumed full agonist at 5-HT_1B sites. The relatively slight stabilization of [¹²⁵I]ICYP and ±CYP binding conferred by G protein/5-HT_1B receptor interaction may reflect the molecular events underlying previous observations that these compounds are partial 5-HT_1B agoinists.     

Positive cooperativity in high affinity binding sites for γ-hydroxybutyric acid in rat brain

High affinity binding sites for -hydroxybutyrate have recently been shown to exist on crude membranes of rat brain. These sites exhibit a dissociation constant of 95 nM and a capacity of 557 fentomoles per mg protein. However, after more extensive washing of the crude membrane fraction and performing binding experiments at a lower concentration of radioactive GHB (below 20 nM), the existence of another binding site for GHB with a higher affinity than previously described was discovered. The data concerning this binding site are in favour of positive cooperative binding characteristics. This binding site may play a role in the mediation of the multiple physiological and pharmacological effects of GHB in the rat CNS and its presence provides additional evidence in favour of a neuromodulator or neurotransmitter role of GHB.This issue is dedicated to Donald B. Tower.     

Lipoxygenation in rat brain?

It has been previously claimed that rodent brain possesses lipoxygenase activity, based upon the structure of products which were formed from arachidonic acid and the inhibition of this activity by "lipoxygenase inhibitors." Our studies confirm that various positional isomers of hydroxyeicosatetraenoic acids (HETE) are formed (e.g., 15-, 12-, 11-, 9-, 8- and 5-HETE) by brain homogenate and that their production is inhibited by certain lipoxygenase inhibitors, such as nordihydroguaiaretic acid (NDGA) but not by cyclooxygenase or cytochrome P-450 inhibitors. However, stereochemical analysis indicated racemic distributions of these products suggesting that they were not formed by a lipoxygenase enzyme but rather by a peroxidative process. It should also be noted that the presence of 12(S)-lipoxygenase activity could be demonstrated by stereochemical analysis only when the brain was not perfused properly, indicating this activity was due to blood cell contamination. It is known that many lipoxygenase inhibitors are also capable of inhibiting peroxidative reactions apparently due to their free radical scavenging properties. For these reasons, it is essential that the stereochemical purity of purported lipoxygenase products be determined and that previous claims of lipoxygenase activity in mammalian brain be reexamined.     

Pharmacological characterization of [³H]CHIBA-3007 binding to glycine transporter 1 in the rat brain

Glycine transporter-1 (GlyT-1) in glial cells regulates extracellular levels of glycine, which acts as an obligatory co-agonist at the N-methyl-D-aspartate (NMDA) receptors in the brain. In the present study, we developed a novel radioligand, [³H]3-chloro-N-((S)-((R)-1-methylpiperidin-2-yl)(thiophen- 3-yl)methyl)-4- (trifluoromethyl)picolinamide ([³H]CHIBA-3007), for studying GlyT-1 in the brain. The presence of a single saturable high-affinity binding component for [³H]CHIBA-3007 binding to the rat brain membranes was detected. Scatchard analysis revealed an apparent equilibrium dissociation constant (K_d) of 1.61±0.16 nM and a maximal number of binding sites (B_max) of 692.8±22.8 fmol/mg protein (mean ± SEM, n = 3). The specific binding of [³H]CHIBA-3007 was inhibited by a number of GlyT-1 inhibitors, such as CHIBA-3007, desmethyl-CHIBA-3007, CHIBA-3008, SSR504734, NFPS/ALX5407, LY2365109 and {"type":"entrez-protein","attrs":{"text":"Org24598","term_id":"1179171570","term_text":"ORG24598"}}Org24598, consistent with the pharmacological profiles of GlyT-1 inhibitors. Interestingly, the potency of eight GlyT-1 inhibitors (CHIBA-3007, desmethyl-CHIBA-3007, NFPS/ALX5407, LY2365109, {"type":"entrez-protein","attrs":{"text":"Org24598","term_id":"1179171570","term_text":"ORG24598"}}Org24598, SSR504734, sarcosine, and glycine) for blocking in vitro specific binding of [³H]CHIBA-3007 was significantly correlated with the potency of these inhibitors for inhibiting [¹⁴C]glycine uptake in the rat brain membranes. In contrast, the GlyT-2 inhibitor ALX1393 exhibited very weak for [³H]CHIBA-3007 binding. Furthermore, the regional distribution of [³H]CHIBA-3007 binding in the rat brain was similar to the previously reported distribution of GlyT-1. The present findings suggest that [³H]CHIBA-3007 would be a useful new radioligand for studying GlyT-1 in the brain.     

