Specific [3H]Piflutixol Binding to CHAPS-Solubilised Rat Striatal Preparations Involves Dopamine D-2 but Not D-1 Binding Sites

[3H]Piflutixol binding to rat striatal membrane preparations identifies both D-1 and D-2 sites. We used [3H]piflutixol to characterise those binding sites present in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)-solubilised rat striatal preparations. The specific binding of [3H]piflutixol, as defined using cis-flupenthixol, to CHAPS-solubilised rat striatal tissue was saturable and of high affinity. Specific [3H]piflutixol binding to the solubilised preparations was displaced stereoselectively by the isomers of butaclamol and to an equal extent by both cis-flupenthixol and (+/-)-sulpiride. A positive correlation was found between the capacity of a range of drugs to displace [3H]piflutixol binding and the displacement of [3H]spiperone to the same preparations. The Bmax of [3H]piflutixol binding was not different from that of [3H]spiperone binding to the same preparation. These studies suggest that, in contrast to specific binding of membrane preparations, the specific binding of [3H]piflutixol to CHAPS-solubilised preparations involves mainly D-2 sites. Specific [3H]piflutixol binding, in contrast to [3H]spiperone binding, showed only slow dissociation from soluble preparations. The binding of [3H]piflutixol to CHAPS-solubilised preparations was retained during passage through a gel filtration column. This prelabelling of solubilised striatal preparations using [3H]piflutixol may aid in the purification of CHAPS-solubilised rat striatal D-2 sites.     

Cell surface antigens on rat islet tumors

To examine the antigenic properties of the rat pancreatic beta cell tumor, RIN, we used a cell surface enzyme-linked immunosorbent assay (CELISA). Monoclonal antibodies of known specificity are used to validate the assay, and the results show that antibodies directed at glycoproteins and glycolipids are detected by this technique. The principal glycolipid targets are found in the ganglio and lacto series of glycosphingolipids, and not in the globo series. When the CELISA was used to study sera from type I diabetics, no differences in binding of control and diabetic samples were detected at low dilutions of serum. However, at higher serum dilutions (1/30 to 1/120) binding of IgG antibodies from type I diabetics was greater than that observed with normal sera. Similar reactivity was not detected in these sera when rat hepatocytes were used as targets in the CELISA. Prolonged culture of RIN cells, however, resulted in the loss of reactivity with sera from type I subjects, and is associated with a corresponding decrease in expression of cell surface gangliosides. Accordingly, subclones selected for ganglioside expression were used to compare anti-RIN binding in type I diabetics with that of normal controls and unaffected siblings. The results indicate that some rat islet tumors express antigenic determinants recognized by anti-islet antibodies associated with type I diabetes. Both nonspecific interaction at low serum dilutions and variable expression of cell surface antigens may explain the difficulties encountered when these cells are used for diagnostic purposes. An objective assay such as the CELISA may help to avoid these problems.     

6-Hydroxydopamine-induced degeneration of nigral dopamine neurons: differential effect on nigral and striatal D-1 dopamine receptors

Dopamine-sensitive adenylate cyclase and 3H-SCH 23390 binding parameters were measured in the rat substantia nigra and striatum 15 days after the injection of 6-hydroxydopamine into the medial forebrain bundle. The activity of nigral dopamine-sensitive adenylate cyclase and the binding of 3H-SCH 23390 to rat nigral D-1 dopamine receptors were markedly decreased after the lesion. On the contrary, 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine pathway enhanced both adenylate cyclase activity and the density of 3H-SCH 23390 binding sites in striatal membrane preparations. The changes in 3H-SCH 23390 binding found in both nigral and striatal membrane preparations were associated with changes in the total number of binding sites with no modifications in their apparent affinity. The results indicate that: within the substantia nigra a fraction (30%) of D-1 dopamine receptors coupled to the adenylate cyclase is located on cell bodies and/or dendrites of dopaminergic neurons; striatal D-1 dopamine receptors are tonically innervated by nigrostriatal afferent fibers.     

