PI3 kinase inhibitor PI828 uncouples aminergic GPCRs and Ca2+ mobilization irrespectively of its primary target.

The phosphoinositide 3-kinase (PI3K) is involved in regulation of multiple intracellular processes. Although the inhibitory analysis is generally employed for validating a physiological role of PI3K, increasing body of evidence suggests that PI3K inhibitors can exhibit PI3K-unrelated activity as well. Here we studied Ca2+ signaling initiated by aminergic agonists in a variety of different cells and analyzed effects of the PI3K inhibitor PI828 on cell responsiveness. It turned out that PI828 inhibited Ca2+ transients elicited by acetylcholine (ACh), histamine, and serotonin, but did not affect Ca2+ responses to norepinephrine and ATP. Another PI3K inhibitor wortmannin negligibly affected Ca2+ signaling initiated by any one of the tested agonists. Using the genetically encoded PIP3 sensor PH(Akt)-Venus, we confirmed that both PI828 and wortmannin effectively inhibited PI3K and ascertained that this kinase negligibly contributed to ACh transduction. These findings suggested that PI828 inhibited Ca2+ responses to aminergic agonists tested, involving an unknown cellular mechanism unrelated to the PI3K inhibition. Complementary physiological experiments provided evidence that PI828 could inhibit Ca2+ signals induced by certain agonists, by acting extracellularly, presumably, through their surface receptors. For the muscarinic M3 receptor, this possibility was verified with molecular docking and molecular dynamics. As demonstrated with these tools, wortmannin could be bound in the extracellular vestibule at the muscarinic M3 receptor but this did not preclude binding of ACh to the M3 receptor followed by its activation. In contrast, PI828 could sterically block the passage of ACh into the allosteric site, preventing activation of the muscarinic M3 receptor.

Agonist-Induced Ca2+ Signaling in HEK-293-Derived Cells Expressing a Single IP3 Receptor Isoform.

In mammals, three genes encode IP3 receptors (IP3Rs), which are involved in agonist-induced Ca2+ signaling in cells of apparently all types. Using the CRISPR/Cas9 approach for disruption of two out of three IP3R genes in HEK-293 cells, we generated three monoclonal cell lines, IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK, with the single functional isoform, IP3R1, IP3R2, and IP3R3, respectively. All engineered cells responded to ACh with Ca2+ transients in an "all-or-nothing" manner, suggesting that each IP3R isotype was capable of mediating CICR. The sensitivity of cells to ACh strongly correlated with the affinity of IP3 binding to an IP3R isoform they expressed. Based on a mathematical model of intracellular Ca2+ signals induced by thapsigargin, a SERCA inhibitor, we developed an approach for estimating relative Ca2+ permeability of Ca2+ store and showed that all three IP3R isoforms contributed to Ca2+ leakage from ER. The relative Ca2+ permeabilities of Ca2+ 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 Ca2+ signals in ER, engineered cells were ranged by resting levels of stored Ca2+ as IP3R3-HEK ≥ IP3R1-HEK > IP3R2-HEK. The developed cell lines could be helpful for further assaying activity, regulation, and pharmacology of individual IP3R isoforms.

Taste Cells of the Type III Employ CASR to Maintain Steady Serotonin Exocytosis at Variable Ca2+ in the Extracellular Medium.

Type III taste cells are the only taste bud cells which express voltage-gated (VG) Ca2+ channels and employ Ca2+-dependent exocytosis to release neurotransmitters, particularly serotonin. The taste bud is a tightly packed cell population, wherein extracellular Ca2+ is expected to fluctuate markedly due to the electrical activity of taste cells. It is currently unclear whether the Ca2+ entry-driven synapse in type III cells could be reliable enough at unsteady extracellular Ca2. Here we assayed depolarization-induced Ca2+ signals and associated serotonin release in isolated type III cells at varied extracellular Ca2+. It turned out that the same depolarizing stimulus elicited invariant Ca2+ signals in type III cells irrespective of bath Ca2+ 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 Ca2+ dependence of intracellular Ca2+ transients produced by VG Ca2+ entry, depolarization-triggered serotonin secretion varied negligibly with bath Ca2+. The evidence implicated the extracellular Ca2+-sensing receptor in mediating the negative feedback mechanism that regulates VG Ca2+ entry and levels off serotonin release in type III cells at deviating Ca2+ in the extracellular medium.

Modeling of Ca2+ transients initiated by GPCR agonists in mesenchymal stromal cells.

