Search PubMed⌕ Search

Biomedical subjects

R C Bruch

Publications and source records attributed to R C Bruch.

At least 37 records · Page 2Linked to original sources
PubMed1993

Bromocriptine, a dopamine D2 receptor agonist, inhibits adenylyl cyclase activity in rat olfactory epithelium.

The presence of large numbers of dopaminergic neurons in the olfactory bulb suggests that dopamine plays an important role in olfaction. Dopamine D2 receptors are produced in olfactory sensory neurons [Shipley et al. (1991) Chem. Senses 16, 5] and found in relatively high concentrations in their terminals in the nerve and glomerular layers of the olfactory bulb [Nickell et al. (1991) NeuroReport 2, 9-12]. In other systems D2 receptors are linked to adenylyl cyclase by an inhibitory G-protein, and activation of the receptors results in inhibition of the enzyme. We examined rat olfactory mucous membrane to determine whether the D2 receptors were linked functionally to adenylyl cyclase as they are in other tissues. Adenylyl cyclase is found in both the olfactory cilia of the sensory epithelium and olfactory nerve terminals in the bulb. Bromocriptine, a D2 receptor agonist, was added to olfactory epithelium membrane preparations from normal and unilaterally bulbectomized adult rats and the preparations were assayed for forskolin-stimulated adenylyl cyclase activity. In unoperated animals bromocriptine significantly inhibited adenylyl cyclase activity, and the inhibition was abolished following pertussis toxin treatment. In mucosa from unilaterally bulbectomized animals we saw significantly lower adenylyl cyclase activity on the operated side and a further decrease in response to bromocriptine. The data indicate that bromocriptine decreases adenylyl cyclase activity in olfactory tissue, specifically in the sensory neurons, and the reaction is dependent on a pertussis toxin-sensitive G-protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclase Inhibitors↗
PubMed1992

Ultrastructural localization of olfactory transduction components: the G protein subunit Golf alpha and type III adenylyl cyclase.

Electron microscopy and postembedding immunocytochemistry on rapidly frozen, freeze-substituted specimens of rat olfactory epithelia were used to study the subcellular localization of the transduction proteins Golf alpha and type III adenylyl cyclase. Antibody binding sites for both of these proteins occur in the same receptor cell compartments, the distal segments of the olfactory cilia. These segments line the boundary between organism and external environment inside the olfactory part of the nasal cavity. Therefore, they are the receptor cell regions that most likely first encounter odorous compounds. The results presented here provide direct evidence to support the conclusion that the distal segments of the cilia contain the sites of the early events of olfactory transduction.

Adenylyl Cyclases↗
PubMed1991

Rat olfactory neurons express a 200 kDa neurofilament.

Neurofilament expression in peripheral olfactory neurons of adult rats was investigated by immunoblotting and immunohistochemistry using monoclonal antibodies specific for each of the 3 neurofilament proteins. Immunoblotting analysis of olfactory epithelium extracts demonstrated the presence of only the 200 kDa (NFH) polypeptide; the 68 kDa (NFL) and 160 kDa (NFM) neurofilaments were not detected. Similarly, no immunoreactivity was observed in tissue sections using the NFL and NFM antibodies. In contrast, when sections were probed with the antibody to NFH, immunoreactivity was localized primarily in the dendritic knobs and near the cell bodies of the receptor cells.

Animals↗
PubMed1991

Amitriptyline-mediated inhibition of neurite outgrowth from chick embryonic cerebral explants involves a reduction in adenylate cyclase activity.

We have previously shown that amitriptyline, a tricyclic antidepressant, inhibited neurite outgrowth from chick embryonic cerebral explants, and that dibutyryl cyclic AMP, 3-isobutyl-1-methylxanthine, or theophylline can enhance neurite outgrowth from embryonic olfactory explants. In the present study, we examined the mechanism(s) underlying amitriptyline-mediated inhibition of neurite outgrowth by studying the effects of amitriptyline on adenylate cyclase activity and cyclic AMP levels. In cultured chick embryonic cerebral explants, dibutyryl cyclic AMP or theophylline, but not dibutyryl cyclic GMP, enhanced neurite outgrowth and partially reduced the inhibitory effects of amitriptyline on neurite outgrowth. Explants treated with amitriptyline for 2 days showed decreased cyclic AMP levels that significantly correlated with the degree of neurite outgrowth. Amitriptyline inhibited both basal and forskolin-stimulated adenylate cyclase activity in vitro, but only in the presence of GTP. Taken together, these data suggest that amitriptyline inhibits the activity of adenylate cyclase via a GTP-dependent mechanism, and that the subsequent decrease in cyclic AMP level may be involved in amitriptyline-mediated inhibition of neurite outgrowth.

