Studies of nicotinic acetylcholine receptor protein from rat brain

Specific binding of 125I-labeled alpha-bungarotoxin to a 34800 X g pellet of a whole rat brain homogenate has been obtained at levels of 2 pmol toxin per g of whole brain with a Kd of 8-10(-9) M. Binding is reduced 90% by 10(-5) M (+)-tubocurarine chloride and 10(-4) M nicotine, whereas concentrations of 10(-4) M choline chloride, atropine sulfate and eserine sulfate have essentially no effect on toxin binding. These results compare closely with those obtained from binding studies with 125I-labeled alpha-bungarotoxin and soluble acetylcholine receptor protein preparations from Torpedo nobiliana; suggesting that this mammalian receptor protein is nicotinic in character. Extraction of the 34800 X g pellet with 1% Emulphogene yields a soluble fraction with specifically binds 125I-labeled alpha-bungarotoxin with a Kd of 5-10(-9) M. Nicotine and alpha-bungarotoxin at concentrations of 10(-5) M abolish toxin-receptor complex formation and carbachol and (+)-tubocurarine chloride reduce complex formation 35-40% at similar concentrations. Eserine sulfate, atropine sulfate, decamethonium, and pilocarpine had no effect on complex formation at concentrations of 10(-5) M.     

Characterization of Clostridium botulinum type B neurotoxin associated with infant botulism in japan

The neurotoxin of strain 111 (111/NT) associated with type B infant botulism showed antigenic and biological properties different from that (Okra/NT) produced by a food-borne botulism-related strain, Okra. The specific toxicity of 111/NT was found to be about 10 times lower than that of Okra/NT. The monoclonal antibodies recognizing the light chain cross-reacted with both neurotoxins, whereas most of the antibodies recognizing the carboxyl-terminal half of the heavy chain of Okra/NT did not react to 111/NT. Binding experiments with rat brain synaptosomes revealed that 125I-labeled 111/NT bound to a single binding site with a dissociation constant (Kd) of 2.5 nM; the value was rather lower than that (0.42 nM) of 125I-Okra/NT for the high-affinity binding site. In the lipid vesicles reconstituted with ganglioside GT1b, 125I-Okra/NT interacted with the amino-terminal domain of synaptotagmin 1 (Stg1N) or synaptotagmin 2 (Stg2N), fused with the maltose-binding protein, in the same manner as the respective full-length synaptotagmins, and the Kd values accorded with those of the low- and high-affinity binding sites in synaptosomes. However, 125I-111/NT only exhibited a low capacity for binding to the lipid vesicles containing Stg2N, but not Stg1N, in the presence of ganglioside GT1b. Moreover, synaptobrevin-2, an intracellular target protein, was digested to the same extent by the light chains of both neurotoxins in a concentration-dependent manner. These findings indicate that the 111/NT molecule possesses the receptor-recognition site structurally different from Okra/NT, probably causing a decreased specific toxicity.     

Astigmatism, accommodation, the oblique effect and meridional amblyopia

Agonist binding to GABAB receptors modulates the activity of the guanine nucleotide binding proteins, Go and Gi. These G proteins are ADP-ribosylated by pertussis toxin and this prevents them from coupling to the GABAB receptor resulting in a reduction in high-affinity GABAB binding. GTP, which binds to a different site on the G protein alpha subunit, also reduces the affinity of the receptor for the G protein, and this can be used as a "marker" for G protein-GABAB receptor linkage. We have examined GABAB binding site distribution in rat brain after unilateral intrahippocampal pertussis toxin injection in vivo, and after incubating brain slices in pertussis toxin in vitro, using the technique of receptor autoradiography. The effect of pertussis toxin was compared with that of GTP gamma S on GABAB binding. Intrahippocampal pertussis toxin administration reduced GABAB but not GABAA receptor binding and the effects appeared to be limited by pertussis toxin diffusion. More widespread reductions in GABAB binding were seen after incubation of brain slices in vitro but the extent varied in different brain regions. No reduction was detected in the corpus striatum. GABAB binding was also reduced in membranes prepared from cerebral cortex, hippocampus and cerebellum but there was no significant reduction in the corpus striatum after pertussis toxin treatment. GTP gamma S reduced GABAB binding to a similar extent in all areas studied irrespective of their sensitivity to pertussis toxin suggesting that while GABAB binding sites are linked to G proteins throughout the rat brain, those in the corpus striatum may be predominantly pertussis toxin insensitive.     

