Citations

Airway inflammation leads to increased intracellular Ca 2+ ([Ca 2+ ] i ) in airway smooth muscle (ASM). There is now growing evidence that the bidrectional plasma membrane Na + /Ca 2+ exchange (NCX) may contribute to ASM [Ca 2+ ] i regulation. However, the roles of NCX‐mediated Ca 2+ influx vs. efflux, or the role of NCX in airway inflammation are largely unknown. In the present study, we examined NCX expression and function in human ASM exposed to the pro‐inflammatory cytokines TNFα and IL‐13. Western blot analysis of human ASM tissue showed significant expression of the NCX1 isoform, with substantial increase in expression following overnight cytokine exposure. In fura‐2 loaded human ASM cells, NCX‐mediated Ca 2+ influx as well as efflux (measured as slopes of changes in [Ca 2+ ] i ) was elicited using techniques previously described for cardiac muscle. Contribution of NCX was verified by measuring [Na + ] i using the fluorescent Na + indicator SBFI. NCX‐mediated influx was verified by demonstrating prevention of rising [Ca 2+ ] i or falling [Na + ] i in the presence of the NCX inhibitor KBR7943. In second set of studies, NCX siRNA transfection of human ASM substantially blunted efflux and influx mode of NCX (siRNA effect verified by Western blot). Exposure to TNF‐α as well as IL‐13 significantly enhanced influx mode of NCX. Although both cytokines also increased efflux mode of NCX, these effects were relatively smaller. These data suggest that NCX may be one mechanism contributing to inflammation‐induced enhancement of [Ca 2+ ] i. Supported by NIH grants HL74309, RR024150, and the Flight Attendants Medical Research Institute (FAMRI). Additional funding from the Department of Anesthesiology, Mayo Clinic.

In airway smooth muscle (ASM), the state of sarcoplasmic reticulum (SR) Ca 2+ stores is an important determinant of [Ca 2+ ] i response to agonist. We have previously shown that acetylcholine (ACh) induces sustained [Ca 2+ ] i oscillations representing repetitive SR Ca 2+ release and reuptake. Thus, SR refilling is a key component in refilling of SR stores. We hypothesized that, like cardiac and vascular smooth muscle, ASM SR refilling is regulated by calmodulin (CaM)‐CaM kinase (CaMKII) dependent phosphorylation of phospholamban (PLB). In ASM cells from fresh porcine trachea, effects of W7 (CaM antagonist) and KN‐93/KN‐62 (CaMKII antagonists) on [Ca 2+ ] i responses to ACh were measured using fluorescence microscopy. Separately, SR‐enriched membranes were isolated to monitor ATP‐energized Ca 2+ uptake and in vitro phosphorylation of PLB in the presence of CaMKII inhibitors. KN‐93 significantly slowed decay of [Ca 2+ ] i responses, and slowed or inhibited ongoing [Ca 2+ ] i oscillations, with visible slowing of fall‐times of individual oscillations. Pre‐exposure to W7 or KN‐62 did not prevent initiation of oscillations. In SR‐enriched membranes, addition of KN‐93/KN‐62 resulted in significantly slower Ca 2+ uptake, accompanied by a matching decrement in the in vitro phosphorylation of Thr 17 ‐PLB, indicating the influence of CaM‐CaMKII on this protein. These data suggest that agonist‐induced [Ca 2+ ] i regulation in ASM involves the CaM‐CaMKII pathway which indirectly influences Ca 2+ release and reuptake, likely through PLB. Supported by NIH grant HL74309 (GCS)

