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I Boekhoff

Publications and source records attributed to I Boekhoff.

At least 37 records · Page 2Linked to original sources
PubMed1994

Probing olfactory receptors with sequence-specific antibodies.

Molecular cloning has revealed the structure of several putative odorant receptors. Chemically synthesized peptides, that correspond to a predicted extracellular domain of the encoded proteins, were employed to generate receptor-specific antibodies. Immunohistological approaches as well as Western-blot analysis confirmed the specificity of the antipeptide sera. Furthermore, deglycosylation experiments explained the observed discrepancy between the molecular mass of odorant receptors, as determined by SDS/PAGE and Western-blot analysis of ciliary proteins (M(r) 50,000), and the predicted protein size based on the deduced primary structure from cloned receptor genes (M(r) 30,000-35,000). Receptor proteins become phosphorylated upon odorant stimulation of olfactory cilia preparations; this was demonstrated by immunoprecipitation experiments employing the sequence-directed, receptor-specific antibodies. Functional assays revealed that the receptor-specific antibodies significantly attenuate second messenger signalling elicited by inositol 1,4,5-trisphosphate-inducing odorants, whereas activation of the cAMP cascade by appropriate odorants was not affected. These observation indicate that the sequence-specific antibodies not only recognize odorant receptors, but also discriminate between receptor subtypes coupling to different second-messenger pathways.

Amino Acid Sequence↗
PubMed1994

Olfactory desensitization requires membrane targeting of receptor kinase mediated by beta gamma-subunits of heterotrimeric G proteins.

Olfaction is mediated by G protein-coupled receptors. In isolated rat olfactory cilia, odorants such as citralva stimulate a burst of cAMP, which peaks in 50 ms and returns almost to base-line level within 150 ms in the continuing presence of odorant. This desensitization is mediated by the cAMP dependent protein kinase and a specialized G protein-coupled receptor kinase originally termed beta ARK2 (GRK3). In vitro experiments suggest that the prenylated beta gamma-subunits of heterotrimeric G proteins target the cytosolic beta ARK1 (GRK2) enzyme to its membrane bound receptor substrate by binding to sites in its carboxyl terminus. Here we demonstrate that odorants stimulate translocation of GRK3 from cytosol to membranes in isolated rat olfactory cilia. We introduced a glutathione S-transferase-GRK3ct fusion protein, containing the carboxyl-terminal 222 amino acid residues of GRK3, which includes the beta gamma binding site, or a 28-amino acid peptide derived therefrom, into permeabilized cilia preparations. These reagents block odorant-mediated enzyme translocation and desensitization while markedly attenuating odorant-stimulated phosphorylation of olfactory proteins. These findings suggest that beta gamma-subunits may physiologically regulate a G protein-coupled receptor kinase and that enzyme translocation may be a general and required feature of the activity of some members of this enzyme family.

Animals↗
PubMed1994

Single odors differentially stimulate dual second messenger pathways in lobster olfactory receptor cells.

Quench-flow measurements are used to determine the subsecond kinetics of odor-induced changes in second messenger concentrations in lobster olfactory receptor neurons. Individual odors transiently and differentially increase the production of both adenosine cAMP and inositol 1,4,5-trisphosphate (IP3) within 50 msec of odor stimulation. The ability of two different odors to stimulate cAMP and IP3 correlates with the odors' ability to excite and inhibit receptor cells physiologically. These results strengthen the proposition, heretofore based largely on evidence from cultured cells, that dual second messenger pathways mediate excitatory and inhibitory input to lobster olfactory receptor cells.

Animals↗
PubMed1993

Transfected cGMP-dependent protein kinase suppresses calcium transients by inhibition of inositol 1,4,5-trisphosphate production.

cGMP is a key regulatory molecule in visual transduction, integration of neuronal response to excitatory neurotransmitters, relaxation of smooth muscle, intestinal secretion of water and salt, and reabsorption of sodium and water in the distal tubules of the nephron. Some of these cellular functions are associated with the activation of cGMP kinase and a decrease in cytosolic calcium levels ([Ca2+]i). The mechanism by which cGMP kinase lowers [Ca2+]i is controversial. We have used CHO cells stably transfected with cGMP kinase to test several of the proposed [Ca2+]i-lowering mechanisms. Thrombin induces a calcium transient in wild-type and cGMP kinase-expressing CHO cells by releasing calcium from intracellular stores. Preincubation of wild-type cells with 8-bromo-cGMP had no effect on the calcium transient, whereas 8-bromo-cGMP prevented the thrombin-stimulated calcium transient in cGMP kinase-expressing CHO cells. In both cell types 8-bromo-cGMP had no effect on [Ca2+]i transients induced by replacing extracellular sodium by tetramethylammonium, ruling out an effect of cGMP kinase on Ca(2+)-ATPases. However, cGMP kinase activation effectively suppressed thrombin-induced stimulation of inositol 1,4,5-trisphosphate production. These results show that cGMP kinase lowers [Ca2+]i by interfering with the inositol 1,4,5-trisphosphate synthesis.

