STIM1-dependent store-operated calcium entry mediates sex differences in macrophage chemotaxis and monocyte recruitment.

Infiltration of monocyte-derived cells to sites of infection and injury is greater in males than in females, due in part, to increased chemotaxis, the process of directed cell movement toward a chemical signal. The mechanisms governing sexual dimorphism in chemotaxis are not known. We hypothesized a role for the store-operated calcium entry (SOCE) pathway in regulating chemotaxis by modulating leading and trailing edge membrane dynamics. We measured the chemotactic response of bone marrow-derived macrophages migrating toward complement component 5a (C5a). Chemotactic ability was dependent on sex and inflammatory phenotype (M0, M1, and M2), and correlated with SOCE. Notably, females exhibited a significantly lower magnitude of SOCE than males. When we knocked out the SOCE gene, stromal interaction molecule 1 (STIM1), it eliminated SOCE and equalized chemotaxis across both sexes. Analysis of membrane dynamics at the leading and trailing edges showed that STIM1 influences chemotaxis by facilitating retraction of the trailing edge. Using BTP2 to pharmacologically inhibit SOCE mirrored the effects of STIM1 knockout, demonstrating a central role of STIM/Orai-mediated calcium signaling. Importantly, by monitoring the recruitment of adoptively transferred monocytes in an in vivo model of peritonitis, we show that increased infiltration of male monocytes during infection is dependent on STIM1. These data support a model in which STIM1-dependent SOCE is necessary and sufficient for mediating the sex difference in monocyte recruitment and macrophage chemotactic ability by regulating trailing edge dynamics.

TASK inhibition by mild acidosis increases Ca2+ oscillations to mediate pH sensing in rat carotid body chemoreceptor cells.

Severe levels of acidosis (pH < 6.8) have been shown to cause a sustained rise in cytosolic Ca2+ concentration in carotid body Type 1 (glomus) cells. To understand how physiologically relevant levels of acidosis regulate Ca2+ signaling in glomus cells, we studied the effects of small changes in extracellular pH (pHo) on the kinetics of Ca2+ oscillations. A decrease in pHo from 7.4 to 7.3 (designated mild) and 7.2 (designated moderate) acidosis produced significant increases in the frequency and amplitude of Ca2+ oscillations. These effects of acidosis on Ca2+ oscillations were not blocked by NS383 and amiloride [acid-sensing ion channel (ASIC) inhibitors]. Mild and moderate levels of acidosis, however, caused a small but significant inhibition of two-pore domain acid-sensing K+ channels (TASK) (TASK-1- and TASK-3-like channels) and depolarized the cell by 6-13 mV. Acidosis-induced increase in Ca2+ oscillations was inhibited by nifedipine (1 µM; L-type Cav inhibitor) and by TTA-P2 (20 µM; T-type Cav inhibitor). Mild inhibition of TASK activity by N-[(2,4-difluorophenyl)methyl]-2'-[[[2-(4methoxyphenyl)acetyl]amino]methyl][1,1'-biphenyl]-2-carboxamide (A1899) (0.3 µM) and 1-[1-[6-[[1,1'-biphenyl]-4-ylcarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine-4-yl]-4-piperidinyl]-1-butanon (PK-THPP) (0.1 µM) increased Ca2+ oscillation frequency to levels similar to those observed with mild-moderate acidosis. Mild acidosis (pHo 7.3) and mild hypoxia (∼5%O2) produced similar levels of changes in the kinetics of Ca2+ oscillations. Block of tetraethylammonium (TEA)-sensitive Kv channels did not affect acid-induced increase in Ca2+ oscillations. Our study shows that mild and moderate levels of acidosis increase the frequency and amplitude of Ca2+ oscillations primarily by inhibition of TASK without involving ASICs, and suggests a major role of TASK for signal transduction in response to a physiological change in pHo.

Ca2+ oscillations in rat carotid body type 1 cells in normoxia and hypoxia.

