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.

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.

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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 in the MSC cytoplasm. A variety of Ca-gated ion channels may couple Ca 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 transients or exhibiting spontaneous Ca oscillations, Ca bursts were associated with hyperpolarization mediated by Ca-gated K channels. The expression analysis revealed transcripts for KCNMA1 and KCNN4 genes encoding for Ca-activated K channels of large (K1.1) and intermediate (K3.1) conductance, respectively. Moreover, transcripts for the Ca-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 stimulated K currents that were inhibited with iberiotoxin. This suggested that K1.1 channels are invariably expressed in MSCs. In ATP- and adenosine-responsive cells, iberiotoxin and TRAM-34 diminished electrical responses, implicating both K1.1 and K3.1 channels in coupling agonist-dependent Ca signals to membrane voltage. Functional tests pointed at the existence of two separate MSC subpopulations exhibiting Ca-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, K1.1 channels are likely to represent the dominant type of Ca-activated K channels in MSCs, which can serve in concert with K3.1 channels as effectors downstream of G-protein-coupled receptor (GPCR)-mediated Ca signaling.