Asthma is a disease characterized by airway hyperresponsiveness and inflammation. One‐third of pregnant women with pre‐existing disease experience a decrease in symptoms during the third trimester, when estrogen levels are highest. Previous studies have shown that estrogens relax vascular smooth muscle, however, little is known about estrogen effects on airway smooth muscle (ASM). We tested the hypothesis that estrogen modulates human ASM contractility. Western blot analysis and immunohistochemical studies showed the presence of both full‐length and truncated isoforms of ERα and β. Furthermore, ER isoforms were localized to caveolae. Caveolin‐1 siRNA downregulated ERα isoform in human ASM. Exposure to the pro‐inflammatory cytokine TNFα substantially increased ERα expression in ASM. In fura‐2 loaded human ASM cells, acute exposure to biologically‐active 17β‐estradiol decreased the intracellular Ca 2+ response to contractile agonists such as histamine and bradykinin; this effect was blunted by the ER antagonist ICI 182, 780. Overall, these data suggest that estrogens may have a bronchodilatory effect. Accordingly, increased ER expression with airway inflammation may actually be protective during pregnancy in that bronchodilation resulting from higher estrogen levels (acting via ERs) may alleviate the increased ASM contractility of asthma. Supported by the Mayo Graduate School and NIH grant UL1RR024150‐01(Mayo Clinic CTSA awards to YSP and CMP)

Store operated calcium entry (SOCE) is important for regulation of intracellular Ca 2+ ([Ca 2+ ] i ) in neurons. Recent studies in other cell type suggest that stromal interaction molecule (STIM1) triggers SOCE. We examined the role of STIM1 in the sensitivity of SOCE to temperature, pH and glutamate in differentiated, fura‐2 loaded rat pheochromocytoma (PC12) cells, a model for neuronal cells. In STIM1 siRNA‐transfected vs. control cells, SOCE was triggered by introduction of extracellular Ca 2+ following endoplasmic reticulum depletion via cyclopiazonic acid in zero Ca 2+ . Cells were washed and exposed to altered pH (6.0–8.0), altered temperature (34–40 o C) or different concentrations of glutamate. In controls, SOCE was progressively inhibited by decreased pH or temperature, while alkalosis and increased temperature stimulated Ca 2+ influx. Increasing glutamate concentrations progressively inhibited SOCE. siRNA inhibition of STIM1 substantially blunted both temperature sensitivity (especially increased temperature) and pH sensitivity (especially acidic pH) of SOCE. These data show that SOCE in PC12 cells is susceptible to acidosis, temperature, and agonist stimulation: factors that can adversely affect neuronal function under pathological conditions. In this regard, the SOCE triggering protein STIM1 may play a role in such susceptibility. This work has been supported by NIH grants UL1RR024150‐01(CR to CMP, CR20 and Early Career Development Award to YSP), and the Department of Anesthesiology (CMP, YSP), Mayo Clinic College of Medicine, Rochester, MN

The mechanisms underlying pulmonary hypertension are still being explored. Recent studies suggest a role for caveolae (pit‐like plasma membrane structures), although it is not entirely clear whether they enhance or blunt pulmonary hypertension. The proteins caveolins ‐1, and ‐2, and polymerase I and transcription release factor (PTRF) are essential to forming caveolae. In other tissues, growth factors called neurotrophins (e.g. brain‐derived neurotrophic factor, BDNF) are thought to affect cell proliferation and structure. This study investigated caveolins and neurotrophins in human pulmonary artery. Western analysis of artery from a patient with primary pulmonary hypertension showed decreased levels of caveolin‐1, caveolin‐2, PTRF and RhoA. Exposure of human pulmonary artery to 1 nM BDNF increased protein levels of caveolin‐1 and PTRF. Isolated pulmonary artery smooth muscle cells exposed to BDNF showed decreased [Ca 2+ ] i response to bradykinin (10 nM) and histamine (10 μM), but no change in caveolin or PTRF levels. These data suggest that decreased caveolin levels may contribute to increased pulmonary vascular contractility, while neurotrophins (potentially by increasing caveolin levels) may alleviate excessive constriction. Further studies are required to determine whether neurotrophins affect caveolae in endothelial cells or smooth muscle. Supported by FAMRI, NIH grant UL1RR024150, and the Department of Anesthesiology, Mayo Clinic.

