Erwin Tareilus

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.

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.

Calcium signals in olfactory neurons.

Laser scanning confocal microscopy in combination with the fluorescent calcium indicators Fluo-3 and Fura-Red was employed to estimate the intracellular concentration of free calcium ions in individual olfactory receptor neurons and to monitor temporal and spatial changes in the Ca(2+)-level upon stimulation. The chemosensory cells responded to odorants with a significant increase in the calcium concentration, preferentially in the dendritic knob. Applying various stimulation paradigma, it was found that in a population of isolated cells, subsets of receptor neurons display distinct patterns of responsiveness.

Presynaptic calcium channels: Pharmacology and regulation

Voltage-dependent Ca2+ channels are considered as molecular trigger elements for signal transmission at chemical synapses. Due to their central role in this fundamental process, function and pharmacology of presynaptic Ca2+ channels have recently been the subject of extensive exploration employing various experimental techniques. Several lines of evidence indicate that, at nerve terminals in higher vertebrates, the evoked influx of Ca2+ -ions is mainly mediated by Ca2+ channels of the P-type. The stringent regulation of presynaptic Ca2+ channels is supposed to be involved in fine-tuning the efficiency of synaptic transmission. Intrinsic control mechanisms, such as voltage- or Ca(2+)-dependent inactivation, or modulation of channel activity, either by G-proteins directly or via phosphorylation by protein kinases, may be of particular functional importance.

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.

RGS21 is a novel regulator of G protein signalling selectively expressed in subpopulations of taste bud cells

G‐protein‐mediated signalling processes are involved in sweet and bitter taste transduction. In particular, the G protein α‐subunit gustducin has been implicated in these processes. One of the limiting factors for the time‐course of cellular responses induced by tastants is therefore the intrinsic GTPase activity of α‐gustducin, which determines the lifetime of the active G protein complex. In several signalling systems specific ‘regulator of G protein signalling’ (RGS) proteins accelerate the GTPase activity of G protein α‐subunits. Using differential screening approaches, we have identified a novel RGS protein termed RGS21, which represents the smallest known member of this protein family. Reverse transcription polymerase chain reaction and in situ hybridization experiments demonstrated that RGS21 is expressed selectively in taste tissue where it is found in a subpopulation of sensory cells. Furthermore, it is coexpressed in individual taste cells with bitter and sweet transduction components including α‐gustducin, phospholipase Cβ2, T1R2/T1R3 sweet taste receptors and T2R bitter taste receptors. In vitro binding assays demonstrate that RGS21 binds α‐gustducin in a conformation‐dependent manner and has the potential to interact with the same Gα subtypes as T1R receptors. These results suggest that RGS21 could play a regulatory role in bitter as well as sweet taste transduction processes.

Neuronal acetylcholine receptor channels from insects: a comparative electrophysiological study.

SummaryThe channel properties of nicotinic acetylcholine receptor subtypes in the nervous system of insects (Locusta migratoria) have been characterized. Single channel measurements were performed using patchclamp techniques as well as planar lipid bilayer reconstitution approaches. In reconstitution experiments using receptor-preparations isolated from neuronal membranes by α-toxin affinity chromatography, a ligand-gated channel type was found, which showed a high conductance and a short mean lifetime. Patch-clamp experiments on synapse-free somata of isolated nerve cells revealed an acetylcholine-gated channel type with a lower conductance but a longer lifetime. The two different agonist-activated channel types are supposed to represent synaptic and extrasynaptic acetylcholine receptors.

Olfactory signalling in antennal receptor neurones of the locust (Locusta migratoria)

The electrical properties of isolated antennal neurones from Locusta migratoria were investigated using patch clamp techniques. The neurones displayed a resting potential of −26±11 mV (n=38) and an input resistance between 1–6 GΩ. Upon application of depolarizing voltage steps a non-inactivating non-specific inward current and a calcium-dependent outward potassium current were identified. Some neurones responded to stimulation by the grass odour hexenoic acid (1–100 μM) with an increase in ion channel activity in cell-attached recordings. This channel was identified as a calcium-activated potassium channel belonging to the maxi-K+-channel types. Odour-stimulation of antennal tissue and antennal cells using a fast quency device leads to a rapid generation of inositol 1,4,5-trisphosphate, supporting the idea that this substance acts as second messenger in insect olfaction. The odour-induced increase in inositol trisphosphate concentration may initiate a rise in intracellular calcium controlling the activity of calcium-dependent ion channels.

Analysis of rapid calcium signals in synaptosomes

A combination of the stopped-flow technology with dual channel spectrofluorometry of Ca(2+)-indicators was utilized for the measurement of rapid Ca(2+)-signals in rat cerebral cortical synaptosomes evoked by K(+)-depolarization. There was no observable contribution of Ca(2+)-ions from intracellular stores to the rise in [Ca2+]i. The kinetics of the fast increase in intracellular Ca2+ concentration was analysed in relation to the depolarization strength. The maximal increase in [Ca2+]i and the time course of Ca(2+)-channel inactivation were determined for depolarizations obtained by different extracellular K(+)-concentrations ([K+]o). An apparent threshold was observed at about 18 mM [K+]o; a maximal Ca(2+)-signal amplitude was estimated at about 40 mM [K+]o. Pharmacological properties of the involved Ca(2+)-channels were determined using selective Ca(2+)-channel blockers (Dihydropyridines, omega-Conotoxin, omega-Agatoxins); the results suggest that a P-type voltage-dependent Ca(2+)-channel is the relevant channel type, generating the evoked Ca(2+)-signals in rat cerebral cortical synaptosomes.

Ca2+-Dependent Inactivation of P-Type Calcium Channels in Nerve Terminals

Abstract: Rapid Ca2+ signals evoked by K+ depolarization of rat cerebral cortical synaptosomes were measured by dual‐channel Ca2+ spectrofluorometry coupled to a stopped‐flow device. Kinetic analysis of the signal rise phase at various extracellular Ca2+ concentrations revealed that the responsible voltage‐dependent Ca2+ channels, previously identified as P‐type Ca2+ channels, inactivate owing to the rise in intracellular Ca2+ levels. At millimolar extracellular Ca2+ concentrations the channels were inactivated very rapidly and the rate was dependent on the high influx rate of Ca2+, thus limiting the Ca2+ signal amplitudes to 500–600 nM. A slower, probably voltage‐dependent regulation appears to be effective at lower Ca2+ influx rates, leading to submaximal Ca2+ signal amplitudes. The functional feedback regulation of calcium channels via a sensor for intracellular Ca2+ levels appears to be responsible for the different inhibition characteristics of Cd2+ versus ω‐agatoxin IVa.