Interactions Between α-Latrotoxin and Trivalent Cations in Rat Striatal Synaptosomal Preparations

The interactions between alpha-latrotoxin (alpha-LTx), a neurosecretagogue purified from the venom of the black widow spider, and the trivalent cations Al3+, Y3+, La3+, Gd3+, and Yb3+ were investigated in rat striatal synaptosomal preparations. All trivalent cations tested were inhibitors of alpha-LTx-induced [3H]dopamine [( 3H]DA) release (order of potency: Yb3+ greater than Gd3+ approximately Y3+ greater than La3+ greater than Al3+). Only with Al3+ could inhibition of [3H]DA release be attributed to a block of 125I-alpha-LTx specific binding to synaptosomal preparations. The inhibitory effect of trivalent ions was reversible provided synaptosomes were washed with buffer containing EDTA. Trivalent ions also inhibited alpha-LTx-induced [3H]DA release at times when alpha-LTx-stimulated release was already evident. alpha-LTx-induced synaptosomal membrane depolarization was blocked by La3+, but not affected by Gd3+, Y3+, and Yb3+. alpha-LTx-stimulated uptake of 45Ca2+ was inhibited by all trivalent cations tested. These results demonstrate that there exist at least three means by which trivalent cations can inhibit alpha-LTx action in rat striatal synaptosomal preparations: (1) inhibition of alpha-LTx binding (Al3+); (2) inhibition of alpha-LTx-induced depolarization (La3+); and (3) inhibition of alpha-LTx-induced 45Ca2+ uptake (Gd3+, Y3+, Yb3+, La3+).     

The measurement of free nitrendipine in human serum by an equilibrium dialysis - radioreceptor assay

A radioreceptor assay using [³H]nitrendipine and rat cerebral cortical membranes, in conjunction with equilibrium dialysis, measures the unbound (free) level of nitrendipine in human sera. The sensitivity of the assay is 0.1–0.2 picomoles/ml and is linear from 4 × 10^?11 to 4 × 10^?9 M nitrendipine. Other dihydropyridine calcium channel antagonists may be measured using this assay if these compounds are used to generate the standard curve. Blank serum interferes with specific [³H]nitrendipine binding (24 percent inhibition per 20 μ1 serum) whereas serum dialysates do not. Total serum nitrendipine levels may be measured, but the sensitivity of the assay is decreased due to interference by serum. Nitrendipine is highly protein bound in serum (93 – 99 percent). This protein binding is essentially unchanged over a serum concentration from 1 to 100 ng/ml. This assay is suitable for pharmacokinetic and pharmacodynamic studies.     

An attempt to analyze various thyroid stimulators by the receptor assay using hTSH radioimmunoassay.

In an attempt to analyze thyroid stimulators in serum we developed an assay procedure using hTSH radioimmunoassay (RIA) in combination with receptor competition. The principle of this method is the determination by RIA of hTSH displaced by other thyroid stimulators from a thyroidal receptor preparation which previously bound unlabelled hTSH. Practically 4 microunits of hTSH were bound with human or bovine receptor, and then hTSH displaced by addition of test serum (0.1 ml) or samples dissolved in serum (0.1 ml) was measured by RIA. This assay can determine the thyroid stimulators other than hTSH in serum that has the displacement activity of 0.5-4.0 microunits of hTSH in the useful range, such as mU/ml level of bovine TSH or rat TSH. Cholera toxin that has the thyroid stimulating activity like TSH also showed the displacement of the bound hTSH. This assay is not applicable for the human serum with more than 5 microunits/ml of TSH, because the assay value is over estimated by the free hTSH derived from the test serum. On the other hand, eighteen sera with high LATS activity and 42 sera with negative LATS activity from patients with untreated hyperthyroidism did not show any displacement. This might be due to the lower binding activity of LATS with hTSH receptor or the lower sensitivity of this assay method. Although it is difficult to use this assay clinically because of its low sensitivity, increased TSH in animal serum can be determined by this assay. The principle of this method may be also useful for examining the receptor binding of other peptide hormone that can be determined by an RIA method.     

Characterization of rodent liver and kidney AVP receptors: pharmacologic evidence for species differences.

Radioligand binding studies with [3H]vasopressin (AVP) were used to determine the affinities of AVP receptor agonists and antagonists for mouse liver and kidney plasma membrane preparations. Both membrane preparations exhibited one class of high-affinity binding site. AVP ligand binding inhibition studies confirmed that mouse liver binding sites belong to the V1A subtype while kidney binding sites belong to the V2 receptor subtype. The affinity of each ligand for mouse V1A receptors was very similar to that for rat V1A receptors, showing differences in Ki values of less than 3-fold. In contrast, several peptide (d(CH2)5Tyr(Me)AVP) and nonpeptide (OPC-21268 and SR 49059) ligands had different affinities for mouse and rat kidney V2 receptors, with differences in Ki values ranging from 14- to 17-fold. These results indicate that mouse and rat kidney V2 receptors show significant pharmacologic differences.     