Adenylyl Cyclases↗
PubMed1990

Signal transducing GTP-binding proteins in olfaction.

1. Several members of the family of heterotrimeric signal transducing GTP-binding proteins have been identified in the olfactory epithelium of vertebrates by biochemical and molecular cloning techniques. 2. Biochemical and electrophysiological evidence indicates that one or more GTP-binding proteins mediate many olfactory responses by coupling stimulus receptors to second messenger systems. 3. Although GTP-binding proteins may function in additional transduction events, a novel GTP-binding protein, expressed only in olfactory neurons, may mediate stimulus activation of adenylate cyclase in olfactory cilia.

Adenylyl Cyclases↗
PubMed1989

In vitro evidence for an inhibitor of lipogenesis in serum from overfed obese rats.

Involvement of a humoral agent in regulation of energy balance has been demonstrated by parabiosis experiments. Overfed obese rats produce a blood-borne factor that inhibits adipose fatty acid synthesis in their partners, resulting in loss of body fat without significant inhibition of food intake. An in vitro bioassay was developed to test small serum samples for antilipogenic activity. Epididymal adipocytes from ad libitum-fed rats were preincubated for 12 h with 2% serum. Basal adipocyte fatty acid synthesis, measured in a subsequent serum-free incubation, was inhibited by obese serum. Insulin response was not changed. Characterization studies indicated that the factor was probably a protein, larger than 30 kDa, but not a protease or a low-density lipoprotein and was not associated with serum albumin. Physiological experiments demonstrated that the agent was produced when body weight was raised substantially above "set-point." Inhibitory activity was neither species specific nor pituitary dependent. Structure, origin, and physiological significance of the factor are unknown, but it may be involved in the control of body fat content.

Adipose Tissue↗
PubMed1988

Ligand binding specificity of a neutral L-amino acid olfactory receptor.

1. The ligand binding specificity of the L-[3H]alanine binding site was investigated in isolated cilia preparations from the olfactory epithelium of channel catfish (Ictalurus punctatus) by competitive binding experiments. 2. Approximately 45 amino acids, derivatives and enantiomers were tested for the ability to compete with radiolabeled L-alanine for common binding sites. 3. Acidic and basic L-amino acids and imino acids did not compete as effectively as L-alanine for the receptor, while long-chain neutral ligands were only partially effective inhibitors of L-alanine binding. 4. D-Alanine and L-alanine derivatives with substituted alpha-amino or carboxyl groups exhibited decreased ability to compete for the receptor, paralleling their lower neurophysiological potency. 5. In combination, the ligand binding results were consistent with previous electrophysiological data in catfish, and suggest the presence of an olfactory receptor site that selectively recognizes short-chain neutral amino acids.

Alanine↗
PubMed1987

Differential interaction of lectins with chemosensory receptors.

L-Alanine and L-arginine bind with similar affinity (Kd 10(-7)-10(-6) M) to receptors in both a sedimentable fraction (P2) from taste epithelium and isolated olfactory cilia from the channel catfish, Ictalurus punctatus. Lectins of differing carbohydrate specificity were used to determine the glycoprotein nature of the chemosensory plasma membranes and to differentially affect receptors for L-alanine and L-arginine. The peroxidase-conjugated lectins concanavalin A (Con A), wheat germ agglutinin (WGA), and peanut agglutinin (PNA) were used to identify the glycoprotein components of the chemosensory plasma membranes after polyacrylamide gel electrophoresis. In both chemosensory membranes, numerous protein components were labelled by Con A and WGA. In contrast, a single predominant component was labeled by PNA in olfactory cilia, whereas several proteins in taste membranes were labeled by this lectin. When unconjugated lectins were preincubated with olfactory cilia, 60-70% of binding to L-alanine and L-arginine receptors was inhibited by Con A and WGA. PNA inhibited L-alanine but not L-arginine binding to olfactory receptors. Inhibition of olfactory receptor binding by lectins was time- and dose-dependent. By contrast, no inhibition of either L-alanine or L-arginine receptor binding in taste membranes was observed with any of the lectins. The differential labeling of the chemosensory membranes and the differential inhibition of receptor binding by lectins suggest that, despite ligand similarity, the chemosensory receptors in these membranes are not identical molecular species.

