Sebastien Hayoz

An IP3R3- and NPY-expressing microvillous cell mediates tissue homeostasis and regeneration in the mouse olfactory epithelium.

Calcium-dependent release of neurotrophic factors plays an important role in the maintenance of neurons, yet the release mechanisms are understudied. The inositol triphosphate (IP3) receptor is a calcium release channel that has a physiological role in cell growth, development, sensory perception, neuronal signaling and secretion. In the olfactory system, the IP3 receptor subtype 3 (IP3R3) is expressed exclusively in a microvillous cell subtype that is the predominant cell expressing neurotrophic factor neuropeptide Y (NPY). We hypothesized that IP3R3-expressing microvillous cells secrete sufficient NPY needed for both the continual maintenance of the neuronal population and for neuroregeneration following injury. We addressed this question by assessing the release of NPY and the regenerative capabilities of wild type, IP3R3+/−, and IP3R3−/− mice. Injury, simulated using extracellular ATP, induced IP3 receptor-mediated NPY release in wild-type mice. ATP-evoked NPY release was impaired in IP3R3−/− mice, suggesting that IP3R3 contributes to NPY release following injury. Under normal physiological conditions, both IP3R3−/− mice and explants from these mice had fewer progenitor cells that proliferate and differentiate into immature neurons. Although the number of mature neurons and the in vivo rate of proliferation were not altered, the proliferative response to the olfactotoxicant satratoxin G and olfactory bulb ablation injury was compromised in the olfactory epithelium of IP3R3−/− mice. The reductions in both NPY release and number of progenitor cells in IP3R3−/− mice point to a role of the IP3R3 in tissue homeostasis and neuroregeneration. Collectively, these data suggest that IP3R3 expressing microvillous cells are actively responsive to injury and promote recovery.

Mechanisms of constitutive and ATP-evoked ATP release in neonatal mouse olfactory epithelium.

BackgroundATP is an extracellular signaling molecule with many ascribed functions in sensory systems, including the olfactory epithelium. The mechanism(s) by which ATP is released in the olfactory epithelium has not been investigated. Quantitative luciferin-luciferase assays were used to monitor ATP release, and confocal imaging of the fluorescent ATP marker quinacrine was used to monitor ATP release via exocytosis in Swiss Webster mouse neonatal olfactory epithelial slices.ResultsUnder control conditions, constitutive release of ATP occurs via exocytosis, hemichannels and ABC transporters and is inhibited by vesicular fusion inhibitor Clostridium difficile toxin A and hemichannel and ABC transporter inhibitor probenecid. Constitutive ATP release is negatively regulated by the ATP breakdown product ADP through activation of P2Y receptors, likely via the cAMP/PKA pathway. In vivo studies indicate that constitutive ATP may play a role in neuronal homeostasis as inhibition of exocytosis inhibited normal proliferation in the OE. ATP-evoked ATP release is also present in mouse neonatal OE, triggered by several ionotropic P2X purinergic receptor agonists (ATP, αβMeATP and Bz-ATP) and a G protein-coupled P2Y receptor agonist (UTP). Calcium imaging of P2X2-transfected HEK293 “biosensor” cells confirmed the presence of evoked ATP release. Following purinergic receptor stimulation, ATP is released via calcium-dependent exocytosis, activated P2X1,7 receptors, activated P2X7 receptors that form a complex with pannexin channels, or ABC transporters. The ATP-evoked ATP release is inhibited by the purinergic receptor inhibitor PPADS, Clostridium difficile toxin A and two inhibitors of pannexin channels: probenecid and carbenoxolone.ConclusionsThe constitutive release of ATP might be involved in normal cell turn-over or modulation of odorant sensitivity in physiological conditions. Given the growth-promoting effects of ATP, ATP-evoked ATP release following injury could lead to progenitor cell proliferation, differentiation and regeneration. Thus, understanding mechanisms of ATP release is of paramount importance to improve our knowledge about tissue homeostasis and post-injury neuroregeneration. It will lead to development of treatments to restore loss of smell and, when transposed to the central nervous system, improve recovery following central nervous system injury.

Aging increases capacitance and spontaneous transient outward current amplitude of smooth muscle cells from murine superior epigastric arteries

Large conductance Ca(2+)-activated K(+) channels (BKCa) contribute to negative feedback regulation of smooth muscle cell (SMC) tone. However, the effects of aging on BKCa function are unclear. We tested the hypothesis that aging alters SMC BKCa function in superior epigastric arteries (SEAs) by using perforated patch recording of enzymatically isolated SMCs from 3- to 4-mo-old male C57BL/6 mice (Young) and 24- to 26-mo-old male C57BL/6 mice (Old). SMC capacitance from Young (15.7 ± 0.4 pF; n = 110) was less than Old (17.9 ± 0.5 pF; n = 104) (P < 0.05). SMCs displayed spontaneous transient outward currents (STOCs) at membrane potentials more positive than -30 mV; depolarization increased STOC amplitude and frequency (P < 0.05; n = 19-24). STOC frequency in Young (2.2 ± 0.6 Hz) was less than Old (4.2 ± 0.7 Hz) at -10 mV (P < 0.05, n = 27-30), with no difference in amplitude (1.0 ± 0.1 vs. 0.9 ± 0.1 pA/pF, respectively). At +30 mV, STOC amplitude in Young (3.2 ± 0.3 pA/pF) was less than Old (5.0 ± 0.5 pA/pF; P < 0.05, n = 61-67) with no difference in frequency (3.9 ± 0.4 vs. 3.2 ± 0.3 Hz, respectively). BKCa blockers (1 μM paxilline, 100 nM iberiotoxin, 1 mM tetraethylammonium) or a ryanodine receptor antagonist (100 μM tetracaine) inhibited STOCs (n ≥ 6; P < 0.05 each). Western blots revealed increased expression of BKCa α-subunit protein in Old. Pressure myography revealed no effect of age on SEA maximal diameter, myogenic tone, or paxilline-induced constriction (n = 10-12; P > 0.05). Enhanced functional expression of SMC BKCa-dependent STOCs in Old may represent an adaptation of resistance arteries to maintain functional integrity.

