Regulation of intracellular cyclic GMP levels in olfactory sensory neurons

Cyclic AMP is the primary second messenger mediating odorant signal transduction in mammals. A number of studies indicate that cyclic GMP is also involved in a variety of other olfactory signal transduction processes, including adaptation, neuronal development, and long-term cellular responses in the setting of odorant stimulation. However, the mechanisms that control the production and degradation of cGMP in olfactory sensory neurons (OSNs) remain unclear. Here, we investigate these mechanisms using primary cultures of OSNs. We demonstrate that odorants increase cGMP levels in intact OSNs in vitro. Different from the rapid and transient cAMP responses to odorants, the cGMP elevation is both delayed and sustained. Inhibition of soluble guanylyl cyclase and heme oxygenase blocks these odorant-induced cGMP increases, whereas inhibition of cGMP PDEs (phosphodiesterases) increases this response. cGMP PDE activity is increased by odorant stimulation, and is sensitive to both ambient calcium and cAMP concentrations. Calcium stimulates cGMP PDE activity, whereas cAMP and protein kinase A appears to inhibit it. These data demonstrate a mechanism by which odorant stimulation may regulate cGMP levels through the modulation of cAMP and calcium level in OSNs. Such interactions between odorants and second messenger systems may be important to the integration of immediate and long-term responses in the setting odorant stimulation.     

Cellular and molecular constituents of olfactory sensation in vertebrates

Since the discovery of odorant-activated adenylate cyclase in the olfactory receptor cilia, research into the olfactory perception of vertebrates has rapidly expanded. Recent studies have shown how the odor discrimination starts at the receptor level: each of 700-1000 types of the olfactory neurons in the neural olfactory epithelium contains a single type of odor receptor protein. Although the receptors have relatively low specific affinities for odorants, excitation of different types of receptors forms an excitation pattern specific to each odorant in the glomerular layer of the olfactory bulb. It was demonstrated that adenosine 3',5'-cyclic monophosphate (cAMP) is very likely the sole second messenger for olfactory transduction. It was also demonstrated that the affinity of the cyclic nucleotide-gated channel for cAMP regulated by Ca(2+)/calmodulin is solely responsible for the adaptation of the cell. However, many other regulatory components were found in the transduction cascade. Regulated by Ca(2+) and/or the protein-phosphorylation, many of them may serve for the adaptation of the cell, probably on a longer time scale. It may be important to consider the resensitization as a part of this adaptation, as well as to collect kinetic data of each reaction to gain further insight into the olfactory mechanism.     

Olfactory transduction mechanisms in sheep

The enzyme adenylyl cyclase from sheep olfactory epithelium is dually regulated by GTP and is highly sensitive to the nucleotide analogues GTPS and GppNHp, as well as to fluoride ions and forskolin. Many, but not all, odorants tested are able to stimulate adenylyl cyclase in a dose-dependent manner and with different potencies. Such an effect is detectable only in the presence of GTP. The odorants belonging to the putrid class are the least effective in stimulating adenylyl cyclase activity, and only furfuryl mercaptan significantly increases cAMP biosynthesis. Mixtures of two odorants, chosen among those able to activate adenylyl cyclase, induce additive or supra-additive effects, suggesting the presence of many different receptor types. The presence of an alternative olfactory signal transduction process, i.e. the inositol phospholipid second messenger system, has been evaluated. Triethylamine, a putrid odorant completely ineffective on cAMP levels, is able to significantly increase inositol phosphate accumulation, indicating the coexistence of both cAMP- and InsP₃-mediated signalling pathways in sheep olfactory epithelium.     