In vivo binding and autoradiographic imaging of (+)−3-[125I]Iodo-MK-801 to the NMDA receptor-channel complex in rat brain

Radioiodinated (+)−3-Iodo-MK-801 is a high affinity radioligand for the N-methyl-d-aspartate (NMDA) receptor-channel complex. We have demonstrated in vivo localization in the CNS of rat which is stereoselective and blocked by coinjection of unlabeled MK-801. Autoradiography indicates localization in vivo which is in concordance with in vitro autoradiographic studies. These results indicate that radioiodinated (+)−3-Iodo-MK-801 is a useful probe for in vitro and in vivo autoradiographic studies and suggest that radioligands for the NMDA receptor may be developed which will provide in vivo images of receptor distribution in man.     

Effect of 17β estradiol on [3H] progesterone binding in rat brain

A progesterone-binding component is reported in the cerebral hemispheres of immature female rat. [³H] progesterone binding in the brain cytosol is increased following two weeks of estradiol administration. The [³H] progesterone binding by this component can be reduced by pretreatment with unlabeled steroid. In addition, the binder from both control and estradiol-treated groups shows inter-action with ATP immobilized on columns of ATP-Sepharose.     

Regional distribution of α-neo-endorphin in rat brain and pituitary

α-Neo-endorphin was isolated as the first form of “big” Leu-enkephalin and its complete amino acid sequence has recently been established. Using an antiserum raised against synthetic α-neo-endorphin, a highly sentitive and specific radioimmunoassay was developed. The antiserum practically possesses no cross-reactivity to Leu-enkephalin, dynorphin[1–13] and PH-8P, and very little to β-neo-endorphin. Distribution of α-neo-endorphin has been determined in rat brain and pituitary by the use of the highly specific antiserum. The highest concentration was observed at posterior lobe of pituitary. Furthermore, immunoreactive α-neo-endorphin was characterized by gel-filtration and high performance liquid chromatography, and shown to be identical with authentic α-neo-endorphin.     

A comparison of the in vitro binding of α-tocopherol to microsomes of lung,liver, heart and brain of the rat

The in vitro binding of α-tocopherol to microsomes of lung, liver, heart and brain of the rat was studied with the insoluble tocopherol ligand presented as a complex with bovine serum albumin. Under these conditions, all microsomes showed nonsaturable binding of α-tocopherol and the amount bound to microsomes was linearly proportional to the concentration of albumin-complexed tocopherol. Increasing the amount of α-tocopherol bound to microsomes in this manner reduced the extent of lipid peroxidation induced by added ferrous iron. The apparent affinities of the microsomes for α-tocopherol, as indicated by the amount bound at a given concentration of albumin-complexed tocopherol, decreased in the order brain > liver ≈ heart > lung. The differences in affinity did not correlate with total fatty acid content (r = − 0.39), total unsaturated fatty acid content (r = − 0.26), or with the content of fatty acids containing two or more double bonds (r = − 0.01). A high positive correlation was found with the content of fatty acids containing three or more double bonds (r = + 0.96). Since lung microsomes contain approx. 6-times the tocopherol levels of liver and brain and about twice that of heart microsomes, these results show that the in vivo levels of microsomal tocopherol do not reflect microsomal affinity for this biological antioxidant.     

Glycolytic enzyme binding in fetal brain — The role of actin

The binding of aldolase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase in fetal calf brain homogenates and extracts has been investigated at both 0° and 37°C under high ionic strength conditions. The results demonstrate far greater enzyme binding at 37°C than at 0°C, which correlates with an increased sedimentation of cytoskeletal actin at the higher temperature. A dependence of enzyme sedimentation on the presence of polymerised actin was also demonstrated, and this indicates that cytoskeletal actin is a major adsorbent of glycolytic enzymes in this non-muscle tissue.     