The human 5-ht5A receptor couples to Gi/Go proteins and inhibits adenylate cyclase in HEK 293 cells

The G protein coupling of human 5-hydroxytryptamine5A (h5-ht5A) receptors was investigated in stably transfected human embryonic kidney (HEK) 293 cells, using radioligand and guanosine-5'[gamma-35S]thiotriphosphate binding to membranes and cyclic adenosine monophosphate measurements in cells. 5-Carboxamido[3H]tryptamine bound to high- and low-affinity sites on h5-ht5A-HEK 293 cell membranes. Guanylyl-imidodiphosphate addition and pertussis toxin pre-treatment abolished high-affinity binding, indicating coupling to G proteins of the Gi/Go family. [N-methyl-3H]Lysergic acid diethylamide bound to a single site; guanylyl-imidodiphosphate and pertussis toxin did not alter lysergic acid diethylamide affinity. 5-Hydroxytryptamine stimulated guanosine-5'[gamma-35S]thiotriphosphate binding to 130% over basal and this effect was completely abolished by pertussis toxin. Various 5-hydroxytryptamine receptor ligands were tested for inhibition of 5-carboxamido[3H]tryptamine binding and in guanosine-5'[gamma-35S]thiotriphosphate binding assays. 5-Hydroxytryptamine consistently inhibited forskolin-induced cyclic adenosine monophosphate formation by 25% in h5-ht5A-HEK 293 cells; no effect was detected on basal cyclic adenosine monophosphate levels, on intracellular Ca2+ concentration or arachidonic acid release. Our studies demonstrate functional coupling of the h5-ht5A receptor to pertussis toxin-sensitive G proteins and to inhibition of adenylate cyclase activity.     

Anti-HBs antibody kinetics--a 4-year follow-up after hepatitis B vaccination

The characteristics of binding sites in rat cerebral cortical synaptic membranes labeled by 125I-ifenprodil, a noncompetitive NMDA receptor antagonist, are described. 125I-ifenprodil was synthesized using Na125I in the presence of chloramine-T and purified by paper chromatography. Binding of the 125I-ligand was optimal at pH 7.7 in 5 mM Tris.HCl buffer. Equilibrium binding of 125I-ifenprodil was displaced by spermine (1 mM) but not by ifenprodil or its analogue, SL 82.0715 (both 16.7 microM). Zn2+, Ca2+, and Mg2+ inhibited specific binding of 125I-ifenprodil in a concentration-dependent manner, with IC50 values of 0.11, 1.1, and 1.7 mM, respectively. The dissociation constant (KD) for unlabeled ifenprodil determined by saturation binding was 205 nM. Scatchard plots of saturation data appeared curvilinear but were best described by a single-binding-site model (Hill coefficient = 0.95), with a density of binding sites (Bmax) of 141 pmol/mg of protein. Binding of 125I-ifenprodil was inhibited by polyamines, with a rank potency order of spermine > spermidine > putrescine = 1,3-diaminopropane. The pattern of inhibition produced by spermidine was apparently competitive. Ifenprodil congeners also fully inhibited polyamine-sensitive binding of 125I-ifenprodil, with a rank potency order of ifenprodil > SL 82.0715 = tibalosine > nylidrin = isoxsuprine. It was found that sigma/antitussive agents partially inhibited specific binding, but inclusion of the sigma drug GBR 12909 had little effect on the binding of 125I-ifenprodil, suggesting this site was not involved. The binding site labeled by 125I-ifenprodil is polyamine sensitive, has a discrete pharmacological profile, and apparently is unrelated to the sigma site.     