The integrative study that included experimentation and mathematical modeling was carried out to analyze dynamic aspects of transient Ca2+ signaling induced by brief pulses of GPCR agonists in mesenchymal stromal cells from the human adipose tissue (AD-MSCs). The experimental findings argued for IP3/Ca2+-regulated Ca2+ release via IP3 receptors (IP3Rs) as a key mechanism mediating agonist-dependent Ca2+ transients. The consistent signaling circuit was proposed to formalize coupling of agonist binding to Ca2+ mobilization for mathematical modeling. The model properly simulated the basic phenomenology of agonist transduction in AD-MSCs, which mostly produced single Ca2+ 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 IP3 uncaging elicited similar Ca2+ transients but IP3 pulses released Ca2+ without pronounced delay. This suggested that IP3 production was rate-limiting in agonist transduction. In a subpopulation of AD-MSCs, brief agonist pulses elicited Ca2+ bursts crowned by damped oscillations. With properly adjusted parameters of IP3R inhibition by cytosolic Ca2+, the model reproduced such oscillatory Ca2+ responses as well. GEM-GECO1 and R-CEPIA1er, the genetically encoded sensors of cytosolic and reticular Ca2+, respectively, were co-expressed in HEK-293 cells that also responded to agonists in an "all-or-nothing" manner. The experimentally observed Ca2+ 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 Ca2+ signaling in AD-MSCs.

Calcium signaling mediated by aminergic GPCRs is impaired by the PI3K inhibitor LY294002 and its analog LY303511 in a PI3K-independent manner.

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 Ca2+ 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 Ca2+ 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 Ca2+ 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.

Arachidonic acid hyperpolarizes mesenchymal stromal cells from the human adipose tissue by stimulating TREK1 K+ channels.

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.

Coupling of P2Y receptors to Ca2+ mobilization in mesenchymal stromal cells from the human adipose tissue.

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 Ca2+. Transcripts for multiple purinoreceptors were detected in MSC preparations, including A1, A2A, A2B, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y13, P2Y14, P2X2, P2X4, and P2X7. Cellular responses to nucleotides were insignificantly sensitive to bath Ca2+, pointing at a minor contribution of Ca2+ entry, and were suppressed by U73122 and 2-APB, implicating the phosphoinositide cascade in coupling P2Y receptors to Ca2+ 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 Ca2+ stimulated Ca2+-induced Ca2+ release (CICR) that was mediated largely by IP3 receptors, and resultant Ca2+ transients were similar to nucleotide responses by magnitude and kinetics. A variety of findings hinted at CICR to be a universal mechanism that finalizes Ca2+ signaling initiated by agonists in MSCs. Individual MSCs responded to nucleotides in an all-or-nothing manner. Presumably just CICR provided invariant Ca2+ responses observed in MSCs at different nucleotide concentrations. The effects of isoform specific agonists and antagonists suggested that both P2Y1 and P2Y13 were obligatory for ADP responses, while P2Y4 and P2Y11 served as primary UTP and ATP receptors, respectively. Extracellular NAD+ stimulated Ca2+ signaling in each ATP-responsive MSC by involving P2Y11. The overall data indicate that extracellular nucleotides and NAD+ can serve as autocrine/paracrine factors regulating MSC functions.

Calcium-gated K+ channels of the KCa1.1- and KCa3.1-type couple intracellular Ca2+ signals to membrane hyperpolarization in mesenchymal stromal cells from the human adipose tissue.

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 Ca2+ in the MSC cytoplasm. A variety of Ca2+-gated ion channels may couple Ca2+ 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 Ca2+ transients or exhibiting spontaneous Ca2+ oscillations, Ca2+ bursts were associated with hyperpolarization mediated by Ca2+-gated K+ channels. The expression analysis revealed transcripts for KCNMA1 and KCNN4 genes encoding for Ca2+-activated K+ channels of large (KCa1.1) and intermediate (KCa3.1) conductance, respectively. Moreover, transcripts for the Ca2+-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 Ca2+ stimulated K+ currents that were inhibited with iberiotoxin. This suggested that KCa1.1 channels are invariably expressed in MSCs. In ATP- and adenosine-responsive cells, iberiotoxin and TRAM-34 diminished electrical responses, implicating both KCa1.1 and KCa3.1 channels in coupling agonist-dependent Ca2+ signals to membrane voltage. Functional tests pointed at the existence of two separate MSC subpopulations exhibiting Ca2+-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, KCa1.1 channels are likely to represent the dominant type of Ca2+-activated K+ channels in MSCs, which can serve in concert with KCa3.1 channels as effectors downstream of G-protein-coupled receptor (GPCR)-mediated Ca2+ signaling.

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 ß2-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.

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.