An immunohistochemical analysis of SERT in the blood–brain barrier of the male rat brain

5-Hydroxytryptamine (5-HT) was originally discovered as a vasoconstrictor. 5-HT lowers blood pressure when administered peripherally to both normotensive and hypertensive male rats. Because the serotonin transporter (SERT) can function bidirectionally, we must consider whether 5-HT can be transported from the bloodstream to the central nervous system (CNS) in facilitating the fall in blood pressure. The blood–brain barrier (BBB) is a highly selective barrier that restricts movement of substances from the bloodstream to the CNS and vice versa, but the rat BBB has not been investigated in terms of SERT expression. This requires us to determine whether the BBB of the rat, the species in which we first observed a fall in blood pressure to infused 5-HT, expresses SERT. We hypothesized that SERT is present in the BBB of the male rat. To test this hypothesis, over 500 blood vessels were sampled from coronal slices of six male rat brains. Immunofluorescence of these coronal slices was used to determine whether SERT and RecA-1 (an endothelial cell marker) colocalized to the BBB. Blood vessels were considered to be capillaries if they were between 1.5 and 23 µm (intraluminal diameter). SERT was identified in the largest pial vessels of the BBB (mean ± SEM = 228.70 ± 18.71 µm, N = 9) and the smallest capillaries (mean ± SEM = 2.75 ± 0.12 µm, N = 369). SERT was not identified in the endothelium of blood vessels ranging from 20 to 135 µm (N = 45). The expression of SERT in the rat BBB means that 5-HT entry into the CNS must be considered a potential mechanism when investigating 5-HT-induced fall in blood pressure.     

Fiber-type specific expression of α-actinin isoforms in rat skeletal muscle

α-Actinins are actin-binding proteins, and two isoforms (α-actinin-2 and -3) are major structural components of the sarcomeric Z line in mammalian skeletal muscle. Based on human and knockout mice studies, α-actinin-3 is thought to be associated with muscle force output and high contraction velocities. However, fiber-type specific expression of α-actinin isoforms is not well understood and may vary among species. In this study, we investigated the expression of α-actinin isoforms and the difference between fiber types in rat skeletal muscle and compared it with those of humans and mice from previous reports. Soleus and plantaris muscles were analyzed immunohistochemically to identify muscle fiber types and α-actinin protein expression. α-Actinin-2 was stained in all muscle fibers in both the soleus and plantaris muscles; i.e., all α-actinin-3 co-expressed with α-actinin-2 in rat skeletal muscles. The proportions of α-actinin-3 expression, regardless of fiber type, were significantly higher in the plantaris (75.8 ± 0.6%) than the soleus (8.0 ± 1.7%). No α-actinin-3 expression was observed in type I fibers, whereas all type IIx+b fibers expressed α-actinin-3. α-Actinin-3 was also expressed in type IIa fibers; however, approximately 75% of type IIa fibers were not stained by α-actinin-3, and the proportion varied between muscles. The proportion of α-actinin-3 expression in type IIa fibers was significantly higher in the soleus muscle than the plantaris muscle. Our results showed that fiber-type specific expression of α-actinin isoforms in rats is more similar to that in humans compared to that of the mouse, whereas the proportion of α-actinin-3 protein varied between muscles.     

Involvement of binding lipoproteins in the absorption and transport of α-tocopherol in the rat

1. Specific lipoproteins binding alpha-tocopherol but not its known metabolites have been isolated and identified from cytosol of rat intestinal mucosa and from serum. 2. A timestudy of the appearance of the orally administered alpha-[(3)H]tocopherol with these lipoproteins indicates that very-low-density lipoprotein of serum acts as a carrier of the vitamin. 3. The involvement of the mucosal lipoprotein in the absorption of the vitamin from the intestine has been inferred from observations on the amounts of alpha-tocopherol in serum of orotic acid-fed rats where release of lipoproteins from the liver to serum is completely inhibited. A considerable decrease in the association of alpha-tocopherol with serum very-low-density lipoprotein under this condition is interpreted to mean that serum lipoproteins are limiting factors for the transport of the vitamin across the intestine and that this is possibly effected by exchange of alpha-tocopherol between serum very-low-density lipoprotein and mucosal lipoprotein.     

