Preferential efflux of phytosterols over cholesterol from macrophages.

Phytosterols are structurally similar to cholesterol. Increased dietary intake of phytosterols effectively lowers LDL-cholesterol. Since phytosterols are incorporated in a growing number of foods and some of the ingested phytosterols reach the circulation, accumulation of phytosterols in foam-cell-prone cells such as macrophages might occur. Therefore we examined the influx and efflux of phytosterols by human THP-1 macrophages. The influx rates of methyl-beta-cyclodextrin delivered phytosterols did not significantly differ from that of cholesterol (approximately 3.8 pmol/min per mg cellular protein), neither did the total influx of oxidised LDL delivered phytosterols differ from that of cholesterol. The efflux of beta-sitosterol and sitostanol from preloaded THP-1 cells to HDL was more efficient than the efflux of campesterol and cholesterol (rate constants of 0.41 +/- 0.04/h, 0.62 +/- 0.08/h, 0.23 +/- 0.05/h and 0.29 +/- 0.03/h, respectively). The efflux of beta-sitosterol was not associated with a dominant transfer to ApoA-I, nor did ABCA1 induction-promoted cholesterol efflux to the level observed for beta-sitosterol. Our data show that THP-1 macrophages take up phytosterols, but have efficient mechanisms to remove phytosterols from their cellular compartments. Consequently, it is less likely that macrophages preferentially accumulate phytosterols over cholesterol and hence promote foam-cell formation in vivo.

G protein betagamma complexes in circumvallate taste cells involved in bitter transduction.

G protein betagamma (Gbetagamma) complexes are considered to play an important role in second messenger signaling of phospholipase C (PLC). Monitoring the inositol 1,4,5-trisphosphate (IP(3)) response in circumvallate tissue homogenates upon stimulation with denatonium benzoate, it was demonstrated that a glutathione S-transferase-GRK3ct fusion protein-a Gbetagamma scavenger-attenuates the bitter tastant-induced second messenger reaction. Towards an identification of the Gbetagamma complex involved in rat bitter taste transduction, it was found that the G protein beta(3) subtype is specifically expressed in taste receptor cells of circumvallate papillae. Gbeta(3)-specific antibodies blocked the denatonium benzoate-induced IP(3) formation in a dose-dependent manner; the inhibitory effect was reversed by preincubation with the antigenic peptide. A less pronounced inhibition was observed using Gbeta(1)-specific antibodies. Analyzing individual taste cells by single cell reverse transcriptase-polymerase chain reaction approaches, overlapping expression patterns for PLCbeta(2), Galpha(gust), Gbeta(3) and Ggamma(3) could be demonstrated. Furthermore, the co-expression of all profiled signal transduction components in individual taste receptor cells could be detected. These data support the concept that the denatonium benzoate-induced IP(3) response is mediated by an activation of PLCbeta(2) via a Gbetagamma complex, possibly composed of Gbeta(3) as the predominant beta subunit and Ggamma(3), and imply that multiple second messenger pathways may exist in individual taste receptor cells.

Structures and functions of calcium channel beta subunits.

Calcium channel beta subunits have profound effects on how alpha1 subunits perform. In this article we summarize our present knowledge of the primary structures of beta subunits as deduced from cDNAs and illustrate their different properties. Upon co-expression with alpha1 subunits, the effects of beta subunits vary somewhat between L-type and non-L-type channels mostly because the two types of channels have different responses to voltage which are affected by beta subunits, such as long-lasting prepulse facilitation of alpha1C (absent in alpha1E) and inhibition by G protein betagamma dimer of alpha1E, absent in alpha1C. One beta subunit, a brain beta2a splice variant that is palmitoylated, has several effects not seen with any of the others, and these are due to palmitoylation. We also illustrate the finding that functional expression of alpha1 in oocytes requires a beta subunit even if the final channel shows no evidence for its presence. We propose two structural models for Ca2+ channels to account for "alpha1 alone" channels seen in cells with limited beta subunit expression. In one model, beta dissociates from the mature alpha1 after proper folding and membrane insertion. Regulated channels seen upon co-expression of high levels of beta would then have subunit composition alpha1beta. In the other model, the "chaperoning" beta remains associated with the mature channel and "alpha1 alone" channels would in fact be alpha1beta channels. Upon co-expression of high levels of beta the regulated channels would have composition [alpha1beta]beta.

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.

A Xenopus oocyte beta subunit: evidence for a role in the assembly/expression of voltage-gated calcium channels that is separate from its role as a regulatory subunit.