Interaction of Escherichia coli heat-stable enterotoxin B with cultured human intestinal epithelial cells

Binding of Escherichia coli heat-stable enterotoxin B (STb) to the human intestinal epithelial cell lines T84 and HT29 and to polarized T84 cells was studied to define the initial interaction of this peptide toxin with target cells. Equilibrium and competitive binding isotherms showed that 125I-STb bound specifically to T84 and HT29 cells; however, the toxin-epithelial cell interactions could be characterized by low-affinity binding (< or = 10(5) M(-1)) to a high number of binding sites (> or = 10(6) per cell). STb binding to T84 and HT29 cells as a function of 125I-STb concentration did not approach saturation at levels well above the effective biological concentration of STb for fluid secretion. Treatment of the 125I-STb-bound T84 and HT29 cells with an acidic saline solution to remove surface-bound toxin revealed that only approximately 55% +/- 10% of 125I-STb could be removed by this treatment at 4 degrees C, suggesting that approximately half of the bound STb was stably associated with the plasma membrane and/or internalized into the cytoplasm. Similar results were obtained when binding and internalization experiments were conducted at 22 and 37 degrees C. Immunofluorescence studies demonstrated that the strongest signal for STb appeared in the plasma membrane even after acid treatment. Toxin-treated cells also displayed diffuse cytoplasmic staining, indicating that once cell bound, STb did not appear to preferentially associate with membrane vesicles or cellular organelles. Binding and subsequent internalization of 125I-STb were not affected by treatment of the cells with trypsin, endoglycosidase F/peptide N-glycosidase F, Vibrio cholerae neuraminidase, tunicamycin, or 5 mM sodium chlorate, which blocks sulfation of surface proteoglycans. In addition, the internalization process was not altered by preincubation of the cells with the cytoskeleton inhibitors cytochalasin D and colchicine or cellular perturbants (i.e., 0.45 M sucrose and 5 mM sodium azide), indicating that cell surface proteins or carbohydrates did not function as STb receptors. The binding of 125I-STb to polarized T84 cells was also examined, and the total and nonspecific binding isotherms were found to overlap, indicating that the apical surface of polarized T84 cells did not contain a specific receptor for STb. In comparison to undifferentiated cells, twice the amount of bound STb (approximately 80% +/- 10%) was removable from polarized T84 cells after treatment with acidic solution. The percentage of surface-bound STb to polarized T84 cells did not vary significantly with the transepithelial electrical resistance of the cells or when STb was applied basolaterally. Together, our results indicate that STb binds with relatively low affinity to the plasma membrane of cultured intestinal epithelial cells and polarized T84 cells, probably to membrane lipids, and becomes stably associated with the lipid bilayer. The fact that a significant portion of the bound STb becomes free in the cytoplasm, even at a low temperature, suggests that the bound toxin may directly traverse the membrane bilayer.     

Binding of pertussis toxin to lipid vesicles containing glycolipids

The binding of pertussis toxin and its B oligomer to lipid vesicles containing glycosphingolipids was studied. Both pertussis toxin and the B oligomer bound to lipid vesicles containing ganglioside GD1a. Binding of pertussis toxin to these vesicles decreased upon treatment of the vesicles with neuraminidase, suggesting that sialic acid residues are important for efficient binding of the toxin to GD1a.     

Characterization of a specific binding site for angiotensin II in chicken liver

We have characterized a specific binding site for angiotensin II (AngII) in chicken liver membranes. Pseudo-equilibrium studies at 22 degrees C for 30 min have shown that this binding site recognizes AngII with a high affinity (pKD of 8.13 +/- 0.21). The binding sites are saturable and relatively abundant (maximal binding capacity varies from 0.318 to 0.88 pmol/mg of protein). Nonequilibrium kinetic analyses at 22 degrees C revealed a calculated kinetic pKD of 8.77 +/- 0.20. The binding site is pharmacologically distinct from the classic AngII receptors AT1 and AT2. Competitive binding studies with chicken liver membranes demonstrated the following rank order of effectiveness: AngII (human; Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) > AngI(Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) > AngIII(Arg-Val-Tyr-Ile-His-Pro-Phe) > AngIV (Val-Tyr-Ile-His-Pro-Phe) > Ang(1-7) (Asp-Arg-Val-Tyr-Ile-His-Pro) > PD123319 (1-[4(dimethylamino)3-methylphenyl] methyl-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo [4,5-c]pyridine-6-carboxylic acid) > DuP753 (2-n-butyl-4-chloro-5 hydroxymethyl-1-[(2'-1H-tetrazol-5-yl)biphenyl-4-yl)methyl] imidazole. This atypical AngII binding site (chicken AT) was sensitive to increasing concentrations of DTT and Mn2+. The structure-activity relationship on position 1 of AngII showed that the primary N-terminal amine was essential for binding affinity ([Asp1]AngII > [Suc1]AngII > or = [Sar1]AngII), but modifications of the side chain in position 1 had less influence on the affinity ([Gly1]AngII > [Cys1]AngII approximately [aminoisobutyryl1]AngII approximately [Ser1]AngII > > > [Sar1]AngII). The presence of substantial quantities of this binding site in chicken liver membranes suggests the possibility that the chicken AT may play an important, yet unrecognized, role in the renin-angiotensin system.     