We studied the mechanisms by which carotid body glomus (type 1) cells produce spontaneous Ca2+ oscillations in normoxia and hypoxia. In cells perfused with normoxic solution at 37°C, we observed relatively uniform, low-frequency Ca2+ oscillations in >60% of cells, with each cell showing its own intrinsic frequency and amplitude. The mean frequency and amplitude of Ca2+ oscillations were 0.6 ± 0.1 Hz and 180 ± 42 nM, respectively. The duration of each Ca2+ oscillation ranged from 14 to 26 s (mean of ∼20 s). Inhibition of inositol (1,4,5)-trisphosphate receptor and store-operated Ca2+ entry (SOCE) using 2-APB abolished Ca2+ oscillations. Inhibition of endoplasmic reticulum Ca2+-ATPase (SERCA) using thapsigargin abolished Ca2+ oscillations. ML-9, an inhibitor of STIM1 translocation, also strongly reduced Ca2+ oscillations. Inhibitors of L- and T-type Ca2+ channels (Cav; verapamil>nifedipine>TTA-P2) markedly reduced the frequency of Ca2+ oscillations. Thus, Ca2+ oscillations observed in normoxia were caused by cyclical Ca2+ fluxes at the ER, which was supported by Ca2+ influx via Ca2+ channels. Hypoxia (2-5% O2) increased the frequency and amplitude of Ca2+ oscillations, and Cav inhibitors (verapamil>nifedipine>>TTA-P2) reduced these effects of hypoxia. Our study shows that Ca2+ oscillations represent the basic Ca2+ signaling mechanism in normoxia and hypoxia in CB glomus cells.

TASK-1 (K2P3) and TASK-3 (K2P9) in Rabbit Carotid Body Glomus Cells.

Glomus cells isolated from rabbit and rat/mouse carotid bodies have been used for many years to study the role of ion channels in hypoxia sensing. Studies show that hypoxia inhibits the inactivating K+ channels (Kv4) in rabbits, but inhibits TASK in rats/mice to elicit the hypoxic response. Because the role of TASK in rabbit glomus cells is not known, we isolated glomus cells from rabbits and studied the expression of TASK mRNA in the whole carotid body (CB), changes in [Ca2+]i and TASK activity. RT-PCR showed that rabbit CB expressed mRNA for TASK-3 and several Kv (Kv2.1, Kv3.1 and Kv3.3). In rabbit glomus cells in which 20 mM KClo elevated [Ca2+], anoxia also elicited a strong rise in [Ca2+]. In cell-attached patches with 140 mM KCl in the pipette, basal openings of ion channels with single-channel conductance levels of 16-pS, 34-pS, and 42-pS were present. TREK-like channels were also observed. In inside-out patches with high [Ca2+]i, BK was activated. The 42-pS channel opened spontaneously and briefly. The 16-pS and 34-pS channels showed properties similar to those of TASK-1 and TASK-3, respectively. TASK activity in cell-attached patches was lower than that in rat glomus cells under identical recording conditions. Hypoxia (~0.5%O2) reduced TASK activity by ~52% and depolarized the cells by ~30 mV. Our results show that the O2-sensitive TASK contributes to the hypoxic response in rabbit glomus cells.

The transcription factor GLI1 modulates the inflammatory response during pancreatic tissue remodeling.