Increased amplitude of inward rectifier K+ currents with advanced age in smooth muscle cells of murine superior epigastric arteries

Inward rectifier K+ channels (KIR) may contribute to skeletal muscle blood flow regulation and adapt to advanced age. Using mouse abdominal wall superior epigastric arteries (SEAs) from either young (3-6 mo) or old (24-26 mo) male C57BL/6 mice, we investigated whether SEA smooth muscle cells (SMCs) express functional KIR channels and how aging may affect KIR function. Freshly dissected SEAs were either enzymatically dissociated to isolate SMCs for electrophysiological recording (perforated patch) and mRNA expression or used intact for pressure myography. With 5 mM extracellular K+ concentration ([K+]o), exposure of SMCs to the KIR blocker Ba2+ (100 μM) had no significant effect (P > 0.05) on whole cell currents elicited by membrane potentials spanning -120 to -30 mV. Raising [K+]o to 15 mM activated Ba2+-sensitive KIR currents between -120 and -30 mV, which were greater in SMCs from old mice than in SMCs from young mice (P < 0.05). Pressure myography of SEAs revealed that while aging decreased maximum vessel diameter by ~8% (P < 0.05), it had no significant effect on resting diameter, myogenic tone, dilation to 15 mM [K+]o, Ba2+-induced constriction in 5 mM [K+]o, or constriction induced by 15 mM [K+]o in the presence of Ba2+ (P > 0.05). Quantitative RT-PCR revealed SMC expression of KIR2.1 and KIR2.2 mRNA that was not affected by age. Barium-induced constriction of SEAs from young and old mice suggests an integral role for KIR in regulating resting membrane potential and vasomotor tone. Increased functional expression of KIR channels during advanced age may compensate for other age-related changes in SEA function.NEW & NOTEWORTHY Ion channels are integral to blood flow regulation. We found greater functional expression of inward rectifying K+ channels in smooth muscle cells of resistance arteries of mouse skeletal muscle with advanced age. This adaptation to aging may contribute to the maintenance of vasomotor tone and blood flow regulation during exercise.

Investigating cyclic nucleotide and cyclic dinucleotide binding to HCN channels by surface plasmon resonance

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels control cardiac and neuronal rhythmicity. HCN channels contain cyclic nucleotide-binding domain (CNBD) in their C-terminal region linked to the pore-forming transmembrane segment with a C-linker. The C-linker couples the conformational changes caused by the direct binding of cyclic nucleotides to the HCN pore opening. Recently, cyclic dinucleotides were shown to antagonize the effect of cyclic nucleotides in HCN4 but not in HCN2 channels. Based on the structural analysis and mutational studies it has been proposed that cyclic dinucleotides affect HCN4 channels by binding to the C-linker pocket (CLP). Here, we first show that surface plasmon resonance (SPR) can be used to accurately measure cyclic nucleotide binding affinity to the C-linker/CNBD of HCN2 and HCN4 channels. We then used SPR to investigate cyclic dinucleotide binding in HCN channels. To our surprise, we detected no binding of cyclic dinucleotides to the isolated monomeric C-linker/CNBDs of HCN4 channels with SPR. The binding of cyclic dinucleotides was further examined with isothermal calorimetry (ITC), which indicated no binding of cyclic dinucleotides to both monomeric and tetrameric C-linker/CNBDs of HCN4 channels. Taken together, our results suggest that interaction of the C-linker/CNBD with other parts of the channel is necessary for cyclic-dinucleotide binding in HCN4 channels.

Protein Kinase A and C Regulate Leak Potassium Currents in Freshly Isolated Vascular Myocytes from the Aorta

We tested the hypothesis that protein kinase A (PKA) inhibits K2P currents activated by protein kinase C (PKC) in freshly isolated aortic myocytes. PDBu, the PKC agonist, applied extracellularly, increased the amplitude of the K2P currents in the presence of the “cocktail” of K+ channel blockers. Gö 6976 significantly reduced the increase of the K2P currents by PDBu suggesting the involvement of either α or β isoenzymes of PKC. We found that forskolin, or membrane permeable cAMP, did not inhibit K2P currents activated by the PKC. However, when PKA agonists were added prior to PDBu, they produced a strong decrease in the K2P current amplitudes activated by PKC. Inhibition of PDBu-elicited K2P currents by cAMP agonists was not prevented by the treatment of vascular smooth muscle cells with PKA antagonists (H-89 and Rp-cAMPs). Zn2+ and Hg2+ inhibited K2P currents in one population of cells, produced biphasic responses in another population, and increased the amplitude of the PDBu-elicited K+ currents in a third population of myocytes, suggesting expression of several K2P channel types. We found that cAMP agonists inhibited biphasic responses and increase of amplitude of the PDBu-elicited K2P currents produced by Zn2+ and Hg2. 6-Bnz-cAMp produced a significantly altered pH sensitivity of PDBu-elicited K2P-currents, suggesting the inhibition of alkaline-activated K2P-currents. These results indicate that 6-Bnz-cAMP and other cAMP analogs may inhibit K2P currents through a PKA-independent mechanism. cAMP analogs may interact with unidentified proteins involved in K2P channel regulation. This novel cellular mechanism could provide insights into the interplay between PKC and PKA pathways that regulate vascular tone.