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In mammals, three genes encode IP 3 receptors (IP 3 Rs), which are involved in agonist-induced Ca 2+ signaling in cells of apparently all types. Using the CRISPR/Cas9 approach for disruption of two out of three IP 3 R genes in HEK-293 cells, we generated three monoclonal cell lines, IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK, with the single functional isoform, IP 3 R1, IP 3 R2, and IP 3 R3, respectively. All engineered cells responded to ACh with Ca 2+ transients in an "all-or-nothing" manner, suggesting that each IP 3 R isotype was capable of mediating CICR. The sensitivity of cells to ACh strongly correlated with the affinity of IP 3 binding to an IP 3 R isoform they expressed. Based on a mathematical model of intracellular Ca 2+ signals induced by thapsigargin, a SERCA inhibitor, we developed an approach for estimating relative Ca 2+ permeability of Ca 2+ store and showed that all three IP 3 R isoforms contributed to Ca 2+ leakage from ER. The relative Ca 2+ permeabilities of Ca 2+ stores in IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK cells were evaluated as 1:1.75:0.45. Using the genetically encoded sensor R-CEPIA1er for monitoring Ca 2+ signals in ER, engineered cells were ranged by resting levels of stored Ca 2+ as IP3R3-HEK ≥ IP3R1-HEK > IP3R2-HEK. The developed cell lines could be helpful for further assaying activity, regulation, and pharmacology of individual IP 3 R isoforms.

The current concept of taste transduction implicates the TASR/PLCβ2/IP 3 R3/TRPM5 axis in mediating chemo-electrical coupling in taste cells of the type II. While generation of IP 3 has been verified as an obligatory step, DAG appears to be a byproduct of PIP 2 cleavage by PLCβ2. Here, we provide evidence that DAG-signaling could play a significant and not yet recognized role in taste transduction. In particular, we found that DAG-gated channels are functional in type II cells but not in type I and type III cells. The DAG-gated current presumably constitutes a fraction of the generator current triggered by taste stimulation in type II cells. Bitter stimuli and DAG analogs produced Ca 2+ transients in type II cells, which were greatly decreased at low bath Ca 2+ , indicating their dependence on Ca 2+ influx. Among DAG-gated channels, transcripts solely for TRPC3 were detected in the taste tissue, thus implicating this channel in mediating DAG-regulated Ca 2+ entry. Release of the afferent neurotransmitter ATP from CV papillae was monitored online by using the luciferin/luciferase method and Ussing-like chamber. It was shown that ATP secretion initiated by bitter stimuli and DAG analogs strongly depended on mucosal Ca 2+ . Based on the overall findings, we speculate that in taste transduction, IP 3 -driven Ca 2+ release is transient and mainly responsible for rapid activation of Ca 2+ -gated TRPM5 channels, thus forming the initial phase of receptor potential. DAG-regulated Ca 2+ entry through apically situated TRPC3 channels extends the primary Ca 2+ signal and preserves TRPM5 activity, providing a needful prolongation of the receptor potential., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Type III taste cells are the only taste bud cells which express voltage-gated (VG) Ca 2+ channels and employ Ca 2+ -dependent exocytosis to release neurotransmitters, particularly serotonin. The taste bud is a tightly packed cell population, wherein extracellular Ca 2+ is expected to fluctuate markedly due to the electrical activity of taste cells. It is currently unclear whether the Ca 2+ entry-driven synapse in type III cells could be reliable enough at unsteady extracellular Ca 2 . Here we assayed depolarization-induced Ca 2+ signals and associated serotonin release in isolated type III cells at varied extracellular Ca 2+ . It turned out that the same depolarizing stimulus elicited invariant Ca 2+ signals in type III cells irrespective of bath Ca 2+ varied within 0.5-5 mM. The serotonin release from type III cells was assayed with the biosensor approach by using HEK-293 cells co-expressing the recombinant 5-HT4 receptor and genetically encoded cAMP sensor Pink Flamindo. Consistently with the weak Ca 2+ dependence of intracellular Ca 2+ transients produced by VG Ca 2+ entry, depolarization-triggered serotonin secretion varied negligibly with bath Ca 2+ . The evidence implicated the extracellular Ca 2+ -sensing receptor in mediating the negative feedback mechanism that regulates VG Ca 2+ entry and levels off serotonin release in type III cells at deviating Ca 2+ in the extracellular medium.