Pancreatic cancer, one of the deadliest human malignancies, is almost uniformly associated with a mutant, constitutively active form of the oncogene Kras. Studies in genetically engineered mouse models have defined a requirement for oncogenic KRAS in both the formation of pancreatic intraepithelial neoplasias, the most common precursor lesions to pancreatic cancer, and in the maintenance and progression of these lesions. Previous work using an inducible model allowing tissue-specific and reversible expression of oncogenic Kras in the pancreas indicates that inactivation of this GTPase at the pancreatic intraepithelial neoplasia stage promotes pancreatic tissue repair. Here, we extend these findings to identify GLI1, a transcriptional effector of the Hedgehog pathway, as a central player in pancreatic tissue repair upon Kras inactivation. Deletion of a single allele of Gli1 results in improper stromal remodeling and perdurance of the inflammatory infiltrate characteristic of pancreatic tumorigenesis. Strikingly, this partial loss of Gli1 affects activated fibroblasts in the pancreas and the recruitment of immune cells that are vital for tissue recovery. Analysis of the mechanism using expression and chromatin immunoprecipitation assays identified a subset of cytokines, including IL-6, mIL-8, Mcp-1, and M-csf (Csf1), as direct GLI1 target genes potentially mediating this phenomenon. Finally, we demonstrate that canonical Hedgehog signaling, a known regulator of Gli1 activity, is required for pancreas recovery. Collectively, these data delineate a new pathway controlling tissue repair and highlight the importance of GLI1 in regulation of the pancreatic microenvironment during this cellular process.

Increase in cytosolic Ca2+ produced by hypoxia and other depolarizing stimuli activates a non-selective cation channel in chemoreceptor cells of rat carotid body.

The current model of O2 sensing by carotid body chemoreceptor (glomus) cells is that hypoxia inhibits the outward K(+) current and causes cell depolarization, Ca(2+) influx via voltage-dependent Ca(2+) channels and a rise in intracellular [Ca(2+)] ([Ca(2+)]i). Here we show that hypoxia (<5% O2), in addition to inhibiting the two-pore domain K(+) channels TASK-1/3 (TASK), indirectly activates an ∼20 pS channel in isolated glomus cells. The 20 pS channel was permeable to K(+), Na(+) and Cs(+) but not to Cl(-) or Ca(2+). The 20 pS channel was not sensitive to voltage. Inhibition of TASK by external acid, depolarization of glomus cells with high external KCl (20 mm) or opening of the Ca(2+) channel with FPL64176 activated the 20 pS channel when 1 mm Ca(2+) was present in the external solution. Ca(2+) (10 µm) applied to the cytosolic side of inside-out patches activated the 20 pS channel. The threshold [Ca(2+)]i for activation of the 20 pS channel in cell-attached patches was ∼200 nm. The reversal potential of the 20 pS channel was estimated to be -28 mV. Our results reveal a sequential mechanism in which hypoxia (<5% O2) first inhibits the K(+) conductance and then activates a Na(+)-permeable, non-selective cation channel via depolarization-induced rise in [Ca(2+)]i. Our results suggest that inhibition of K(+) efflux and stimulation of Na(+) influx both contribute to the depolarization of glomus cells during moderate to severe hypoxia.

THIK-1 (K2P13.1) is a small-conductance background K(+) channel in rat trigeminal ganglion neurons.

The goal of this study was to determine the molecular identity of a small-conductance (~5-pS) background K(+) channel expressed in trigeminal ganglion (TG) neurons. We tested the hypothesis that the 5-pS channel is a K2P channel by comparing the pharmacological and single-channel properties of THIK-1 expressed in HEK293 cells. As reported earlier, whole-cell THIK-1 current was inhibited by halothane and activated by arachidonic acid. Among 25 additional modulators tested, bupivacaine (100 µM), quinidine (50 µM) and Ba(2+) (3 mM) and cold (10 °C) were most effective inhibitors of THIK-1 current (>50 % inhibition). In cell-attached patches with high KCl in the pipette and bath solutions, THIK-1 produced a small-conductance (~5 pS) channel with a weak inwardly rectifying current-voltage relationship. Halothane, bupivacaine and cold inhibited the single-channel activities of both THIK-1 and the 5-pS channel in TG neurons, whereas arachidonic acid augmented them. THIK-1 expressed in HEK293 cells and the 5-pS channels in TG neurons were insensitive to hypoxia. Reverse transcriptase-PCR, Western blot and immunocytochemical analyses suggested that THIK-1 mRNA and protein were expressed in TG neurons. These results show that THIK-1 is functionally expressed in TG neurons and contributes to the background K(+) conductance.

