Stanislav S. Kolesnikov

Afferent neurotransmission mediated by hemichannels in mammalian taste cells

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 Ca2+ 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, PLCβ2, 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.

Voltage dependence of ATP secretion in mammalian taste cells.

Mammalian type II taste cells release the afferent neurotransmitter adenosine triphosphate (ATP) through ATP-permeable ion channels, most likely to be connexin (Cx) and/or pannexin hemichannels. Here, we show that ion channels responsible for voltage-gated (VG) outward currents in type II cells are ATP permeable and demonstrate a strong correlation between the magnitude of the VG current and the intensity of ATP release. These findings suggest that slowly deactivating ion channels transporting the VG outward currents can also mediate ATP secretion in type II cells. In line with this inference, we studied a dependence of ATP secretion on membrane voltage with a cellular ATP sensor using different pulse protocols. These were designed on the basis of predictions of a model of voltage-dependent transient ATP efflux. Consistently with curves that were simulated for ATP release mediated by ATP-permeable channels deactivating slowly, the bell-like and Langmuir isotherm–like potential dependencies were characteristic of ATP secretion obtained for prolonged and short electrical stimulations of taste cells, respectively. These observations strongly support the idea that ATP is primarily released via slowly deactivating channels. Depolarizing voltage pulses produced negligible Ca2+ transients in the cytoplasm of cells releasing ATP, suggesting that ATP secretion is mainly governed by membrane voltage under our recording conditions. With the proviso that natural connexons and pannexons are kinetically similar to exogenously expressed hemichannels, our findings suggest that VG ATP release in type II cells is primarily mediated by Cx hemichannels.

The ATP permeability of pannexin 1 channels in a heterologous system and in mammalian taste cells is dispensable.

Summary 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.

Functional expression of the extracellular-Ca2+-sensing receptor in mouse taste cells

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 Ca2+ transients, whereas type I and type II cells were not specifically responsive. Consistent with these findings, certain amino acids stimulated PLC-dependent Ca2+ 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 Ca2+ 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 Ca2+ 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.

Electrophysiologically identified subpopulations of taste bud cells

The heterogeneous population of mammalian taste cells includes several cellular subtypes specializing in distinct physiological functions. They are poorly understood at the single cell level because the available physiological data have generally been obtained from unidentified taste cells. We recorded them from individual taste cells isolated from circumvallate, foliate, and fungiform papilla of the mouse, employing the patch clamp technique, and tried to elucidate whether universal electrophysiological criteria may be established for the identification of functionally different cellular subpopulations. It was found that irrespective of the papillae type, most ( approximately 96%) of robust taste cells could be categorized into three distinct subgroups on the basis of families of whole-cell (WC) currents exhibited in response to membrane polarization. The validity of this quite simple criterion was further confirmed by using different voltage clamp protocols, ion substitutions, and channel blockers to record different ionic currents, including voltage-gated (VG) Ca(2+), inward-rectifying K(+), and hyperpolarization-activated currents. Given that our findings are based on the statistically significant number of recordings, we believe that the electrophysiological identification of taste cells presented here may be effective for further studies on single taste cell physiology, including taste transduction.

Polypeptide Modulators of TRPV1 Produce Analgesia without Hyperthermia

Transient receptor potential vanilloid 1 receptors (TRPV1) play a significant physiological role. The study of novel TRPV1 agonists and antagonists is essential. Here, we report on the characterization of polypeptide antagonists of TRPV1 based on in vitro and in vivo experiments. We evaluated the ability of APHC1 and APHC3 to inhibit TRPV1 using the whole-cell patch clamp approach and single cell Ca2+ imaging. In vivo tests were performed to assess the biological effects of APHC1 and APHC3 on temperature sensation, inflammation and core body temperature. In the electrophysiological study, both polypeptides partially blocked the capsaicin-induced response of TRPV1, but only APHC3 inhibited acid-induced (pH 5.5) activation of the receptor. APHC1 and APHC3 showed significant antinociceptive and analgesic activity in vivo at reasonable doses (0.01–0.1 mg/kg) and did not cause hyperthermia. Intravenous administration of these polypeptides prolonged hot-plate latency, blocked capsaicin- and formalin-induced behavior, reversed CFA-induced hyperalgesia and produced hypothermia. Notably, APHC3’s ability to inhibit the low pH-induced activation of TRPV1 resulted in a reduced behavioural response in the acetic acid-induced writhing test, whereas APHC1 was much less effective. The polypeptides APHC1 and APHC3 could be referred to as a new class of TRPV1 modulators that produce a significant analgesic effect without hyperthermia.

Adenosine triphosphate mobilizes cytosolic calcium and modulates ionic currents in mouse taste receptor cells

By using photometry and the patch clamp technique, we identified P(2Y)-like receptors in mouse taste receptor cells (TRCs) and found them to be coupled to Ca(2+) mobilization and ionic current modulation. Particularly, adenosine triphosphate (ATP) and the P(2Y) agonist 2-methylthio-ATP increased intracellular Ca(2+) by stimulating the phosphoinositide pathway, whereas beta, gamma-methylene-D-ATP, a P(2X) agonist, was ineffective. In a distinctive TRC subpopulation, ATP closed Ca(2+) channels. This regulation may underlie the negative feedback tuning neurotransmitter release. By mobilizing intracellular Ca(2+), ATP activated Ca(2+)-dependent Cl(-) channels, the intracellular event that may universally occur upon taste stimulation triggering IP(3) formation and Ca(2+) release in the TRC cytoplasm.

P2Y isoforms operative in mouse taste cells

Recent functional evidence indicates that mouse taste cells express P2Y receptors coupled to IP3 production and Ca2+ 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 Ca2+ at saturating concentrations, suggesting that common receptors for both nucleotides, i.e., P2Y2 and P2Y4 subtypes, might be involved. Reverse transcription/polymerase chain reaction and immunohistochemistry have confirmed the presence of P2Y2 and P2Y4 receptors in a population of taste bud cells from the circumvallate and foliate papillae. Transcripts for the P2Y1 and P2Y6 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 P2Y2 and P2Y4 receptors play a predominant role in mediating taste cell responses to ATP and UTP.

Functional expression of adrenoreceptors in mesenchymal stromal cells derived from the human adipose tissue.

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.

Stimulation of the extracellular Ca2+-sensing receptor by denatonium

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.