The integrative study that included experimentation and mathematical modeling was carried out to analyze dynamic aspects of transient Ca 2+ signaling induced by brief pulses of GPCR agonists in mesenchymal stromal cells from the human adipose tissue (AD-MSCs). The experimental findings argued for IP 3 /Ca 2+ -regulated Ca 2+ release via IP 3 receptors (IP 3 Rs) as a key mechanism mediating agonist-dependent Ca 2+ transients. The consistent signaling circuit was proposed to formalize coupling of agonist binding to Ca 2+ mobilization for mathematical modeling. The model properly simulated the basic phenomenology of agonist transduction in AD-MSCs, which mostly produced single Ca 2+ spikes upon brief stimulation. The spike-like responses were almost invariantly shaped at different agonist doses above a threshold, while response lag markedly decreased with stimulus strength. In AD-MSCs, agonists and IP 3 uncaging elicited similar Ca 2+ transients but IP 3 pulses released Ca 2+ without pronounced delay. This suggested that IP 3 production was rate-limiting in agonist transduction. In a subpopulation of AD-MSCs, brief agonist pulses elicited Ca 2+ bursts crowned by damped oscillations. With properly adjusted parameters of IP 3 R inhibition by cytosolic Ca 2+ , the model reproduced such oscillatory Ca 2+ responses as well. GEM-GECO1 and R-CEPIA1er, the genetically encoded sensors of cytosolic and reticular Ca 2+ , respectively, were co-expressed in HEK-293 cells that also responded to agonists in an "all-or-nothing" manner. The experimentally observed Ca 2+ signals triggered by ACh in both compartments were properly simulated with the suggested signaling circuit. Thus, the performed modeling of the transduction process provides sufficient theoretical basis for deeper interpretation of experimental findings on agonist-induced Ca 2+ signaling in AD-MSCs., Competing Interests: The authors disclose no conflict of interest., (© 2021 The Author(s).)

The phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (LY294) and its much less active analog LY303511 (LY303) constitute the paired probe that is commonly used to demonstrate the involvement of PI3K in intracellular signaling. We studied effects of LY294 and LY303 on Ca 2+ signaling initiated by certain GPCR agonists in cells of several lines, including CHO cells expressing the recombinant serotonin receptor 5-HT2C and mesenchymal stromal cells derived from the human adipose tissue (AD-MSCs) and umbilical cord (UD-MSCs). The LY294/LY303 pair exerted apparently specific effects on responsiveness of AD-MSCs to ATP, suggesting the involvement of PI3K in ATP transduction. Surprisingly, LY303 inhibited Ca 2+ transients elicited by histamine in the same cells, while LY294 was ineffective. This observation and other findings implicated a PI3K-unrelated mechanism in mediating effects of the LY compound on AD-MSC responsiveness to histamine. With LY303 in the bath, the dose dependence of histamine responses was shifted positively at the invariable number of responsive cells, as would be the case with a competitive antagonist of histamine receptors. Moreover, LY303 and LY294 inhibited Ca 2+ transients elicited by acetylcholine and serotonin in UD-MSCs and CHO/5-HT2C cells, respectively. Our overall results argued for the possibility that LY294 and LY303 could directly affect activity of aminergic GPCRs. Thus, LY303511 and LY294002 should be used cautiously in studies of PI3K as a factor of GPCR signaling., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The current knowledge of electrogenesis in mesenchymal stromal cells (MSCs) remains scarce. Earlier, we demonstrated that in MSCs from the human adipose tissue, transduction of certain agonists involved the phosphoinositide cascade. Its pivotal effector PLC generates DAG that can regulate ion channels directly or via its derivatives, including arachidonic acid (AA). Here we showed that AA strongly hyperpolarized MSCs by stimulating instantly activating, outwardly rectifying TEA-insensitive K + channels. Among AA-regulated K + channels, K2P channels from the TREK subfamily appeared to be an appropriate target. The expression of K2P channels in MSCs was verified by RT-PCR, which revealed TWIK-1, TREK-1, and TASK-5 transcripts. The TREK-1 inhibitor spadin antagonized the electrogenic action of AA, which was simulated by the channel activator BL 1249. This functional evidence suggested that TREK-1 channels mediated AA-dependent hyperpolarization of MSCs. Being mostly silent at rest, TREK-1 negligibly contributed to the "background" K + current. The dramatic stimulation of TREK-1 channels by AA indicates their involvement in AA-dependent signaling in MSCs.