Animals↗
PubMed1993

A beta-adrenergic receptor kinase-like enzyme is involved in olfactory signal termination.

We have previously shown that second-messenger-dependent kinases (cAMP-dependent kinase, protein kinase C) in the olfactory system are essential in terminating second-messenger signaling in response to odorants. We now document that subtype 2 of the beta-adrenergic receptor kinase (beta ARK) is also involved in this process. By using subtype-specific antibodies to beta ARK-1 and beta ARK-2, we show that beta ARK-2 is preferentially expressed in the olfactory epithelium in contrast to findings in most other tissues. Heparin, an inhibitor of beta ARK, as well as anti-beta ARK-2 antibodies, (i) completely prevents the rapid decline of second-messenger signals (desensitization) that follows odorant stimulation and (ii) strongly inhibits odorant-induced phosphorylation of olfactory ciliary proteins. In contrast, beta ARK-1 antibodies are without effect. Inhibitors of protein kinase A and protein kinase C also block odorant-induced desensitization and phosphorylation. These data suggest that a sequential interplay of second-messenger-dependent and receptor-specific kinases is functionally involved in olfactory desensitization.

Animals↗
PubMed1993

Cloning and expression of odorant receptors.

Myriads of odorous molecules that vary widely in structure are nevertheless readily detected and discriminated by the sense of smell, but how this is achieved by the olfactory system has been a long-standing puzzle. Several different models have been proposed, and previous observations indicate that the recognition sites for odorous molecules could be G-protein-coupled receptor proteins, an idea supported by the discovery of a new gene family that probably encodes a diversity of odorant receptors. Here we report the identification of new members of the gene family encoding putative odorant receptors and demonstrate that they are indeed transcribed in olfactory receptor neurons. Furthermore, the receptor-encoding complementary DNA is expressed in non-neuronal surrogate cells, which generate second messenger responses upon stimulation with appropriate odorants, indicating that the receptors recognize odorants and couple to G proteins of the host cells.

Amino Acid Sequence↗
PubMed1993

Rapid kinetic measurements of second messenger formation in olfactory cilia from channel catfish.

The effect of stimulating olfactory cilia from the channel catfish (Ictalurus punctatus) with odorant amino acids on the formation of adenosine 3',5'-cyclic monophosphate, guanosine 3',5'-cyclic monophosphate, and inositol 1,4,5-trisphosphate (IP3) was studied in the subsecond time scale using a quenched flow technique. L-Alanine (L-Ala) and L-cysteine (L-Cys) (100 microM) elicited a transient elevation in IP3 levels that peaked at 25 ms. In contrast, even at high concentration a mixture of odorant amino acids (1 mM L-Ala, L-Cys, L-norleucine, L-glutamate, L-proline, and L-arginine) did not elicit a change in cAMP levels in this time scale and caused only relatively slow and minor increases in cGMP. The dose-response relationship for the IP3 response of L-Cys and L-Ala in the range from 10 nM to 1 mM is consistent with previous electrophysiological and ligand binding experiments. Odorant amino acid-stimulated IP3 formation was GTP dependent and was inhibited by guanosine 5'-O-(2-thiodiphosphate), suggesting that the response was G protein mediated. These results are consistent with a mediatory role for IP3 in amino acid olfactory signal transduction in catfish.

Alanine↗
PubMed1993

Generation of monoclonal antibodies detecting specific epitopes in locust antennae.

1. Following a tissue-specific screening paradigm, monoclonal antibodies have been generated that interact with distinct subpopulations of cells in locust antennae. 2. Antigens were identified as high molecular weight components. 3. Immunoreactivity was not detectable during embryonic development, but rapidly appeared within a few hours of hatching. 4. The time course of antigen expression in antennal cells could be followed in situ as well as in vitro. 5. Expression of monoclonal antibody B14/6D2-like immunoreactivity was prevented by blocking protein synthesis with cycloheximide.

Animals↗
PubMed1993

Towards an identification of odorant receptors.

The molecular nature and diversity of receptive sites for odorous molecules is a central unanswered issue in olfaction. Based on the enormous resolving power of the olfactory system, which enables the stereospecific discrimination of numerous compounds at low concentrations specific receptor proteins have been proposed. Due to the central role of G-proteins in olfactory signalling it has been predicted that odorant receptors might be members of the superfamily of receptor proteins with seven transmembrane domains. Upon application of degenerated oligonucleotides and the PCR-technology a number of putative odorant receptors have been cloned and sequenced. In situ hybridisation studies using receptor-specific probes have been performed and olfactory neurons specifically expressing a particular receptor subtype are topologically identified in the nasal epithelium of rats. Clones of interest are now being expressed in heterologous systems in order to demonstrate functional activity of these putative receptor proteins and to match defined odorants to identified receptors.

Amino Acid Sequence↗
PubMed1992

Odor-induced phosphorylation of olfactory cilia proteins.