Two closely related beta subunit mRNAs (xo28 and xo32) were identified in Xenopus oocytes by molecular cloning. One or both appear to be expressed as active proteins, because: (i) injection of Xenopus beta antisense oligonucleotides, but not of sense or unrelated oligonucleotides, significantly reduced endogenous oocyte voltage-gated Ca2+ channel (VGCC) currents and obliterated VGCC currents that arise after injection of mammalian alpha1 cRNAs (alpha(1C) and alpha(1E)); (ii) coinjection of a Xenopus beta antisense oligonucleotide and excess rat beta cRNA rescued expression of alpha1 Ca2+ channel currents; and (iii) coinjection of mammalian alpha1 cRNA with cRNA encoding either of the two Xenopus beta subunits facilitated both activation and inactivation of Ca2+ channel currents by voltage, as happens with most mammalian beta subunits. The Xenopus beta subunit cDNAs (beta3xo cDNAs) predict proteins of 484 aa that differ in only 22 aa and resemble most closely the sequence of the mammalian type 3 beta subunit. We propose that "alpha1 alone" channels are in fact tightly associated alpha1beta3xo channels, and that effects of exogenous beta subunits are due to formation of higher-order [alpha1beta]beta(n) complexes with an unknown contribution of beta3xo. It is thus possible that functional mammalian VGCCs, rather than having subunit composition alpha1beta, are [alpha1beta]beta(n) complexes that associate with alpha2delta and, as appropriate, other tissue-specific accessory proteins. In support of this hypothesis, we discovered that the last 277-aa of alpha(1E) have a beta subunit binding domain. This beta binding domain is distinct from the previously known interaction domain located between repeats I and II of calcium channel alpha1 subunits.

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.

Ca(2+)-dependent inactivation of P-type calcium channels in nerve terminals.

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 omega-agatoxin IVa.

GABAB-receptor-mediated inhibition of calcium signals in isolated nerve terminals.

Time-resolved fluorometric monitoring of rapid changes in intracellular Ca(2+)-concentrations [Ca2+]i was employed to analyze the effect of gamma-aminobutyric acid (GABA) on evoked Ca(2+)-signals in rat hippocampal synaptosomes. The inhibitory action of GABA was mimicked by Baclofen, the selective agonist for GABAB-receptors, and also by the nonhydrolyzable GTP-derivative GTP-gamma-S. Preincubation of synaptosomes with GABA or baclofen for up to 250 ms before depolarization resulted in a maximal inhibition. The GABA-induced attenuation of the evoked rise in [Ca2+]i was maximal during the first milliseconds after depolarization. The inhibitory action of GABA apparently does not involve second messengers, such as cAMP or IP3/DAG; the GABA-induced inhibition of presynaptic voltage-dependent Ca(2+)-channels may be mediated directly by G-proteins.

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.

Rapid kinetics of depolarization-induced changes in intrasynaptosomal calcium concentrations.

The rapid kinetics of depolarization-evoked calcium influxes in isolated nerve terminals from rat cortex were monitored by stopped-flow spectrofluorimetry using specific indicators (Fluo-3, Indo-1). A very rapid increase in the intrasynaptosomal Ca(2+)-level was detected within the subsecond time range after depolarizing synaptosomes by mixing with physiological saline containing elevated K(+)-concentrations. About 15 mM [K+]o was determined as threshold concentration for inducing Ca(2+)-influx, which increased with higher concentration and saturated at [K+]o-concentrations of about 40 mM [K+]o.

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

The channel properties of nicotinic acetylcholine receptor subtypes in the nervous system of insects (Locusta migratoria) have been characterized. Single channel measurements were performed using patch-clamp techniques as well as planar lipid bilayer reconstitution approaches. In reconstitution experiments using receptor-preparations isolated from neuronal membranes by alpha-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.

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.

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.

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.

Identification of a cationic channel in synaptosomal membranes.

Synaptosomal membranes were fused with liposomes using the 'hydration technique' to produce giant proteoliposomes amenable to patch clamp recordings. Single channel currents of a cationic channel with particular properties were detected. In a solution of 150 mM NaCl, the channel displayed a unit conductance of 136 pS and a mean open state lifetime of 1.1 ms. The gating of the channel was shown to be voltage as well as calcium dependent. Pharmacological studies revealed that the channel was insensitive to a variety of channel blockers, but was inactivated by ruthenium red. Presumably, this channel may play a role in regulating the evoked release of neurotransmitters.