Glucagon binding to hepatocytes isolated from two teleost fishes, the American eel and the brown bullhead

We have characterized the specific binding of glucagon in hepatocytes isolated from two teleost species, the American eel (Anguilla rostrata) and the brown bullhead (Ictalurus nebulosus). Specific glucagon binding was 9.3 and 10.7% in bullhead and eel hepatocytes respectively, after a 2-h incubation at 12 degrees C. Curvilinear Scatchard plots suggest the presence of two classes of binding sites with apparent dissociation constants (Kd) of 1.97 nM (high affinity) and 17.3 nM (low affinity) for bullhead and 2.68 and 22.9 nM for eel cells. The number of high-affinity binding sites per cell was significantly higher in the eel (10,413) than in the bullhead (3811). The number of high-affinity insulin-binding sites was approximately two times higher than that for glucagon in bullheads and the opposite in the eel hepatocytes. In competition experiments, insulin did not displace 125I-labelled glucagon binding in the hepatocytes of either species, while glucagon-like peptide-1(7-37) (GLP-1) displaced glucagon but only at high concentrations, suggesting separate glucagon- and GLP-1-binding sites. The rate of dissociation of hepatocyte-bound 125I-labelled glucagon was similar for both species. Preincubation of hepatocytes in 100 nM glucagon decreased the number of high-affinity glucagon-binding sites by approximately 55% in both species, while the Kd values remained unchanged. Glucagon bound to the cell surface is internalized by fish hepatocytes. These properties indicate that the glucagon binding to hepatocytes of these two teleost species is similar to that reported for mammalian hepatocytes.     

Ricin A chain can be chemically cross-linked to the mammalian ribosomal proteins L9 and L10e

Indirect immunofluorescence studies revealed that when fixed, permeabilized cultured human cells were incubated with ricin A chain, the toxin molecule localized in a staining pattern indicative of binding to the endoplasmic reticulum and to nucleoli. Chemical cross-linking experiments were performed to identify the cellular components that mediated the binding of ricin A chain. Conjugates were formed between 125I-labeled ricin A chain and two proteins present in preparations of total cell membranes and in samples of purified mammalian ribosomes. Specificity of the ricin A chain-ribosome interaction was demonstrated by inhibition of formation of the complexes by excess unlabeled ricin A chain, but not by excess unlabeled gelonin, another ribosome-inactivating protein. Complexes of ricin A chain cross-linked to the ribosomal proteins were purified and subjected to proteolytic digestion with trypsin. Amino acid sequencing of internal tryptic peptides enabled identification of the ricin A chain-binding proteins as L9 and L10e of the mammalian large ribosomal subunit.     

Purification and kinetic properties of Mus booduga (Gray) hepatic arginase

Hepatic L-arginase from the Mus booduga (Gray) was purified and its kinetic characteristics were investigated. The enzyme was not adsorbed on DEAE-cellulose, but was retained on CM-cellulose column at pH 7.2. The Michaelis-Menten constant was 8.3 mM for L-arginine and was independent of pH in the range of 7.5-10.5. L-arginine concentrations as high as 0.4 M did not exert substrate inhibition in the pH range 7.4-10.0. Manganese was required at a concentration of 0.05 M for full activation of the enzyme. L-ornithine and L-lysine inhibited the enzyme competitively with inhibitory constants of 1.9 mM and 3.7 mM respectively. Several properties of the L-arginase from Mus booduga clearly identify it as an enzyme similar to ureotelic basic arginases from mammalian liver.     