Maxadilan activates PAC1 receptors expressed in Xenopus laevis xelanophores

Functional interactions between ligands and their cognate receptors can be investigated using the ability of melanophores from Xenopus laevis to disperse or aggregate their pigment granules in response to alterations in the intracellular levels of second messengers. We have examined the response of long-term lines of cultured melanophores from X. laevis to pituitary adenylate cyclase activating peptide (PACAP), a neuropeptide with vasodilatory activity, and maxadilan, a vasodilatory peptide present in the salivary gland extracts of the blood feeding sand fly. Pituitary adenylate cyclase activating peptide increased the intracellular levels of cyclic adenosine monophosphate (cAMP) and induced pigment dispersion in the pigment cells, confirming that melanophores express an endogenous PACAP receptor. Maxadilan did not induce a response in non-transfected melanophores. When the melanophores were transfected with complementary DNA (cDNA) from the three different members of the PACAP receptor family, maxadilan induced pigment dispersion specifically and cAMP accumulation in melanophores transfected with the cDNA for PAC1 receptors but not VPAC1 or VPAC2 receptors. A melanophore line was generated that stably expresses the PAC1 receptor.     

Heterogeneity of molecular mechanisms the olfactor reception

In experiments on the frog isolated olfactory epithelium by using vital fluorescent microscope, odorants with fruit, rank, flower and camphor smell were shown to involve intracellular signaling systems in olfactory transduction. The odorants with different qualitative smells have different messenger and activity mechanisms. Intracellular messengers do not participate in reception of odorants with piquant and rotten smells. Thus the perception of different odour substances is maintained by physical and chemical processes. Hence, not only taste, carotid, medullar, but olfactory reception as well are characterised by heterogeneity of biophysical mechanisms.     

Protein kinase A, which regulates intracellular transport, forms complexes with molecular motors on organelles

Major signaling cascades have been shown to play a role in the regulation of intracellular organelle transport . Aggregation and dispersion of pigment granules in melanophores are regulated by the second messenger cAMP through the protein kinase A (PKA) signaling pathway ; however, the exact mechanisms of this regulation are poorly understood. To study the role of signaling molecules in the regulation of pigment transport in melanophores, we have asked the question whether the components of the cAMP-signaling pathway are bound to pigment granules and whether they interact with molecular motors to regulate the granule movement throughout the cytoplasm. We found that purified pigment granules contain PKA and scaffolding proteins and that PKA associates with pigment granules in cells. Furthermore, we found that the PKA regulatory subunit forms two separate complexes, one with cytoplasmic dynein ("aggregation complex") and one with kinesin II and myosin V ("dispersion complex"), and that the removal of PKA from granules causes dissociation of dynein and disruption of dynein-dependent pigment aggregation. We conclude that cytoplasmic organelles contain protein complexes that include motor proteins and signaling molecules involved in different components of intracellular transport. We propose to call such complexes 'regulated motor units' (RMU).     

Calcium Requirement for α-MSH Action on Melanophores: Studies with Forskolin

Abstract

α-MSH-induced pigment dispersion in melanophores shows an absolute requirement for extracellular Ca²⁺. To localize Ca²⁺ sites involved in the mechanism of action of α-MSH we studied the effects of Ca²⁺ deprivation on α-MSH and forskolin-induced melanophore responses. In an in vitro melanophore system employing ventral tailfins of Xenopus tadpoles, melanophore responses were assayed in terms of pigment dispersion and the phosphorylation state of a 53 kDa melanophore-specific protein. In the same melanophore system α-MSH has been shown to specifically increase the phosphorylation of this 53 kDa protein.

Forskolin induces a dose-dependent pigment dispersion (EC₅₀ 7 × 10^?7 M). In contrast to the dispersion induced by α-MSH forskolin-induced dispersion does not require extracellular Ca²⁺. Moreover, in a Ca²⁺-free medium melanophores with permanently activated MSH-receptors aggregate, but can be redispersed by the addition of forskolin. Forskolin increases 53 kDa phosphorylation in a dosedependent manner. Maximal stimulation with forskolin (10^?5 M) is four-fold and equals maximal 53 kDa phosphorylation obtainable with α-MSH. The MSH-induced increase in 53 kDa phosphorylation is inhibited by Ca²⁺ deprivation, whereas the forskolin-induced increase is unaffected. Our results suggest that α-MSH and forskolin stimulate melanophores through a common pathway and confirm that cAMP is a second messenger in α-MSH action in this system. We conclude that the Ca²⁺ sites in the mechanism of α-MSH action on melanophores precede adenylate cyclase activation.     