Conventional chemical synapses in the nervous system involve a presynaptic accumulation of neurotransmitter-containing vesicles, which fuse with the plasma membrane to release neurotransmitters that activate postsynaptic receptors. In taste buds, type II receptor cells do not have conventional synaptic features but nonetheless show regulated release of their afferent neurotransmitter, ATP, through a large-pore, voltage-gated channel, CALHM1. Immunohistochemistry revealed that CALHM1 was localized to points of contact between the receptor cells and sensory nerve fibers. Ultrastructural and super-resolution light microscopy showed that the CALHM1 channels were consistently associated with distinctive, large (1- to 2-μm) mitochondria spaced 20 to 40 nm from the presynaptic membrane. Pharmacological disruption of the mitochondrial respiratory chain limited the ability of taste cells to release ATP, suggesting that the immediate source of released ATP was the mitochondrion rather than a cytoplasmic pool of ATP. These large mitochondria may serve as both a reservoir of releasable ATP and the site of synthesis. The juxtaposition of the large mitochondria to areas of membrane displaying CALHM1 also defines a restricted compartment that limits the influx of Ca 2+ upon opening of the nonselective CALHM1 channels. These findings reveal a distinctive organelle signature and functional organization for regulated, focal release of purinergic signals in the absence of synaptic vesicles., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The purinergic transduction was examined in mesenchymal stromal cells (MSCs) from the human adipose tissue, and several nucleotides, including ATP, UTP, and ADP, were found to mobilize cytosolic Ca 2+ . Transcripts for multiple purinoreceptors were detected in MSC preparations, including A 1 , A 2A , A 2B , P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 13 , P2Y 14 , P2X 2 , P2X 4 , and P2X 7 . Cellular responses to nucleotides were insignificantly sensitive to bath Ca 2+ , pointing at a minor contribution of Ca 2+ entry, and were suppressed by U73122 and 2-APB, implicating the phosphoinositide cascade in coupling P2Y receptors to Ca 2+ release. While individual cells were sensitive to several P2Y agonists, responsiveness to a given nucleotide varied from cell to cell, suggesting that particular MSCs could employ different sets of purinoreceptors. Caged Ca 2+ stimulated Ca 2+ -induced Ca 2+ release (CICR) that was mediated largely by IP 3 receptors, and resultant Ca 2+ transients were similar to nucleotide responses by magnitude and kinetics. A variety of findings hinted at CICR to be a universal mechanism that finalizes Ca 2+ signaling initiated by agonists in MSCs. Individual MSCs responded to nucleotides in an all-or-nothing manner. Presumably just CICR provided invariant Ca 2+ responses observed in MSCs at different nucleotide concentrations. The effects of isoform specific agonists and antagonists suggested that both P2Y 1 and P2Y 13 were obligatory for ADP responses, while P2Y 4 and P2Y 11 served as primary UTP and ATP receptors, respectively. Extracellular NAD + stimulated Ca 2+ signaling in each ATP-responsive MSC by involving P2Y 11 . The overall data indicate that extracellular nucleotides and NAD + can serve as autocrine/paracrine factors regulating MSC functions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Electrogenesis in mesenchymal stromal cells (MSCs) remains poorly understood. Little is known about ion channels active in resting MSCs and activated upon MSC stimulation, particularly, by agonists mobilizing Ca 2+ in the MSC cytoplasm. A variety of Ca 2+ -gated ion channels may couple Ca 2+ signals to polarization of the plasma membrane. Here, we studied MSCs from the human adipose tissue and found that in cells responsive to ATP and adenosine with Ca 2+ transients or exhibiting spontaneous Ca 2+ oscillations, Ca 2+ bursts were associated with hyperpolarization mediated by Ca 2+ -gated K + channels. The expression analysis revealed transcripts for KCNMA1 and KCNN4 genes encoding for Ca 2+ -activated K + channels of large (K Ca 1.1) and intermediate (K Ca 3.1) conductance, respectively. Moreover, transcripts for the Ca 2+ -gated cation channel TRPM4 and anion channels Ano1, Ano2, and bestrophin-1, bestrophin-3, and bestrophin-4 were revealed. In all assayed MSCs, a rise in cytosolic Ca 2+ stimulated K + currents that were inhibited with iberiotoxin. This suggested that K Ca 1.1 channels are invariably expressed in MSCs. In ATP- and adenosine-responsive cells, iberiotoxin and TRAM-34 diminished electrical responses, implicating both K Ca 1.1 and K Ca 3.1 channels in coupling agonist-dependent Ca 2+ signals to membrane voltage. Functional tests pointed at the existence of two separate MSC subpopulations exhibiting Ca 2+ -gated anion currents that were mediated by Ano2-like and bestrophin-like anion channels, respectively. Evidence for detectable activity of Ano1 and TRPM4 was not obtained. Thus, K Ca 1.1 channels are likely to represent the dominant type of Ca 2+ -activated K + channels in MSCs, which can serve in concert with K Ca 3.1 channels as effectors downstream of G-protein-coupled receptor (GPCR)-mediated Ca 2+ signaling.