Alanine↗
PubMed1987

Interaction of GTP-binding regulatory proteins with chemosensory receptors.

GTP-binding regulatory proteins (G-proteins) were identified in chemosensory membranes from the channel catfish, Ictalurus punctatus. The common G-protein beta-subunit was identified by immunoblotting in both isolated olfactory cilia and purified taste plasma membranes. A cholera toxin substrate (Mr 45,000), corresponding to the G-protein that stimulates adenylate cyclase, was identified in both membranes. Both membranes also contained a single pertussis toxin substrate. In taste membranes, this component co-migrated with the alpha-subunit of the G-protein that inhibits adenylate cyclase. In olfactory cilia, the Mr 40,000 pertussis toxin substrate cross-reacted with antiserum to the common amino acid sequence of G-protein alpha-subunits, but did not cross-react with antiserum to the alpha-subunit of the G-protein from brain of unknown function. The interaction of G-proteins with chemosensory receptors was determined by monitoring receptor binding affinity in the presence of exogenous guanine nucleotides. L-Alanine and L-arginine bind with similar affinity to separate receptors in both olfactory and gustatory membranes from the catfish. GTP and a nonhydrolyzable analogue decreased the affinity of olfactory L-alanine and L-arginine receptors by about 1 order of magnitude. In contrast, the binding affinities of the corresponding taste receptors were unaffected. These results suggest that olfactory receptors are functionally coupled to G-proteins in a manner similar to some hormone and neurotransmitter receptors.

Adenosine Diphosphate Ribose↗
PubMed1987

Properties of phospholipase C in isolated olfactory cilia from the channel catfish (Ictalurus punctatus).

1. Cilia were isolated from the olfactory epithelium of the channel catfish (Ictalurus punctatus) with improved yield. The isolated preparations were enriched in cilia as indicated by electron microscopy, tubulin immunoblotting and identification of a ciliary-specific glycoprotein. 2. The isolated cilia preparations exhibited phospholipase C (EC 3.1.4.11) activity. The enzyme was maximally active at pH 6.7. 3. Analysis of inositol phosphates resulting from the hydrolysis of exogenous radiolabeled phosphatidylinositol-4,5-bisphosphate in isolated cilia, indicated that inositol triphosphate was the major (90%) inositol phosphate produced. 4. Three molecular forms of the enzyme, Mr greater than or equal to 100,000, 82,000 and 60,000 were resolved by gel filtration chromatography from a cytosolic fraction from the olfactory epithelium.

Animals↗
PubMed1986

The rat ovarian lutropin receptor. Purification, hormone binding properties, and subunit composition.

The luteinizing hormone/human choriogonadotropin (hCG) receptor from superovulated rat ovary was purified to homogeneity. A novel scheme based on reverse immunoaffinity chromatography using immobilized antibodies to membrane proteins from receptor down-regulated ovary and subsequent two-step affinity purification on hCG-Sepharose was used to isolate homogeneous receptor. The purification method was also compared to an alternate scheme involving lectin affinity chromatography followed by hCG affinity chromatography. The purified receptor obtained by the latter method was heterogeneous and highly aggregated. The hormone binding properties, molecular size, and subunit composition of the purified receptor obtained by either method were identical. The stability of the receptor during and following solubilization was markedly improved by using 20% glycerol. The pure receptor consists of four nonidentical subunits of molecular weight 79,300 (alpha), 66,400 (beta), 55,300 (gamma), and 46,700 (delta) as indicated by polyacrylamide gel electrophoresis under reducing conditions. All receptor subunits generally, but occasionally excepting the alpha-subunit, were specifically labeled with iodinated hCG in membrane and soluble receptor preparations using bifunctional cross-linking agents. Analysis of the cross-linked hormone-receptor complexes under nonreducing conditions showed the molecular mass of the undissociated receptor to be 268,000 daltons. Hormone binding studies demonstrated that the isolated receptor retained all of the specific binding characteristics expected for the luteinizing hormone/hCG receptor. In combination, these results indicate that the functional and structural properties of the receptor were not altered during purification.