Voltage- and receptor-mediated activation of a non-selective cation channel in rat carotid body glomus cells.

A recent study showed that hypoxia activates a Ca2+-sensitive, Na+-permeable non-selective cation channel (NSC) in carotid body glomus cells. We studied the effects of mitochondrial inhibitors that increase Ca2+ influx via Ca2+ channel (Cav), and receptor agonists that release Ca2+ from endoplasmic reticulum (ER) on NSC. Mitochondrial inhibitors (NaCN, FCCP, H2S, NO) elevated [Ca2+]i and activated NSC. Angiotensin II and acetylcholine that elevate [Ca2+]i via the Gq-IP3 pathway activated NSC. However, endothelin-1 (Gq) and 5-HT (Gq) showed little or no effect on [Ca2+]i and did not activate NSC. Adenosine (Gs) caused a weak rise in [Ca2+]i but did not activate NSC. Dopamine (Gs) and γ-aminobytyric acid (Gi) were ineffective in raising [Ca2+]i and failed to activate NSC. Store-operated Ca2+ entry (SOCE) produced by depletion of Ca2+ stores with cyclopiazonic acid activated NSC. Our results show that Ca2+ entry via Cav, ER Ca2+ release and SOCE can activate NSC. Thus, NSC contributes to both voltage- and receptor-mediated excitation of glomus cells.

Role of cystathionine-γ-lyase in hypoxia-induced changes in TASK activity, intracellular [Ca2+] and ventilation in mice.

Cystathionine-γ-lyase (CSE) is a multifunctional enzyme, and hydrogen sulfide (H2S) is one of its products. CSE and H2S have recently been proposed to be critical signaling molecules in hypoxia-induced excitation of carotid body (CB) glomus cells and the chemosensory response. Because the role of H2S in arterial chemoreception is still debated, we further examined the role of CSE by studying the effects of hypoxia on TASK K+ channel activity, cell depolarization, [Ca2+]i and ventilation using CSE+/+ and CSE-/- mice. As predicted, hypoxia reduced TASK activity and depolarized glomus cells isolated from CSE+/+ mice. These effects of hypoxia were not significantly altered in glomus cells from CSE-/- mice. Basal [Ca2+]i and hypoxia-induced elevation of [Ca2+] were also not significantly different in glomus cells from CSE+/+ and CSE-/- mice. In whole-body plethysmography, hypoxia (10%O2) increased minute ventilation in both CSE+/+ and CSE-/- mice equally well, and no significant differences were found in either males or females when adjusted by body weight. Together, these results show that deletion of the CSE gene has no effects on hypoxia-induced changes in TASK, cell depolarization, [Ca2+]i and ventilation, and therefore do not support the idea that CSE/H2S signaling is important for CB chemoreceptor activity in mice.

Species differences and molecular determinant of TRPA1 cold sensitivity.

TRPA1 is an ion channel and has been proposed as a thermosensor across species. In invertebrate and ancestral vertebrates such as fly, mosquito, frog, lizard and snakes, TRPA1 serves as a heat receptor, a sensory input utilized for heat avoidance or infrared detection. However, in mammals, whether TRPA1 is a receptor for noxious cold is highly controversial, as channel activation by cold was observed by some groups but disputed by others. Here we attribute the discrepancy to species differences. We show that cold activates rat and mouse TRPA1 but not human or rhesus monkey TRPA1. At the molecular level, a single residue within the S5 transmembrane domain (G878 in rodent but V875 in primate) accounts for the observed difference in cold sensitivity. This residue difference also underlies the species-specific effects of menthol. Together, our findings identify the species-specific cold activation of TRPA1 and reveal a molecular determinant of cold-sensitive gating.