Ingrid Boekhoff

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.

G Protein-coupled Receptor Kinase 3 (GRK3) Gene Disruption Leads to Loss of Odorant Receptor Desensitization

G protein-coupled receptor kinases (GRKs) 2 and 3 (β-adrenergic receptor kinases 1 and 2 (βARK1 and -2)) mediate the agonist-dependent phosphorylation and uncoupling of many G protein-coupled receptors. These two members of the GRK family share a high degree of sequence homology and show overlapping patterns of substrate specificity in vitro. To define their physiological roles in vivo we have generated mice that carry targeted disruption of these genes. In contrast to GRK2-deficient mice, which die in utero (Jaber, M., Koch, W. J., Rockman, H., Smith, B., Bond, R. A., Sulik, K. K., Ross, J. JR., Lefkowitz, R. J. Caron, M. G., and Giros, B. (1996)Proc. Natl. Acad. Sci. U. S. A. 93, 12974–12979), GRK3 deletion allows for normal embryonic and postnatal development. GRK3 is expressed to a high degree in the olfactory epithelium, where GRK2 is absent. Here we report that cilia preparations derived from GRK3-deficient mice lack the fast agonist-induced desensitization normally seen after odorant stimulation. Moreover, total second messenger (cAMP) generation in these cilia preparations following odorant stimulation is markedly reduced when compared with preparations from wild-type littermates. This reduction in the ability to generate cAMP is evident even in the presence of nonodorant receptor stimuli (GTPγS and forskolin), suggesting a compensatory dampening of the G protein-adenylyl cyclase system in the GRK3 (−/−) mice in the olfactory epithelium. These findings demonstrate the requirement of GRK3 for odorant-induced desensitization of cAMP responses.

Selective Activation of G Protein Subtypes in the Vomeronasal Organ upon Stimulation with Urine-derived Compounds

Chemosensory neurons in the vomeronasal organ (VNO) detect pheromones related to social and reproductive behavior in most terrestrial vertebrates. Current evidence indicate that the chemoelectrical transduction process is mediated by G protein-coupled second messenger cascades. In the present study, attempts were made to identify the G protein subtypes which are activated upon stimulation with urinary pheromonal components. G protein-specific antibodies were employed to interfere specifically with inositol 1,3,4-trisphosphate formation induced by urinary stimuli and to immunoprecipitate Gα-subunits, activation dependently labeled with [α-32P]GTP azidoanilide. The results of both experimental approaches indicate that stimulation of female VNO membrane preparations with male urine samples induces activation of Gi as well as Go subtypes. Experiments using different fractions of urine revealed that upon stimulation with lipophilic volatile odorants, only Gi proteins were activated, whereas Go activation was elicited by α2u-globulin, a major urinary protein, which is a member of the lipocalin superfamily. Since each G protein subtype is stereotypically coexpressed with one of the two structurally different candidate pheromone receptors (V1R and V2R), the results provide the first experimental evidence that V1Rs coexpressed with Gimay be activated by lipophilic probably volatile odorants, whereas V2Rs coexpressed with Go seem to be specialized to interact with pheromonal components of proteinaceous nature.

Second messenger signalling in olfaction.

Odorous molecules are recognized by specific receptor proteins located in the ciliary membrane of olfactory receptor neurons. These receptors have been identified using molecular cloning--they are members of the seven-transmembrane-domain G protein-coupled receptor superfamily. Specific receptor subtypes are expressed in subsets of olfactory neurons spatially segregated within certain areas of the olfactory epithelium. Interaction of odorants with receptors initiates the primary reaction of olfactory signalling. Intracellular reaction cascades are activated via specific G proteins, leading to a rapid and transient rise in second messenger levels; odorous compounds elicit mutually exclusive cAMP or inositol 1,4,5-trisphosphate responses. Odorant-induced second messenger signalling is terminated via kinase-mediated negative feedback loops uncoupling the reaction cascades by phosphorylation of receptor proteins. Strong odour stimuli elicit a delayed response of another messenger system, the nitric oxide/cGMP cascade. cGMP may control some adaptive reactions in olfactory receptor neurons.

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.

Pheromone-induced second-messenger signaling in insect antennae

Antennal preparations from Heliothis virescens respond to a blend of sex-pheromone components with a rapid and transient inositol trisphosphate signal in the subsecond time range that is supposed to be the primary reaction of the chemo-electrical transduction process. The pheromone-induced second messenger response is species- and sex-specific. The major component of the blend (Z-11-hexadecenal) is much more potent than one of the minor components (Z-9-tetradecenal). Application of high pheromone doses elicited a delayed and sustained increase of cyclic GMP. Elevated cGMP-levels significantly reduced the pheromone-induced IP3-response, indicating a considerable cross-talk between both second messenger pathways and suggesting that cGMP may be involved in adaptation of antennal receptor cells.

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.

Perireceptor Events and Transduction Mechanisms in Insect Olfaction

The first Chap. of this book has described the morphological properties of insect antennal sense organs, the pheromone-sensitive as well as the general odour-sensitive sensilla. This Chap. follows the odour molecules as they pass through the pores in the cuticle of the antennal sensilla, and on their way through the aqueous sensillum lymph, until they reach the outer dendrites of the olfactory receptor neurons (ORNs). Here, the transduction cascade is initiated that finally causes changes in the membrane potential, and in the rate of action potentials that encode the biologically relevant properties of the olfactory stimuli.

Sodium/calcium exchanger in rat olfactory neurons.

The chemo-electrical transduction process in olfactory neurons is accompanied by a rapid and transient increase in intracellular calcium concentrations. The notion that Na+/Ca2+ exchanger activities may play a major role in extruding calcium ions out of the cell and maintaining Ca2+ homeostasis in olfactory receptor cells was assessed by means of laser scanning confocal microscopy in combination with the fluorescent indicators Fluo-3 and Fura-Red. The data indicate that high exchanger activity, which was inhibited by amiloride derivatives, is located in the dendritic knob and probably in the olfactory cilia. This result was supported by experiments using specific antiserum raised against retinal Na+/Ca2+ exchanger protein which labelled an immunoreactive protein of 230 kDa in Western blots from olfactory tissue and strongly stained the ciliary layer of the olfactory epithelium.

Pheromone-induced stimulation of inositol-trisphosphate formation in insect antennae is mediated by G-proteins

SummaryPhospholipase C in the antennae of Periplaneta americana was stimulated by pheromones in a GTP-dependent manner. The enzyme activity was potentiated by hydrolysis-resistant analogs of GTP and decreased by GDP analogs. Guanine nucleotide binding regulatory proteins (G-proteins) in antennal preparations were identified by bacterial toxin-catalyzed ADP-ribosylation and immunoreactivity with antibodies of designed specificity. The stimulatory effect of pheromones on inositol phosphate formation was completely blocked by pertussis toxin suggesting that the pheromone action was mediated via specific G-proteins.