Cell-to-cell communications and autocrine/paracrine regulations are mediated by an extracellular signaling network involving secretion of a variety of different factors, hormones, neurotransmitters, and other signaling molecules that are recognized in an extracellular medium by multiple molecular receptors operating in the plasma membrane of cells. Most of plasma membrane receptors belong to the superfamily of heptahelical receptors, many of which are coupled by G-proteins to adenylate cyclase responsible for cAMP production in the cell cytoplasm. The canonical role of cAMP in cell physiology is to serve as a second messenger and universal regulator of intracellular processes. Meanwhile, increasing body of evidence leaves little doubts that stimulated cells can release cAMP into intercellular space, where it may serve as signaling molecule in cell-to-cell communications and autocrine regulations. This review considers the basic concept on mechanisms of intracellular and extracellular signaling with cAMP as the second and first messenger.

Specialized Ca(2+)-dependent ion channels ubiquitously couple intracellular Ca(2+) signals to a change in cell polarization. The existing physiological evidence suggests that Ca(2+)-activated Cl(-) channels (CaCCs) are functional in taste cells. Because Ano1 and Ano2 encode channel proteins that form CaCCs in a variety of cells, we analyzed their expression in mouse taste cells. Transcripts for Ano1 and Ano2 were detected in circumvallate (CV) papillae, and their expression in taste cells was confirmed using immunohistochemistry. When dialyzed with CsCl, taste cells of the type III exhibited no ion currents dependent on cytosolic Ca(2+). Large Ca(2+)-gated currents mediated by TRPM5 were elicited in type II cells by Ca(2+) uncaging. When TRPM5 was inhibited by triphenylphosphine oxide (TPPO), ionomycin stimulated a small but resolvable inward current that was eliminated by anion channel blockers, including T16Ainh-A01 (T16), a specific Ano1 antagonist. This suggests that CaCCs, including Ano1-like channels, are functional in type II cells. In type I cells, CaCCs were prominently active, blockable with the CaCC antagonist CaCCinh-A01 but insensitive to T16. By profiling Ano1 and Ano2 expressions in individual taste cells, we revealed Ano1 transcripts in type II cells only, while Ano2 transcripts were detected in both type I and type II cells. P2Y agonists stimulated Ca(2+)-gated Cl(-) currents in type I cells. Thus, CaCCs, possibly formed by Ano2, serve as effectors downstream of P2Y receptors in type I cells. While the role for TRPM5 in taste transduction is well established, the physiological significance of expression of CaCCs in type II cells remains to be elucidated.