Maxadilan Activates PAC1 Receptors Expressed in Xenopus laevis Melanophores

Functional interactions between ligands and their cognate receptors can be investigated using the ability of melanophores from Xenopus laevis to disperse or aggregate their pigment granules in response to alterations in the intracellular levels of second messengers. We have examined the response of long‐term lines of cultured melanophores from X. laevis to pituitary adenylate cyclase activating peptide (PACAP), a neuropeptide with vasodilatory activity, and maxadilan, a vasodilatory peptide present in the salivary gland extracts of the blood feeding sand fly. Pituitary adenylate cyclase activating peptide increased the intracellular levels of cyclic adenosine monophosphate (cAMP) and induced pigment dispersion in the pigment cells, confirming that melanophores express an endogenous PACAP receptor. Maxadilan did not induce a response in non‐transfected melanophores. When the melanophores were transfected with complementary DNA (cDNA) from the three different members of the PACAP receptor family, maxadilan induced pigment dispersion specifically and cAMP accumulation in melanophores transfected with the cDNA for PAC1 receptors but not VPAC1 or VPAC2 receptors. A melanophore line was generated that stably expresses the PAC1 receptor.     

Second messenger signaling in olfactory transduction

Olfactory receptor neurons respond to odorants with G-protein mediated increases in the concentration of cyclic adenosine 3′,5′-monophosphate (cAMP) and/or inositol 1,4,5-triphosphate (InsP₃). These two second messengers directly regulate opening of cAMP- and InsP₃-regulated conductances localized to the apical transduction compartments of the cell (cilia and olfactory knob). In the presence of physiological concentrations of extracellular Ca²⁺, these second messenger regulated conductances mediate influx of Ca²⁺ into the olfactory neuron resulting in large, localized increases in intracellular Ca²⁺ ([Ca²⁺]_i). A significant advance in our understanding of the molecular mechanisms of olfaction is the recent realization that this increase in [Ca²⁺]_i plays an important role as a “third messenger” in olfactory transduction. Second messenger dependent increases in [Ca²⁺]_i cause opening of ciliary Ca²⁺-activated Cl⁻, cation and/or K⁺ channels that can carry a large percentage of the generator current, thus amplifying the signal substantially. As a result of this sequence of events, the generator potential in olfactory neurons can be depolarizing, leading to excitation of the neuron, or hyperpolarizing, leading to suppression of basal action potential firing rate. This dual effect of odorants on olfactory neurons may play an important role in quality coding and in the ability to detect low concentrations of odorants, particularly in complex mixtures. © 1996 John Wiley & Sons, Inc.     

Olfactory transduction: cross-talk between second-messenger systems

Chemosensory cilia of olfactory receptor neurons contain an adenylate cyclase which is stimulated by high concentrations of odorants. Cyclic AMP produced by this enzyme has been proposed to act as second messenger in olfactory transduction. Here we report that olfactory cilia contain calmodulin and that calmodulin potently activates olfactory adenylate cyclase by a mechanism additive to and independent from direct stimulation by odorants. Activation by calmodulin is calcium dependent and enhanced by GTP. Thus, olfactory transduction may involve a second-messenger cascade in which an odorant-induced increase in intracellular calcium concentration leads to activation of adenylate cyclase by calmodulin.     