Kinetic characteristics of the effect of GTP on properties of opioid receptors

Specific interactions of guanine nucleotides with rat brain membrane preparations were investigated; a two-step scheme of this interaction was proposed. The effect of GTP on the agonist binding to rat brain opioid receptors was studied. Nucleotides were shown to inhibit the ligand interactions with mu- and sigma-receptors. It was found that the GTP-induced inhibition differs from that of the GTP stable analog, GppNp, i.e., GTP does not initiate any noticeable dissociation of the receptor system component and its liberation from the membrane into solution as is the case with GppNp. A kinetic model of the GTP interactions with the opioid receptor system stipulating that the affinity of high affinity centers for mu- and delta-ligands changes after nucleotide hydrolysis on the N-protein, was proposed. The differences in the mechanisms of GTP effect on mu- and sigma-receptors were revealed, i.e., the inhibition of the ligand binding to mu-receptors may occur just after GTP binding to the N-protein. The ligand-receptor interactions in the presence of GTP were modelled according to the proposed scheme.     

4-Aminopyridine Increases Acetylcholine Release Without Diminishing Membrane Phosphatidylcholine

4-Aminopyridine (10(-4)-10(-5) M) increased severalfold the release of acetylcholine from rat striatal slices superfused with an eserine-containing, choline-free medium, and caused stoichiometric decreases in the release of choline. It had no effect on tissue acetylcholine and choline levels. Electrical stimulation of the striatal slices increased acetylcholine release without affecting that of choline. Superfusion of the stimulated slices with 4-aminopyridine decreased choline release and increased the ratio of acetylcholine to choline in superfusates. As shown previously, electrical stimulation of the striatal slices decreased their contents of phospholipids, principally phosphatidylcholine; 4-aminopyridine fully protected against these membrane changes. In synaptosomal preparations, 4-aminopyridine was found to enhance the high-affinity uptake of [14C]choline and its conversion to [14C]acetylcholine. This effect on choline uptake may underlie 4-aminopyridine's ability to enhance acetylcholine release in the absence of supplemental choline while suppressing the "autocannibalism" of membrane phospholipids.     

Characterization of a Human Serum Inhibitor of Clostridium histolyticum Proteinase(s)

Normal animal sera inhibit at least one Clostridium histolyticum proteinase. An assay procedure based on immune hemolysis was developed for the estimation of this inhibition. This inhibitory activity occurs in various levels in the sera of different animal species. The highest titers have been obtained with rat sera. The inhibitory activity from human serum was isolated and purified 16- to 27-fold by Sephadex G-200 gel filtration and diethylaminoethyl cellulose or hydroxylapatite chromatography. The properties of the human serum inhibitor of the clostridial proteinase were compared with a trypsin inhibiting factor found in the partially purified preparations. Identical behavior of the two inhibitory factors was observed when measured by heat inactivation, beta-mercaptoethanol sensitivity, pH stability, and sucrose gradient centrifugation. The inhibitory factor has an approximate sedimentation coefficient (S(20,w)) of 17. Goat anti-alpha-2-macroglobulin specifically precipitated the clostridial proteinase inhibitor from a partially purified preparation.     

Differential binding of folates by rat renal cortex brush border and basolateral membrane preparations

The binding of radioactive 5-methyltetrahydrofolate and folic acid was found to be greater in brush border than in basolateral membrane preparations of rat renal cortex. This appeared to be due to an increased amount of a specific folate binding protein in the brush border membrane preparations as compared to those of the basolateral membrane. The binding was saturable and inhibited by nonradioactive folic acid and, therefore, a specific, rather than nonspecific process. The Km's for folic acid binding in brush border and basolateral membrane preparations were similar and involved a single high-affinity binding site. In contrast, methotrexate was found to bind equally well to both brush border and basolateral membrane preparations. Moreover, folic acid binding was not inhibited by an equimolar amount of methotrexate. A folate binding protein could be extracted from either membrane preparation with 1% Triton X-100 and, to a lesser extent, with 0.6 M NaCl. These different extraction procedures resulted in different apparent molecular weights for folate binding protein (greater than 160,000 for Triton X-100-extracted samples and 40,000 for NaCl-extracted samples). The membrane preparation pellets remaining after NaCl extraction were able to rebind tritiated folic acid and also the 40,000-Da folate binding protein. On the other hand, membrane preparations extracted with Triton X-100 lost the ability to bind folic acid or the 40,000-Da folate binding protein. These differences in molecular weight and rebinding capacity may be explained by the existence of a receptor for folate binding protein which was extracted by Triton X-100, but not by NaCl. The greater concentration of folate binding protein in the renal tubule cell brush border membrane preparations as compared to those from basolateral membranes ascribes, for the first time, a functional role for folate binding protein in the renal reabsorption of folates which is required to prevent loss of folate in the urine and perhaps in the membrane transport of folates in general.     