Cultured mesenchymal stromal cells (MSCs) from different sources represent a heterogeneous population of proliferating non-differentiated cells that contains multipotent stem cells capable of originating a variety of mesenchymal cell lineages. Despite tremendous progress in MSC biology spurred by their therapeutic potential, current knowledge on receptor and signaling systems of MSCs is mediocre. Here we isolated MSCs from the human adipose tissue and assayed their responsivity to GPCR agonists with Ca(2+) imaging. As a whole, a MSC population exhibited functional heterogeneity. Although a variety of first messengers was capable of stimulating Ca(2+) signaling in MSCs, only a relatively small group of cells was specifically responsive to the particular GPCR agonist, including noradrenaline. RT-PCR and immunocytochemistry revealed expression of α1B-, α2A-, and β2-adrenoreceptors in MSCs. Their sensitivity to subtype-specific adrenergic agonists/antagonists and certain inhibitors of Ca(2+) signaling indicated that largely the α2A-isoform coupled to PLC endowed MSCs with sensitivity to noradrenaline. The all-or-nothing dose-dependence was characteristic of responsivity of robust adrenergic MSCs. Noradrenaline never elicited small or intermediate responses but initiated large and quite similar Ca(2+) transients at all concentrations above the threshold. The inhibitory analysis and Ca(2+) uncaging implicated Ca(2+)-induced Ca(2+) release (CICR) in shaping Ca(2+) signals elicited by noradrenaline. Evidence favored IP3 receptors as predominantly responsible for CICR. Based on the overall findings, we inferred that adrenergic transduction in MSCs includes two fundamentally different stages: noradrenaline initially triggers a local and relatively small Ca(2+) signal, which next stimulates CICR, thereby being converted into a global Ca(2+) signal., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Gene expression analysis facilitates the study of physiological processes operating at the single-cell level. It allows for the identification of key molecules that are involved in distinct intracellular processes in individual cells. The review focuses on the modern techniques and strategies that are used to establish gene expression profile of a single cell. Particularly, it describes how analysis of gene expression patterns at the single-cell level had provided significant insight into understanding of molecular and cellular mechanisms of odor and taste transduction in mammals.

Afferent output in type II taste cells is mediated by ATP liberated through ion channels. It is widely accepted that pannexin 1 (Panx1) channels are responsible for ATP release in diverse cell types, including taste cells. While biophysical evidence implicates slow deactivation of ion channels following ATP release in taste cells, recombinant Panx1 activates and deactivates rapidly. This inconsistency could indicate that the cellular context specifies Panx1 functioning. We cloned Panx1 from murine taste tissue, and heterologously expressed it in three different cell lines: HEK-293, CHO and neuroblastoma SK-N-SH cells. In all three cell lines, Panx1 transfection yielded outwardly rectifying anion channels that exhibited fast gating and negligible permeability to anions exceeding 250 Da. Despite expression of Panx1, the host cells did not liberate ATP upon stimulation, making it unclear whether Panx1 is involved in taste-related ATP secretion. This issue was addressed using mice with genetic ablation of the Panx1 gene. The ATP-biosensor assay revealed that, in taste cells devoid of Panx1, ATP secretion was robust and apparently unchanged compared with the control. Our data suggest that Panx1 alone forms a channel that has insufficient permeability to ATP. Perhaps, a distinct subunit and/or a regulatory circuit that is absent in taste cells is required to enable a high ATP-permeability mode of a native Panx1-based channel.