Membrane-bound calmodulin in Xenopus laevis oocytes as a novel binding site for melatonin

Melatonin has been suggested as a physiological antagonist of calmodulin. In this work, we have characterized melatonin binding sites in Xenopus laevis oocyte membranes. Binding of [125I]melatonin by X. laevis oocyte membranes fulfills all criteria for binding to a receptor site. Binding was dependent on time, temperature, and membrane concentration and was stable, reversible, saturable, and specific. The binding site was also pharmacologically characterized. Stoichiometric studies showed a high-affinity binding site with a Kd of 1.18 nM. These data are in close agreement with data obtained from kinetic studies (Kd=0.12 nM). In competition studies, we observed a low-affinity binding site (Kd=63.41 microM). Moreover, the binding site was characterized as calmodulin. Thus, binding was dependent on calcium and blocked by anti-CaM antibodies in a concentration-dependent manner. Calmodulin inhibitor chlorpromazine also inhibited binding of the tracer. From these results, it is suggested that membrane-bound calmodulin acts as a melatonin binding site in Xenopus laevis oocytes, where it might couple cellular activities to rhythmic circulating levels of melatonin. This hypothesis correlates with the previous findings describing melatonin as a physiological antagonist of calmodulin.     

Expression of a functional endothelin (ETA) receptor in human meningiomas

Endothelin (ET) receptor subtypes (ETA and ETB) in human meningiomas were characterized using quantitative receptor autoradiography. A single class of high-affinity 125I-ET-1 binding sites was localized in all meningioma tissue studied (dissociation constant: 2.4 +/- 0.3 nM, maximum binding capacity: 319 +/- 66 fmol/mg (mean +/- standard error of the mean for 13 tumors)). Unlabeled ET-1 showed a strong affinity for 125I-ET-1 binding to tissue sections of the tumors with a 50% inhibiting concentration (IC50) of 2.9 +/- 0.7 x 10(-9) M, whereas ET-3 showed a much lower affinity (IC50: 8.4 +/- 2.5 x 10(-6) M). Sarafotoxin S6c, a selective agonist for the ETB receptor, could not compete for 125I-ET-1 binding to meningiomas. Endothelin-1 significantly stimulated deoxyribonucleic acid (DNA) synthesis in a dose-dependent manner in cultured human meningioma cells. In contrast, no significant stimulation of DNA synthesis occurred with an S6c concentration up to 10(-7) M. Pretreatment of the meningioma cells with pertussis toxin, a bacterial toxin that adds adenosine 5'-diphosphate-ribose to the alpha subunit of guanine nucleotide binding (G) proteins such as Gi or G(o), induced a concentration-dependent reduction in ET-stimulated DNA synthesis in meningioma cells, but did not affect the epidermal growth factor-induced DNA synthesis. These observations suggest that the ETA receptor is predominantly expressed in human meningioma tissue and that ET may act as a growth factor on the meningioma cells by interacting with the ETA receptor and by pertussis toxin-sensitive mechanisms.     

Melatonin binding sites in the harderian gland of the rat and Syrian hamster

Specific melatonin binding sites in the harderian gland of both rat and Syrian hamster were studied using [125I]melatonin. In both species, binding of [125I]melatonin by harderian gland membranes exhibited properties such as dependence on time, temperature, membrane concentration, saturability, and high specificity. Only one class of high-affinity binding sites was found with a Kd of 0.19 and 6.47 nM for the rat and Syrian hamster, respectively. The binding capacity in the rat harderian gland was 4.00 fmol/mg protein; in the Syrian hamster it was 7.58 fmol/mg protein. In the rat, no sex differences were found in the binding of the tracer to the membranes. However, in the Syrian hamster, binding of [125I]melatonin by the harderian gland was twice higher in the female than in the male. No changes were found in the Kd values (6.47 vs. 6.94 nM), while binding capacity was significantly increased in the female (13.50 fmol/mg protein) when compared to the male hamster (7.58 fmol/mg protein). Binding of [125I]melatonin by the harderian gland of male hamsters was modified by castration but not by melatonin treatment. Castration induced an increase of binding up to the level of females. However, chronic melatonin administration did not alter the [125I]melatonin binding in either intact or gonadectomized male hamsters. Binding studies also showed diurnal variations. There was a diurnal rhythm of [125I]melatonin binding by Syrian hamster harderian glands with the peak at the end of the light period and the trough late in the dark period. This rhythm in the binding is observed in both male and female hamsters, although binding in females was always higher than that in males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid uptake and binding of estradiol-17beta-6-(O-carboxymethyl)oxime:125I-labeled BSA by female rat liver

