Calcium Signaling Initiated by Agonists in Mesenchymal Stromal Cells from the Human Adipose Tissue

Mesenchymal stromal cells (MSCs) from different sources represent a heterogeneous population of proliferating non-differentiated cells that contain multipotent stem cells capable of originating a variety of mesenchymal cell lineages. By using Ca2+ imaging and the Ca2+ dye Fluo-4, we studied MSCs from the human adipose tissue and examined Ca2+ signaling initiated by a variety of GPCR ligands, focusing primarily on adrenergic and purinergic agonists. Being characterized by a relative change of Fluo-4 fluorescence, agonist-induced Ca2+ responses were generated in an “all-or-nothing” fashion. Specifically, at relatively low doses, agonists elicited undetectable responses but initiated quite similar Ca2+ transients at all concentrations above the threshold. The inhibitory analysis and Ca2+/IP3 uncaging pointed at the phosphoinositide cascade as a pivotal pathway responsible for agonist transduction and implicated Ca2+-induced Ca2+ release (CICR) in shaping agonists-dependent Ca2+ signals. Altogether, our data suggest that agonist transduction in MSCs includes two fundamentally different stages: an agonist initially triggers a local, gradual, and relatively small Ca2+ signal, which next stimulates CICR to accomplish transduction with a large and global Ca2+ transient. By involving the trigger-like mechanism CICR, a cell is capable of generating Ca2+ responses of virtually universal shape and magnitude at different agonist concentrations above the threshold

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

Chemical Synapses without Synaptic Vesicles: Purinergic Neurotransmission through a CALHM1 Channel-mitochondrial Signaling Complex

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 Ca2+ 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

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 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, 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