Recently, the novel peptide named purotoxin-1 (PT1) has been identified in the venom of the spider Geolycosa sp. and shown to exert marked modulatory effects on P2X3 receptors in rat sensory neurons. Here we studied another polypeptide from the same spider venom, purotoxin-2 (PT2), and demonstrated that it also affected activity of mammalian P2X3 receptors. The murine and human P2X3 receptors were heterologously expressed in cells of the CHO line, and nucleotide-gated currents were stimulated by CTP and ATP, respectively. Both PT1 and PT2 negligibly affected P2X3-mediated currents elicited by brief pulses of the particular nucleotide. When subthreshold CTP or ATP was added to the bath to exert the high-affinity desensitization of P2X3 receptors, both spider toxins strongly enhanced the desensitizing action of the ambient nucleotides. At the concentration of 50nM, PT1 and PT2 elicited 3-4-fold decrease in the IC(50) dose of ambient CTP or ATP. In contrast, 100nM PT1 and PT2 negligibly affected nucleotide-gated currents mediated by mP2X2 receptors or mP2X2/mP2X3 heteromers. Altogether, our data point out that the PT1 and PT2 toxins specifically target the fast-desensitizing P2X3 receptor, thus representing a unique tool to manipulate its activity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The extracellular Ca(2+)-sensing receptor (CASR) is a promiscuous G-protein-coupled receptor closely related to the taste receptors T1R1-T1R3. Here we analyzed the possibility that apart from being stimulated by external Ca(2+) and amino acids, the substances effective as tastants, CASR might serve as a receptor for other sapid compounds. CASR was heterologously expressed in HEK-293 cells, and their responsivity to a variety of bitter and sweet substances was examined. Among them, solely denatonium was found to stimulate Ca(2+) signaling in CASR-positive HEK-293 cells. Apparently, these Ca(2+) responses were specific, as those were inhibited by the CASR antagonist NSP-4123. Altogether, our findings indicate that denatonium stimulates CASR by shifting a dose-response curve for the principal CASR agonist Ca(2+) to lower concentrations., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Three types of morphologically and functionally distinct taste cells operate in the mammalian taste bud. We demonstrate here the expression of two G-protein-coupled receptors from the family C, CASR and GPRC6A, in the taste tissue and identify transcripts for both receptors in type I cells, no transcripts in type II cells and only CASR transcripts in type III cells, by using the SMART-PCR RNA amplification method at the level of individual taste cells. Type I taste cells responded to calcimimetic NPS R-568, a stereoselective CASR probe, with Ca(2+) transients, whereas type I and type II cells were not specifically responsive. Consistent with these findings, certain amino acids stimulated PLC-dependent Ca(2+) signaling in type III cells, but not in type I and type II cells, showing the following order of efficacies: Phe~Glu>Arg. Thus, CASR is coupled to Ca(2+) mobilization solely in type III cells. CASR was cloned from the circumvallate papilla into a pIRES2-EGFP plasmid and heterologously expressed in HEK-293 cells. The transfection with CASR enabled HEK-293 cells to generate Ca(2+) transients in response to the amino acids, of which, Phe was most potent. This observation and some other facts favor CASR as the predominant receptor subtype endowing type III cells with the ability to detect amino acids. Altogether, our results indicate that type III cells can serve a novel chemosensory function by expressing the polymodal receptor CASR. A role for CASR and GPRC6A in physiology of taste cells of the type I remains to be unveiled.

Peroxiredoxins (Prx) are a family of nonselenium peroxidases that are involved in cell defense against oxidative stress and in redox regulation of intracellular signaling. Mammalian peroxiredoxin 6 (Prx6) belongs to the 1-Cys Prx subfamily. The protein--protein interactions of human Prx6 were studied using a yeast two-hybrid system. Hybrid plasmid pHybLex/Zeo/Prx6, which directed synthesis of a chimeric protein consisting of the DNA-binding domain (BD) of LexA and a Prx6 sequence, was used to screen a two-hybrid cDNA library Hybrid Hunter (Invitrogen). The screening identified two potential interaction partners of Prx6: the calcium-activated cysteine endopeptidase calpain and the p50RhoGAP protein of the family of Sec14-like proteins. The possibility for the interactions observed in the two-hybrid system to occur in oxidative stress in vivo is discussed.