Determination of adenosine 3′:5′-cyclic monophosphate in cerebral tissues by saturation analysis. Assessment of a method using a binding protein from ox muscle

1. The Gilman (1970) procedure for determining cyclic AMP (adenosine 3':5'-cyclic monophosphate) by saturation analysis gave erroneous results when applied to the analysis of extracts of whole brain or preparations of membrane fragments from brain. 2. The extracts contained a non-diffusible factor, which enhanced the binding of cyclic AMP by the muscle protein fraction. 3. Extracts also contained material which inhibited binding, but net inhibition of binding was only observed when relatively concentrated extracts were analysed. 4. The error introduced by the factors modifying binding could be eliminated by incorporation of unlabelled internal standards in the unknowns. The design adopted enables a statistical estimate to be made of the standard error of a single assay. 5. The modified assay was used to determine bound cyclic AMP and adenylate cyclase activity in cerebral membrane fragments. Five preparations of synaptic membrane fragments contained less than 3.5pmol of cyclic AMP/mg of protein; a microsomal fraction from rat contained 65pmol of cyclic AMP/mg of protein.     

Action of interferon on cell membrane of mouse lymphocytes : Inhibition of ligand-induced redistribution of surface receptors

Mouse interferon at 50–100 U/ml or higher concentrations inhibited cap formation of surface components on mouse lymphocytes induced by anti-lymphocyte serum, concanavalin A (ConA), phytohemagglutinin (PHA) and soybean agglutinin (SBA). Interferon from species other than mouse were not effective, suggesting that interferon itself in the preparations used was responsible for the observed effect. The inhibition was observed immediately after addition of interferon, without preincubation of cells with it. The effect was reversible, disappearing after a short lag when interferon was removed. The effect may thus be a reflection of a rapid change in cell membrane upon binding of interferon molecules. On the other hand, the cap formation by anti-immunoglobulin, anti-thy 1, 2, anti-H-2 sera and wheat germ agglutinin (WGA) were not influenced by interferon at all, indicating that the inhibition by interferon was not a general phenomenon. The cap formations susceptible to interferon are those that are enhanced by pretreatment of cells with colchicine and are accompanied by marked uropod formation, but those insusceptible to interferon occur readily, becoming sharp spots which are then shed. It is suggested that interferon may modify the microtubule-containing structure, it it may selectively affect those membrane components that are anchored to it.     

Studies with anti-growth hormone receptor antibodies.

Antisera against a partially purified growth hormone receptor derived from rabbit liver were generated in guinea pigs. The antisera specifically inhibited the binding of 125I-ovine growth hormone (oGH) to liver membranes but had no effect on the binding of 125I-ovine prolactin to rabbit mammary gland receptors. These antisera did not bind or destroy 125I-oGH. Moreover, the binding of labeled growth hormone to membrane particles derived from liver of several species was also inhibited by the antisera, thus suggesting that immunological determinants of the growth hormone receptor of several species are similar. gamma-Globulin fractions derived from the antisera were responsible for the inhibition. In addition 125I-gamma-globulin derived from one antiserum bound to membrane pellets with a corresponding decline in 125I-oGH binding. Kinetic analysis of inhibition of 125I-oGH binding suggested a hyperbolic competitive inhibition, a point of view which is favored by the demonstration of a hormone receptor . antibody complex. The availability of the antireceptor sera confirmed previous data that differential affinity chromatography separated growth hormone and prolactin receptors in solubilized rabbit liver membrane preparations. The antireceptor sera will be useful probes in further characterization of the growth hormone receptor.     

Guanine nucleotides do not inhibit tritiated dopamine agonist binding to bovine striatal membranes

The addition of GTP (50 M), MnCl₂ (1 mM) or EDTA (2 mM) had no effect on the affinity or capacity of bovine striatal plasma membranes for [³H]spiperone. However, GTP caused a decrease in the potency of dopamine as an inhibitor of [³H]spiperone binding under all conditions tested. Manganese enhanced the potency of dopamine both in the presence and absence of GTP, but NaCl (100 mM) had no effect. Neither manganese nor GTP caused any change in the affinity or capacity of bovine striatal membranes for the tritiated agonists dopamine, apomorphine or ADTN. GPPNHP, a nonhydrolyzable analog of GTP, was also ineffective. However, in identical experiments using rat striatal membranes, 50 M GTP caused a decrease in affinity for all three tritiated agonists and this effect was observed both in the presence and absence of manganese (1 mM). In addition, binding capacities for [³H]dopamine and [³H]ADTN were doubled when manganese was present. In light of this and other reports that GTP inhibits tritiated agonist binding in rat striatum, it is suggested that the absence of such inhibition in bovine striatal membranes may reflect a fundamental difference between the two species with regard to their receptors for dopamine agonists.     