Stimulation of isolated rat olfactory cilia in the presence of [gamma-32P]ATP leads to a significantly enhanced incorporation of [32P]phosphate. Depending on the type of odorants applied, the induced phosphorylation is completely blocked by specific inhibitors of either protein kinase A or protein kinase C. Time-course experiments indicate that the odor-induced modification of ciliary proteins is transient; the intensity of labeling decayed over time (1-10 sec). Separation of ciliary proteins by SDS/polyacrylamide gel electrophoresis followed by autoradiography demonstrated that upon stimulation with lilial, a single polypeptide (50,000 Da) was phosphorylated; the size of the modified protein is in line with the hypothesis that odorant receptors are phosphorylated subsequent to activation by specific odors.

Animals↗
PubMed1992

Termination of second messenger signaling in olfaction.

By using isolated rat olfactory cilia and a fast kinetics methodology, it has been demonstrated that odorant-induced second messenger signaling in the millisecond time range is terminated via phosphorylation reactions catalyzed by specific protein kinases. The cyclic adenosine nucleotide pathway is turned off by kinase A activity, whereas the inositol trisphosphate cascade is terminated by kinase C. The data support the concept that desensitization of odorant responses involves phosphorylation of key elements in the transduction cascade.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Second messenger signalling in olfaction.

The primary reactions of the chemo-electrical signal transduction pathway in olfactory receptor neurons are mediated by two alternative second messengers, cAMP and inositol 1,4,5-trisphosphate. The rapid and transient intracellular signalling is terminated by the action of negative-feedback loops which uncouple the reaction cascades (desensitization). Recent evidence suggests that secondary reactions in olfaction (adaptation) may also be controlled by second messengers.

Animals↗
PubMed1992

Nitric oxide mediated formation of cyclic GMP in the olfactory system.

Olfactory cilia preparation from rats contain considerable activity of soluble guanylate cyclase as indicated by the formation of cyclic GMP (cGMP) upon application of nitroprusside, a nitric oxide generating agent. Stimulation of olfactory cilia with high doses of odorants elicited a delayed and sustained elevation of the cGMP-concentration. The odorant-induced cGMP-response was abolished by L-NG-nitro-arginine, a selective inhibitor of nitric oxide synthesis, as well as by haemoglobin which efficiently binds and inactivates nitric oxide. These observations suggest that the NO/cGMP cascade may plan an important role in signal processing of the olfactory system.

Animals↗
PubMed1990

Rapid kinetics of second messenger formation in olfactory transduction.

Olfactory transduction is thought to be mediated by a membrane-bound receptor protein initiating a multistep reaction cascade which ultimately leads to a depolarizing generator current. There is considerable evidence for the involvement of adenylate cyclase in vertebrate olfactory transduction, and some data indicate that phospholipase C may have a central role in insect olfaction. However, one must show that odorants not only stimulate enzyme activity but also induce changes in concentrations of relevant second messengers. One important criterion for a candidate second messenger of chemo-electrical transduction is that its formation must precede the onset of the odorant-induced membrane permeability changes which proceed on a subsecond time-scale. Here we report an odorant-induced, transient accumulation of cyclic AMP in isolated olfactory cilia from rats, and the generation of inositol trisphosphate in antennal preparations from insects, both of which show subsecond time courses that are sufficiently rapid to mediate the odorant-regulated permeability of olfactory receptor cells.

Animals↗
PubMed1990

Rapid activation of alternative second messenger pathways in olfactory cilia from rats by different odorants.

The molecular mechanisms mediating the chemo-electrical signal transduction in olfactory receptor cells are still elusive. In this study odor induced formation of second messengers in rat olfactory cilia was monitored in a subsecond time range using a rapid kinetic device. Application of micromolar concentration of citralva induced a rapid, transient elevation of the cyclic adenosine monophosphate level, whereas the concentration of inositol trisphosphate was not affected. In contrast, pyrazine caused a rise in the concentration of inositol trisphosphate, not affecting the level of cyclic adenosine monophosphate. Analysis of the kinetic parameter for the odorant induced reaction indicated that apparently two systems are operating simultaneously. The activating effects of odorants appear to be mediated via different G-proteins. Thus, at least two different second messenger pathways appear to be involved in olfactory signal transduction.

Animals↗
PubMed1990

Odorant-sensitive phospholipase C in insect antennae.

Exogenous tritiated phosphatidylinositol bisphosphate added to antennal preparations from locust and cockroach was hydrolysed releasing inositol trisphosphate. High activity of phospholipase C was detected in the soluble as well as in the membrane fraction. At low free calcium concentrations hydrolysis of the labelled lipid was stimulated by odorants and pheromones in a GTP-dependent manner. Consequently the level of inositol trisphosphate in antennal preparations increased upon odorant stimulation.

Animals↗
PubMed1989

Isolation and reconstitution of the high-affinity choline carrier.

Monoclonal antibodies, which block the high-affinity uptake of choline in synaptosomal ghosts, have been used to purify a membrane polypeptide (80 kDa) from insect synaptosomal membranes. This isolated protein was found to catalyse the sodium-dependent, hemicholinium-sensitive accumulation of choline after reconstitution into liposomes, thus, apparently represents the high-affinity choline transporter.

Animals↗