Peroxiredoxins (Prx) are a family of antioxidant proteins with peroxidase activity. The ability of 1-Cys Prx to self-associate was studied with the use of native PAGE and Western blotting. Two protein bands corresponding to monomeric and dimeric forms were detected in the preparation of the recombinant 1-Cys Prx subjected to native PAGE, with dimers being more abundant. The third band corresponding to the oligomeric form was detected after incubation of the recombinant 1-Cys Prx with DTT, although monomers and dimers were also observed. These results indicate that monomeric, dimeric, and oligomeric states of the protein are likely to be interchangeable. Native PAGE in combination with Western blot analysis revealed that self-association of 1-Cys Prx also occurred at physiologically relevant concentrations in vivo. The native 1-Cys Prx existed in the monomeric and dimeric forms in rat olfactory epithelium, with monomers being more common. The structural sensitivity of the recombinant 1-Cys Prx to imidazole was shown.

In mammalian taste buds, ionotropic P2X receptors operate in gustatory nerve endings to mediate afferent inputs. Thus, ATP secretion represents a key aspect of taste transduction. Here, we characterized individual vallate taste cells electrophysiologically and assayed their secretion of ATP with a biosensor. Among electrophysiologically distinguishable taste cells, a population was found that released ATP in a manner that was Ca(2+) independent but voltage-dependent. Data from physiological and pharmacological experiments suggested that ATP was released from taste cells via specific channels, likely to be connexin or pannexin hemichannels. A small fraction of ATP-secreting taste cells responded to bitter compounds, indicating that they express taste receptors, their G-protein-coupled and downstream transduction elements. Single cell RT-PCR revealed that ATP-secreting taste cells expressed gustducin, TRPM5, PLCbeta2, multiple connexins and pannexin 1. Altogether, our data indicate that tastant-responsive taste cells release the neurotransmitter ATP via a non-exocytotic mechanism dependent upon the generation of an action potential.

Recent functional evidence indicates that mouse taste cells express P2Y receptors coupled to IP(3) production and Ca(2+) mobilization. Our studies of the expression profile of particular P2Y isoforms in the taste tissue of the mouse have revealed that ATP and UTP equipotently mobilize intracellular Ca(2+) at saturating concentrations, suggesting that common receptors for both nucleotides, i.e., P2Y(2) and P2Y(4) subtypes, might be involved. Reverse transcription/polymerase chain reaction and immunohistochemistry have confirmed the presence of P2Y(2) and P2Y(4) receptors in a population of taste bud cells from the circumvallate and foliate papillae. Transcripts for the P2Y(1) and P2Y(6) isoforms have also been detected in taste tissue preparations, this observation being consistent with the ADP and UDP responsiveness of taste cells. Together, our data suggest that P2Y(2) and P2Y(4) receptors play a predominant role in mediating taste cell responses to ATP and UTP.

Peroxiredoxins are a novel family of antioxidant proteins that specifically prevent enzymes from metal-catalyzed oxidation. The localization of a member of the mono-cystein subfamily of peroxiredoxins, the 28-kDa protein, in different rat tissues and its antioxidant properties were investigated. By immunoblotting, the 28-kDa peroxiredoxin was found to be most highly concentrated in olfactory epithelium and present in all tissues tested (skin, lung, trachea, kidney, womb, and brain). Immunostaining with rabbit polyclonal antibody raised against the 28-kDa peroxiredoxin revealed the particularly high level of the 28-kDa peroxiredoxin immunoreactivity in air-contacting areas (apical regions and mucus of the olfactory and respiratory epithelium and skin epidermis), which are continually exposed to numerous air-borne reactive oxygen species. In the apical regions of the olfactory and respiratory epithelium, the 28-kDa-peroxiredoxin immunogold labeling outlined microvilli and cilia and was mainly located in sustentacular cells and in respiratory and goblet cells, as electron-microscopic analysis revealed. In skin epidermis, the 28-kDa peroxiredoxin immunoreactivity was confined to the granular layer and specifically concentrated in sebaceous glands of hair follicle. In situ hybridization with 33P-labeled antisense RNA probe revealed the expression of the 28-kDa peroxiredoxin mRNA in tissues with a high level of the 28-kDa peroxiredoxin immunoreactivity. Immunodepletion of the 28-kDa peroxiredoxin profoundly decreased the antioxidant activity of the olfactory tissue extract.