Animals↗
PubMed1986

Odorant- and guanine nucleotide-stimulated phosphoinositide turnover in olfactory cilia.

Isolated olfactory cilia from the channel catfish (Ictalurus punctatus) exhibited phosphatidylinositol-4,5-bisphosphate phosphodiesterase (E.C.3.1.4.11) activity. The phosphodiesterase activity was stimulated in the presence of an odorant for the catfish, namely the amino acid L-alanine. The enzyme activity was also stimulated in the presence of GTP and its nonhydrolyzable analogues. The activation of the phosphodiesterase by guanine nucleotides, in combination with the identification of guanine nucleotide-binding protein(s) in the isolated cilia, indicate the probable participation of a guanine nucleotide-binding protein in stimulation of phosphoinositide turnover in the olfactory receptor neuron.

Animals↗
PubMed1984

Characterization of virtual coupling in the proton nuclear magnetic resonance spectrum of N,N'-diacetylchitobiose by two-dimensional J-resolved spectroscopy.

The anomeric proton of the reducing N-acetylglucosamine residue of beta-N,N'-diacetylchitobiose exhibited an unusual lineshape indicative of virtual coupling in the 250 MHz proton NMR spectrum. The two-dimensional J-resolved spectrum contained four lines associated with this proton, and this pattern can be used to distinguish virtual coupling from other effects. Anomeric protons of previously studied asparagine-linked glycopeptides which exhibited unusual lineshapes in the conventional NMR spectra did not exhibit the four line patterns indicative of virtual coupling in the two-dimensional J-resolved spectra. Although virtual coupling may partially account for the unusual lineshapes observed in the normal proton NMR spectra, the anomalous behavior observed in the two-dimensional J-resolved spectra of these glycopeptides cannot be explained solely by this phenomenon.

Disaccharides↗
PubMed1983

Differential effect of lipid peroxidation on membrane fluidity as determined by electron spin resonance probes.

The effect of lipid peroxidation on membrane fluidity was examined in sonicated soybean phospholipid vesicles. Following iron/ascorbate dependent peroxidation, the vesicles were labeled with a series of doxyl stearate spin probes which differed in the site of attachment of the nitroxide free radical to the fatty acid. Comparison of motional and partitioning parameters derived from electron spin resonance spectra of the probes indicated that the membranes were less fluid following peroxidation. However, the magnitude of the fluidity decrease was markedly dependent on the intramembrane location, as well as on the extent of lipid peroxidation. The effect of lipid peroxidation on fluidity was maximal in the membrane microenvironment sampled by 12-doxyl stearate, whereas other regions of the bilayer were less affected. These findings indicate that lipid peroxidation leads to an alteration of the transbilayer fluidity gradient.

Electron Spin Resonance Spectroscopy↗
PubMed1982

Two-dimensional J-resolved proton NMR spectroscopy of oligomannosidic glycopeptides.

Individual anomeric protons that are unresolved in the one-dimensional 250-MHz spectra of oligomannosidic glycopeptides can be separated and characterized by two-dimensional J-resolved NMR spectroscopy. Homogeneous preparations of ovalbumin glycopeptides Man6GlcNAc2Asn and Man5GlcNAc2Asn were characterized by chemical methods, conventional proton NMR, and two-dimensional J-resolved NMR. Due to characteristic differences in coupling constants, mannose (J1,2 less than or equal to 2 Hz) and N-acetylglucosamine (J1,2 approximately 9 Hz) anomeric signals of similar chemical shift were readily separated and identified in the two-dimensional spectra. It is shown that two-dimensional J-resolved NMR spectroscopy, in combination with conventional NMR and limited chemical analysis, is a rapid and reliable technique for the determination of glycopeptide primary structure.

Animals↗