Lyophilized membranes as a test system for analyzing the properties of neuromediator receptors and activity of neurotropic agents

The effect of lyophilization and prolonged storage of rat brain membrane preparations on the properties of receptors of cholinergic and adrenergic neuromediator systems has been studied by the radioreceptor assay. Lyophilized membrane preparations have been shown to be highly stable as compared with fresh membranes and retain their binding properties during storage for 1 year.     

Rabbit latent group a allotypes: characterization and relationship to nominal group a allotypic specificities.

Latent group a allotypes were detected with a sensitive radioimmune inhibition assay. Sera, IgG preparations, and antibody fractions containing these allotypes inhibited the binding of insolubilized allotypic antisera to various radiolabeled antigens including IgG pools, homogeneous antibodies, and, in the case of a3, a VH fragment from a3/b4 IgG. Several different group a antiallotypic sera were used in the assays and all gave similar results. Comparison of inhibition curves for nominal and latent allotypes indicated that the full spectrum of allotypic subspecificities may be expressed in latent allotypes. Hemagglutination studies carried out with five sera containing high levels of latent allotypes confirmed the results obtained with the radioimmunoassay and indicated that inhibition values did not, at least in four of the five samples studied, reflect the presence of antiallotype antibodies.     

Species specificity of radioreceptor assay and radioimmunoassay for rat FSH

Species specificity of the radioreceptor assay (RRA) for rat FSH, in which pregnant mare serum gonadotropin (PMSG)-treated immature rat ovary was employed as the receptor, was compared with that of NIAMDD rat FSH radioimmunoassay (RIA). In the RIA system, pituitary preparations from mammals only showed significant crossreaction. Their inhibition curves, however, were not always parallel to the standard curve. On the other hand, in the RRA system, the pituitary preparations from mammals, avians, lizard and amphibians competitively inhibited the binding of radioactive rat FSH to the ovarian receptor. Only the pituitary preparation from dog salmon failed to show any crossreaction in the RRA system. These results indicated that this RRA system would be useful for the measurement of FSH or gonadotropins of the pituitaries from mammals to amphibians.     

Change in the charactertistics of 3H-spiperone binding to rat striatal membranes after acute chlorpromazine administration: effects of buffer washing of membranes.

After intraperitoneal injections of ³H-spiperone into the rat, brain membrane preparations retain the majority of the radioactivity even after several buffer washes. With ³H-spiperone as ligand, dissociation constants were significantly elevated and maximum binding unchanged in rat striatal membranes after acute intraperitoneal injection of chlorpromazine (14 mg/kg). It is suggested that in studies of post-mortem brains of schizophrenics that contain neuroleptics specific ³H-spiperone binding will be lowered by competition from residual drug in membrane preparations and valid comparisons of ³H-spiperone binding to preparations from control and schizophrenic brains can only be made if maximum binding values are determined.     

High-Affinity Choline Transport Regulation by Drug Administration During Postnatal Development

High-affinity choline transport sites specifically bind [3H]hemicholinium-3. Hemicholinium-3 binding sites are regulated by in vivo drug treatments in the same manner as these drugs alter acetylcholine release and high-affinity choline transport. The current study examines regulation of binding sites by in vivo drug administration for adult, day 15, and day 5 rats. Drugs or saline were administered intraperitoneally, and striatal and cortical membrane preparations were assayed. Control [3H]hemicholinium-3 binding increases twofold between postnatal days 5 and 15 only in striatum. After day 15, binding increases 2.7-fold in cortex and striatum. Nicotine treatment increases striatal and cortical hemicholinium-3 binding at all three ages, with greater percent increases at day 5. Haloperidol increases binding only in striatum, again with larger effects at day 5. Both striatal and cortical binding are reduced by oxotremorine; however, the magnitude of this effect is unchanged during development. Pentobarbital reduces binding only in striatum, with no developmental change. Atropine and apomorphine do not change binding from control values. In summary, all drug treatments effective in adults were already effective by day 5. Cholinergic terminals present early in development are regulated by similar nicotinic and muscarinic cholinergic, dopaminergic, and sedative-hypnotic mechanisms as the adult. Changes in magnitude may be due to changes in drug metabolism or to developmental differences in regulation.