To investigate potential membrane-mediated responses to estrogen, a membrane-impermeant, radioiodinated, steroid-BSA conjugate--estradiol-17beta-6-(O-carboxymethyl)oxime:125I-labeled BSA (17beta-E-6-125I-BSA)--or related steroid conjugates, or 125I-BSA was injected into female Sprague-Dawley rats, and tissues were collected at varying times postinjection. The liver, adrenal, and spleen displayed the most prominent uptake of 17beta-E-6-125I-BSA, reaching a maximum of 43 times blood levels in sonicated liver samples at 5 min postinjection, but no uptake of 125I-BSA. Isolation of liver membranes by differential centrifugation showed that over 50% of recovered radioactivity was in association with microsomes and plasmalemma (P3 fraction) at 30 sec postinjection. By 60 min postinjection, over 75% of recovered radioactivity was in association with mitochondrial and lysosomal membranes (P2 fraction), and less than 10% remained in the P3 fraction. In vitro competition assays demonstrated two binding sites in liver P3 fractions. The spleen and liver also showed saturable binding in vivo. These data suggest the presence of at least one membrane-binding protein for estrogen in liver, adrenal, and spleen. Initial studies of affinity-purified liver P3 fractions using ligand blots indicated the presence of two binding proteins. These potential membrane estrogen-binding proteins may be involved in a very rapid shuttling of estrogen from the plasmalemma to mitochondria and lysosomes.     

Studies of nicotinic acetylcholine receptor protein from rat brain. II. Partial purification

The pharmacological specificity of the binding of 125I-labeled alpha-bungarotoxin to a 1% Emulphogene BC-720 extract of a rat brain particulate fraction has been investigated. The extract contains a component which possesses the binding characteristics of a nicotinic acetylcholine receptor protein. The crude soluble acetylcholine receptor protein was purified by affinity chromatography utilizing the alpha-neurotoxin of Naja naja siamensis as ligand and 1.0 M carbamylcholine chloride as eluant. A single, batch-wise, affinity chromatography procedure yields an average purification of 510-fold. When this purified material is treated a second time by affinity chromatography, purification as high as 12600-fold has been obtained. Binding of 125I-labeled alpha-bungarotoxin to this purified acetylcholine receptor protein is saturable with a Kd of 1 - 10(-8) M. Nicotine and acetylcholine iodide at concentrations of 10(-5) M inhibit 125I-labeled toxin-acetylcholine receptor protein complex formation by 41 and 61% respectively. At 10(-4) M, carbamylcholine chloride and (+)-tubocurarine chloride give respectively 52 and 82% inhibition. Eserine sulfate and atropine sulfate have no effect on complex formation at a concentration of 10(-4) M. These data support the isolation of a partially purified nicotinic acetylcholine receptor protein.     

Selective reconstitution of gastrin-releasing peptide receptor with G alpha q

Identification of the molecular mechanisms that determine specificity of coupling interactions between gastrin-releasing peptide receptors (GRPrs) and their cognate heterotrimeric GTP-binding proteins is a fundamental step in understanding the signal transduction cascade initiated by receptor-ligand interaction. To explore these mechanisms in greater detail, we have developed an in situ reconstitution assay in chaotrope-extracted membranes from mouse fibroblasts expressing the GRPr, and we have used it to measure GRPr-catalyzed binding of GTP gamma S to purified G protein alpha subunits. Binding studies with 125I-labeled [D-Tyr6]bombesin(6-13) methyl ester (125I-Tyr-ME), a GRPr specific antagonist, show a single binding site with a Kd = 1.4 nM +/- 0.4 (mean +/- SD, n = 3) and capacity of 15-22 pmol of receptor per mg of protein in the extracted membrane preparations, representing a 2- to 3-fold enrichment of binding sites compared with the membranes before extraction. Quantitative ligand displacement analysis using various unlabeled GRPr agonists shows a rank order of potency characteristic of the GRPr: bombesin > or = GRP > > neuromedin B. Reconstitution of urea extracted membranes with a purified G alpha q showed that receptor-catalyzed binding of GTP gamma S was dependent on agonist (GRP) and G beta gamma subunits. The EC50 for GRP was 3.5 nM, which correlates well with the reported Kd of 3.1 nM for GRP binding to GRPr expressed in mouse fibroblasts [Benya, R. V., et al. (1994) Mol. Pharmacol. 46, 235-245]. The apparent Kd for bovine brain G beta gamma in this assay was 60 nM, and the Km for squid retinal G alpha q was 90 nM. The GRPr-catalyzed binding of GTP gamma S is selective for G alpha q, since we did not detect receptor-catalyzed exchange using either G alpha i/o or G alpha t. These data demonstrate that GRPr can functionally couple to G alpha q but not to the pertussis toxin-sensitive G alpha i/o or retinal specific G alpha t. This in situ receptor reconstitution method will allow molecular characterization of G protein coupling to other heptahelical receptors.     