β-d-glucosidase in fractions from rat brain

-Glucosidase activity has been determined in homogenate and in centrifugation fractions of 7-day-old and adult rat brain; maximum activity was found at pH 4 and pH 5. Of the adult brain, more than 50% of the activity was concentrated in the 800-g sediment fraction (P₁), while in the brain of 7-day-old rat about 20% was found in the corresponding fraction. The activity maximum in all fractions after a 2% Triton X-100 treatment occurs at pH 5. Addition of Triton to adult brain homogenate enhances the activity, but this stimulation is less than the sum of the activities observed at pH 4 and pH 5 in the absence of Triton. Triton addition to brain homogenate of 7-day-old rat results in a fall in activity at pH 4 and in a maximum at pH 5. In rat brain homogenate subjected to sonication, a loss of activity is observed at pH 4, scarcely at pH 5; the activity loss is completely abolished and turned into an increase under the influence of Triton. This increase equals the level obtained when Triton is added to an untreated brain homogenate. Sonication of rat brain homogenate leads to changes in the distribution pattern; about 25% of the activity of the adult brain is found in the P₁ fraction compared to 50% in the corresponding fraction of the untreated brain. Fractionation of a sonicated brain homogenate from adult rat reveals that at pH 4 most activity (52%) is concentrated in the 20,000-g pellet (P₂), 23% in supernatant fluid (S₂); at pH 5 the opposite is observed: most activity (49%) is found in the 20,000-g supernatant (S₂) and 23% in the 20,000-g pellet (P₂). In the presence of Triton the activity of the sonicated brain homogenate of adult rat increases; this stimulation roughly equals the sum of the corresponding activities measured at pH 4 and pH 5 in the absence of Triton.     

Localization of Ca2+-binding proteins of rat cortex on two-dimensional gels—II. analysis of Ca2+ binding proteins in ammonium sulfate fractions of rat brain

A total of seven high-affinity calcium-binding proteins have been detected in rat brain. This was accomplished using a combination of ammonium sulfate fractionation, two-dimensional gel electrophoresis, western blotting and ⁴⁵Ca²⁺-autoradiography. Of these seven proteins, three are detectable in a crude tissue punch of rat cortex while four are seen only after protein enrichment with ammonium sulfate. Four of the seven proteins detected in this study have been identified: calmodulin, the B subunit of calcineurin, the intestinal vitamin D-dependent calcium-binding protein and parvalbumin. The identities of the other three proteins visualized by ⁴⁵Ca²⁺-autoradiography in this study are unknown.     

Biosynthesis of serotonin in Raphé nuclei of rat brain: effect of p-chlorophenylalanine

The activity of tryptophan hydroxylase (EC 1.99.1.4) in the region of the raphé nuclei of rat brain was higher than that of any other brain area. The content of serotonin and the rate of serotonin synthesis were also highest in the raphé nuclei. Following the administration of p-chlorophenylalanine the injection of tryptophan and pargyline increased the content of serotonin in the region of the raphé nuclei of rat brain. The results suggest that the raphé nuclei retained the capacity to hydroxyl-late tryptophan to some extent after the injection of p-chlorophenylalanine.     

Effect of morphine and abstinence syndrome on [3H]bromoxidine binding to α2-adrenoreceptors in rat brain

At 4 days after the implantation of two subcutaneous 75 mg morphine pellets in the back skin, rats were morphine-dependent. In the three layers studied in the occipital cortex we found that the values of the ₂-adrenergic agonist [³H]bromoxidine binding increased with respect to animals implanted with placebo pellets. Typical behavioral and physiological symptoms of the abstinence syndrome appeared 30 minutes after administration of naloxone, [³H]bromoxidine binding values being similar to those obtained in animals implanted with placebo pellets. The pattern of response of the [³H]bromoxidine binding was similar in the hippocampus and the superficial gray layer of the superior colliculus of the mesencephalon, but the differences were not statistically significant in these areas. This paper concludes that exist brain regional differences in the ₂-adrenoreceptors response under morphine-treatment and possibly under naloxone-induced morphine abstinence syndrome.