Inward currents and increases in cytosolic Ca2+ concentration induced by cyclic ADP-ribose in turtle olfactory receptor cells

In olfactory receptor cells, it is well established that cyclic AMP (cAMP) and inositol-1,4,5-trisphosphate (IP(3)) act as second messengers during odor responses. In previous studies, we have shown that cAMP-increasing odorants induce odor responses even after complete desensitization of the cAMP-mediated pathway. These results suggest that at least one cAMP-independent pathway contributes to the generation of odor responses. In an attempt to identify a novel second messenger, we investigated the possible role of cyclic ADP-ribose (cADPR) in olfactory transduction. Turtle olfactory receptor cells were isolated using an enzyme-free procedure and loaded with fura-2/AM. The cells responded to dialysis with cADPR with an inward current and an increase of the intracellular Ca(2+) concentration, [Ca(2+)](i). Flooding of cells with 100 microM cADPR from the pipette also induced an inward current without changes in [Ca(2+)](i) in Na(+)-containing and Ca(2+)-free Ringer solution. In an Na(+)-free and Ca(2+)-containing Ringer solution, cADPR induced only a small inward current with a concomitant increase in [Ca(2+)](i). Inward currents and increases in [Ca(2+)](i) induced by cADPR were completely inhibited by removal of both Na(+) and Ca(2+) from the outer solution. The experiments suggest that cADPR activates a cation channel at the plasma membrane, allowing inflow of Na(+) and Ca(2+) ions. The magnitudes of the inward current responses to cAMP-increasing odorants were greatly reduced by prior dialyses of a high concentration of cADPR or 8-bromo-cyclic ADP-ribose (8-Br-cADPR), an antagonist. It is possible that the cADPR-dependent pathway contributes to the generation of olfactory responses.     

Cross-adaptation between olfactory responses induced by two subgroups of odorant molecules

It has long been believed that vertebrate olfactory signal transduction is mediated by independent multiple pathways (using cAMP and InsP3 as second messengers). However, the dual presence of parallel pathways in the olfactory receptor cell is still controversial, mainly because of the lack of information regarding the single-cell response induced by odorants that have been shown to produce InsP3 exclusively (but not cAMP) in the olfactory cilia. In this study, we recorded activities of transduction channels of single olfactory receptor cells to InsP3-producing odorants. When the membrane potential was held at -54 mV, application of InsP3-producing odorants to the ciliary region caused an inward current. The reversal potential was 0 +/- 7 mV (mean +/- SD, n = 10). Actually, InsP3-producing odorants generated responses in a smaller fraction of cells (lilial, 3.4%; lyral, 1.7%) than the cAMP-producing odorant (cineole, 26%). But, fundamental properties of responses were surprisingly homologous; namely, spatial distribution of the sensitivity, waveforms, I-V relation, and reversal potential, dose dependence, time integration of stimulus period, adaptation, and recovery. By applying both types of odorants alternatively to the same cell, furthermore, we observed cells to exhibit symmetrical cross-adaptation. It seems likely that even with odorants with different modalities adaptation occurs completely depending on the amount of current flow. The data will also provide evidence showing that olfactory response generation and adaptation are regulated by a uniform mechanism for a wide variety of odorants.     

Control of the melanophores of the crab,Pachygrapsus marmoratus: Release of pigment dispersing and pigment concentrating neurohormones by amines

1. The black pigment, in the melanophores, of Pachygrapsus marmoralus, a crab, disperses in specimens on a black background and concentrates in specimens on a white background.2. Bilateral eyestalk ablation results in black pigment concentration.3. These melanophores are regulated by pigment dispersing and concentrating hormones.4. In intact Pachygrapsus, 5-hydroxytryptamine produces black pigment dispersion whereas dopamine produces black pigment concentration.5. Neither 5-hydroxytryptamine nor dopamine affects melanophores in isolated legs. Presumably, therefore, these amines affect melanophores of intact Pachygrapsus indirectly only; 5-hydroxytryptamine by stimulating release of black pigment dispersing hormone and dopamine by stimulating release of black pigment concentrating hormone.     