Ultrasound-guided neurosurgery: a feasibility study in the 3-30 MHz frequency range

We have investigated the response of adenylate cyclase to GTP and to dopamine (DA) in striatal membranes of rats treated for 3 weeks with chlorpromazine or haloperidol, and further measured the level of Gi (an inhibitory GTP-binding protein) or Go (a similar GTP-binding protein of unknown function) in 3 areas (cerebral cortex, striatum and hippocampus) utilizing pertussis toxin-catalyzed ADP ribosylation. In saline-treated control membranes, GTP exerted a biphasic effect on basal and DA-stimulated enzyme activity--peak levels of stimulation by DA plus GTP were observed at 1 microM GTP. Conversely, dopaminergic inhibitory effects at 10-100 microM GTP were completely attenuated in chlorpromazine or haloperidol-treated membranes. D2 inhibition of adenylate cyclase by the selective D2 agonist PPHT was also attenuated due to these neuroleptic treatments, while an increase in D2 receptor binding was observed. The pertussis toxin ADP-ribosylation of G-proteins (Gi/Go) did not differ significantly in any area. This indicates that long-term neuroleptic treatments increased D2 receptor binding, but attenuated D2 inhibition of adenylate cyclase, and exercised no influence on pertussis toxin ADP-ribosylation.     

Antibodies against high-density lipoprotein binding proteins enhance high-density lipoprotein uptake but do not affect cholesterol efflux from rat hepatoma cells

High-density lipoprotein plays a key role in the reverse cholesterol transport pathway as well as in the delivery of cholesterol to the liver and steroidogenic tissues. Metabolism of high-density lipoprotein is determined by one of its apolipoproteins, apolipoprotein A-I; however, the identity and function of cellular protein which binds high-density lipoprotein remains unclear. The effect of antibodies against rat high-density lipoprotein binding proteins, HB1 and HB2, on high-density lipoprotein metabolism in a rat hepatoma cell line were studied. Cells were preincubated with the antibodies and 125I-labeled high-density lipoprotein binding and uptake as well as cholesterol biosynthesis and cholesterol efflux to human plasma or isolated high-density lipoprotein were studied. Both antibodies reacted specifically with HB1 and HB2 on the ligand and Western blots, but their binding was not blocked by high-density lipoprotein. Both antibodies inhibited 125I-labeled high-density lipoprotein binding to cells by 20-40%, but stimulated 125I-labeled high-density lipoprotein uptake by up to 2.5-fold. The antibodies had no effect on cholesterol efflux or on cholesterol synthesis. It is concluded that high-density lipoprotein binding proteins, HB1 and HB2, may be involved in high-density lipoprotein uptake in the liver rather than in mediating cholesterol efflux.     

Memory for pain and affect in chronic pain patients

Radioligand binding studies were performed to determine the effect of various cations on the characteristics of [3H]glibenclamide binding to its high-affinity receptor in rat cerebral cortex membranes. Mg2+ was shown specifically to reduce radioligand binding to membranes pretreated with 5 mM EDTA. CaCl2 enhanced [3H]glibenclamide binding whilst MnCl2, KCl and NaCl were without significant effect. 2 mM MgCl2 induced a statistically significant rightward shift in the dissociation constant for glibenclamide obtained from both saturation and competition studies. These results suggest that Mg2+ may participate in the regulation of the sulphonylurea receptor in the rat cerebral cortex.