The 28 kDa peroxiredoxin from rat exhibited peroxidase activity only in the presence of dithiothreitol. Both organic and nonorganic peroxidases were found to be substrates for the 28-kDa peroxiredoxin activity. Analysis of the protective antioxidant activity of the 28-kDa peroxiredoxin revealed that it is accounted for by its peroxidase activity.

cDNA clones encoding the 45 kDa protein were isolated from a rat olfactory epithelium cDNA library and their inserts were sequenced. The reconstructed protein sequence comprises 400 amino acids with a calculated molecular mass of 46,026 Da. A homology was revealed between the amino acid sequence of the 45 kDa protein and the proteins involved in the transfer of hydrophobic ligands. Using in situ hybridisation, the 45 kDa protein mRNA expression was detected in the layer of supportive cells of olfactory epithelium, apical region of trachea, surface layer of the ciliated bronchial epithelium in lung and in skin epidermis.

The secretory 28 kD protein, an abundant water-soluble protein from rat olfactory epithelium, belongs to the 1-Cys subfamily of thiol-specific antioxidants (peroxiredoxins). The 28 kD protein contains a single cysteine residue at the position 46 which accounts for the antioxidant activity. Here we studied the effects of N-ethyilmaleimide and t-butyl hydroperoxide on the antioxidant activity of the 28 kD protein and that of the 23 kD protein from rat erythrocyte which is a member of 2-Cys subfamily of peroxiredoxines. N-ethylmaleimide, modifier for cysteine residues, had no effect on antioxidant activity of the dithiothreitol-treated 28 kD protein but irreversibly inhibited activity of the 23 kD protein under reducing conditions. The 28 kD protein was sensitive to treatment with peroxides: t-butyl hydroperoxide at micromolar concentrations was shown to irreversibly inactivate 28 kD protein. In the presence of dithiothreitol, the lower level of peroxide concentrations was required to inhibit 28 kD protein activity. The mechanism of this effect may be mediated through conversion of sulfhydryl group of 46Cys to oxidized states (46Cys-SO2H and 46Cys-SO3H). Antioxidant property of 23 kD protein was impaired by t-butyl hydroperoxide only in the presence of dithiothreitol. The concentrations of t-butyl hydroperoxide needed to affect the 23 kD protein were at least one order of magnitude higher than were required for the 28 kD protein inhibition. The given results suggest the essential differences between catalytic site of 28 kD protein and that of 2-Cys peroxiredoxins.

Microwave radiation decreased specific camphor binding to a membrane fraction of rat epithelium but not to a Triton X-100 extract of this fraction. Inhibition of the ligand binding did not depend on the modulation frequency of the microwave field in the region 1-100 Hz and was not a linear function of specific absorption rate (SAR). The decreased ligand binding was due to a shedding or release of the specific camphor-binding protein from the membrane into solution. It is highly probable that several other membrane proteins may be shed into solution during microwave exposure.

The specific membrane glycoproteins with high affinity for camphor and decanal were isolated from rat olfactory epithelium. Antibodies to these glycoproteins inhibited both the electroolfactogram and the binding of odorants. The enzyme immunoassay has shown these glycoproteins to be present in the olfactory epithelium of rat, mouse, guinea-pig and hamster but not in that of frog and carp. The molecular mass of the odour-binding glycoproteins from rat olfactory epithelium solubilized by Triton X-100 was approx. 140 kDa. They consisted of two subunits (88 and 55 kDa). The 88 kDa subunit was capable of binding odorants. The data obtained suggest that the glycoproteins isolated have some properties that make them plausible candidates for olfactory receptor molecules.

The specific odor-binding glycoproteins have been isolated from rat olfactory epithelium. They consist of two subunits, gp88 and gp55. Subunit gp88 is capable of odorant binding.