Identification of second messenger mediating signal transduction in the olfactory receptor cell

One of the biggest controversial issues in the research of olfaction has been the mechanism underlying response generation to odorants that have been shown to fail to produce cAMP when tested by biochemical assays with olfactory ciliary preparations. Such observations are actually the original source proposing a possibility for the presence of multiple and parallel transduction pathways. In this study the activity of transduction channels in the olfactory cilia was recorded in cells that retained their abilities of responding to odorants that have been reported to produce InsP3 (instead of producing cAMP, and therefore tentatively termed "InsP3 odorants"). At the same time, the cytoplasmic cNMP concentration ([cNMP]i) was manipulated through the photolysis of caged compounds to examine their real-time interactions with odorant responses. Properties of responses induced by both InsP3 odorants and cytoplasmic cNMP resembled each other in their unique characteristics. Reversal potentials of currents were 2 mV for InsP3 odorant responses and 3 mV for responses induced by cNMP. Current and voltage (I-V) relations showed slight outward rectification. Both responses showed voltage-dependent adaptation when examined with double pulse protocols. When brief pulses of the InsP3 odorant and cytoplasmic cNMP were applied alternatively, responses expressed cross-adaptation with each other. Furthermore, both responses were additive in a manner as predicted quantitatively by the theory that signal transduction is mediated by the increase in cytoplasmic cAMP. With InsP3 odorants, actually, remarkable responses could be detected in a small fraction of cells ( approximately 2%), explaining the observation for a small production of cAMP in ciliary preparations obtained from the entire epithelium. The data will provide evidence showing that olfactory response generation and adaptation are regulated by a uniform mechanism for a wide variety of odorants.     

Receptor mechanisms in fish chromatophores--VI. Adenosine receptors mediate pigment dispersion in guppy and catfish melanophores

Using the guppy, Lebistes reticulatus, and the siluroid catfish, Parasilurus asotus , the effects of purine and pyrimidine derivatives on the movement of melanophores were studied. All the substances tested did not aggregate pigment within melanophores. Adenosine and adenine nucleotides were very effective in dispersing melanosomes within the cell, although adenine itself lacked such action. Derivatives of other purines than adenine and of pyrimidines did not disperse melanosomes. The pigment dispersion induced by adenine derivatives was specifically antagonized by methylxanthines. It was concluded that adenosine receptors are present on the melanophore membrane, which take part in the darkening reaction of fishes.     

The protein kinase A-anchoring protein moesin is bound to pigment granules in melanophores

Major signaling cascades have been shown to play a role in the regulation of intracellular transport of organelles. In Xenopus melanophores, aggregation and dispersion of pigment granules are regulated by the second messenger cyclic AMP through the protein kinase A (PKA) signaling pathway. PKA is bound to pigment granules where it forms complexes with molecular motors involved in pigment transport. Association of PKA with pigment granules occurs through binding to A-kinase-anchoring proteins (AKAPs), whose identity remains largely unknown. In this study, we used mass spectrometry to examine an 80 kDa AKAP detected in preparations of purified pigment granules. We found that tryptic digests of granule protein fractions enriched in the 80 kDa AKAP contained peptides that corresponded to the actin-binding protein moesin, which has been shown to function as an AKAP in mammalian cells. We also found that recombinant Xenopus moesin interacted with PKA in vitro , copurified with pigment granules and bound to pigment granules in cells. Overexpression in melanophores of a mutant moesin lacking conserved PKA-binding domain did not affect aggregation of pigment granules but partially inhibited their dispersion. We conclude that Xenopus moesin is an AKAP whose PKA-scaffolding activity plays a role in the regulation of pigment dispersion in Xenopus melanophores.     

The second messenger cascade in olfactory receptor neurons.

The molecular cloning of components involved in the cAMP second messenger cascade has allowed their biochemical characterization and revealed properties that are important for their role in sensory transduction. Recent evidence suggests inositol 1,4,5-trisphosphate functions as an additional second messenger in olfactory signalling. The interaction of these two pathways may contribute to the sensitivity of the olfactory system.     

Pigment movements in fish melanophores: Morphological and physiological studies

Summary Cyclic adenosine monophosphate (cAMP) has been shown to cause pigment dispersion in amphibian and fish melanophores. Since pigment displacements in melanophores of Pterophyllum scalare are known to be accompanied by assembly and disassembly of microtubules, the effect of cAMP on this process was investigated. Melanophores of isolated scales were treated with cAMP in the presence of vinblastine, a potent antimicrotubular agent. During the initial phase of vinblastine action, cAMP as well as its dibutyryl derivative are capable of counteracting the inhibitory effects of vinblastine on pigment dispersion. In addition, cAMP retains the velocity of pigment dispersion at about the maximum level during 1 hour experiments. Pigment aggregation was unaffected by cAMP. Since pigment dispersion in Pterophyllum-melanophores is accompanied by assembly of microtubules, it is concluded that cAMP influences, at least in part, melanosome dispersion through facilitation of microtubule assembly.     

Olfactory perception in honeybees: Concatenated and mixed odorant stimuli,concentration, and exposure effects

Summary Here we present results obtained from 7 different series of experiments, all employing odor conditioning of proboscis extension in worker honeybees and each designed to address a particular question involving olfactory perception. The questions relate to: temporal complexity of odor cues; effects of concentration, suppression, and/or potentiation in mixture perception; acquisition and extinction rates, as well as levels of generalization associated with aliphatic compounds that have the same functional groups or same alkyl radical length; and the effects of continuous exposure to odorants in the first several days of adult life on various learning and discrimination tasks involving olfactory perception. From the data obtained in these experiments we were able to conclude the following: First, worker honeybees have a limited ability to perceive complex temporal odor-quality patterns in olfactory stimuli — they learn to associate the quality of only the last part of the stimulus with a sucrose reward. Second, we confirm that citral is qualitatively different in several perceptual contexts involving odor learning and conditioning and our results help elucidate the nature of these differences as they relate to learning, discrimination, mixture perception, and continuous exposure to particular odorants. Third, we appear to have uncovered some important perceptual differences between functional groups attached to the first as opposed to the second carbon atom of alkyl radicals. Finally, we failed to uncover any significant effects relating to continuous exposure to odorants during the first several days of a worker's adult life, despite evidence that considerable sensory development takes place during this period. Thus ontogenetic changes to the peripheral system due to environmental effects appear to leave basic perceptual systems unaltered.     

Reactivated melanophore motility: differential regulation and nucleotide requirements of bidirectional pigment granule transport

To study the molecular basis for organized pigment granule transport, procedures were developed to lyse melanophores of Tilapia mossambica under conditions in which pigment granule movements could be reactivated. Gentle lysis of the melanophores resulted in a permeabilized cell model, which, in the absence of exogenous ATP, could undergo multiple rounds of pigment granule aggregation and dispersion when sequentially challenged with epinephrine and cAMP. Both directions of transport required ATP, since aggregation or dispersion in melanophores depleted of nucleotides could be reactivated only upon addition of MgATP or MgATP plus cAMP, respectively. Differences between the nucleotide sensitivities for aggregation and dispersion were demonstrated by observations that aggregation had a lower apparent Km for ATP than did dispersion and could be initiated at a lower ATP concentration. Moreover, aggregation could be initiated by ADP, but only dispersion could be reactivated by the thiophosphate ATP analog, ATP gamma S. The direction of pigment transport was determined solely by cAMP, since pigment granules undergoing dispersion reaggregated when cAMP was removed, and those undergoing aggregation dispersed when cAMP was added. These results provide evidence that pigment granule motility may be based on two distinct mechanisms that are differentially activated and regulated to produce bidirectional movements.