There is increasing interest in the role of hydrogen sulfide (H 2 S) as a gasotransmitter similar to nitric oxide and carbon monoxide. In this study, we examined the role of H 2 S in airway smooth muscle (ASM) inflammation. H 2 S is produced by the enzymes cystathionine beta‐synthase (CBS) and cystathionine gamma‐lysase (CTH). Both of these enzymes were detected in human ASM by Western blot analysis. In tissues exposed to the pro‐inflammatory cytokine tumor necrosis factor‐a (TNFα) both CTH and CBS expression were upregulated. In ASM strips contracted with 10 µM ACh, only exposure to 1 mM of the H 2 S donor NaHS induced relaxation. Human ASM cells loaded with fura‐2 demonstrated a dose‐dependent decrease in intracellular calcium concentration ([Ca 2+ ] i ) to NaHS (50‐250 μM), in the presence of either 1 μM ACh or 10 μM histamine. However, sodium sulfide (Na 2 S, 100 µM up to 1 mM) showed no effect on [Ca 2+ ] i . Exposure to 60 mM KCl in the presence of either NaHS or Na 2 S (50 μM‐3 mM) did not affect [Ca 2+ ] i . Relaxant effects of NaHS or Na 2 S on ACh‐induced [Ca 2+ ] i elevation were absent when experiments were conducted in zero extracellular Ca 2+ . These results suggest that H 2 S relaxes ASM and reduces [Ca 2+ ] i by affecting Ca 2+ influx, potentially via voltage‐gated channels, but does not affect sarcoplasmic reticulum Ca 2+ release. The relevance of endogenous H 2 S generation in ASM, and its role in infalmmation needs to be explored further. Supported by the Flight Attendants Medical Research Institute (FAMRI), NIH grant HL74309, and the Department of Anesthesiology, Mayo Clinic.

Sarcoplasmic reticulum (SR) Ca 2+ release and reuptake are important mechanisms in airway smooth muscle (ASM) intracellular Ca 2+ [Ca 2+ ] i regulation. In other tissues, there is increasing evidence for a role of mitochondria in [Ca 2+ ] i buffering, especially following SR Ca 2+ release. Mitochondrial buffering may also influence Ca 2+ influx. The purpose of the present study was to examine the role of mitochondria in [Ca 2+ ] i buffering in human ASM. Cells were loaded with fura‐2 and exposed to 10 µM histamine in "0" Ca 2+ HBSS containing 1mM lanthanum and 1µM nifedipine (to block Ca 2+ influx/efflux) and in the additional presence of 1µM RU360 (mitochondrial Ca 2+ uniporter inhibitor) or 1µM CGP37157 (CGP; mitochondrial Na + /Ca 2+ exchange inhibitor). Compared to controls, peak [Ca 2+ ] i levels were increased by 30% in the presence of CGP, while RU360 had not effect. Both CGP and RU360 significantly increased store‐operated Ca 2+ entry. In cells co‐loaded with fura‐2 and rhod‐2 (mitochondrial Ca 2+ indicator), exposure to 5 µM caffeine showed instantaneous SR Ca 2+ release followed by an increase in [Ca 2+ ] mito . Exposure to 1 µM ACh resulted in ACh‐induced [Ca 2+ ] i oscillations that were reflected by mitochondria (but with a time delay). In ASM cells co‐labeled for mitochondria (MitoTracker) and SR (fluorescent ryanodine) migration of mitochondria towards SR was observed upon exposure to ACh or cyclopiazonic acid. This migration was completely inhibited by phalloidin (actin inhibitor). These data demonstrate an important role of mitochondria in [Ca 2+ ] i buffering in ASM. Supported by NIH grant RR024150, and the Flight Attendants Medical Research Institute (FAMRI). Additional funding from the Department of Anesthesiology, Mayo Clinic.

Intracellular Ca 2+ ([Ca 2+ ] i ) levels are increased in inflamed airway smooth muscle (ASM). Sarcoplasmic reticulum (SR) Ca 2+ release and reuptake are key components of ASM [Ca 2+ ] i regulation. Ca 2+ reuptake occurs via the ATPase SERCA. We hypothesized that inflammation disrupts SERCA function, contributing to increased [Ca 2+ ] i . We examined SERCA expression and regulation in human ASM exposed to pro‐inflammatory cytokines TNFα and IL‐13. Western blot analysis showed significantly decreased SERCA expression following overnight cytokine exposure. In other tissues, SERCA is regulated by the inhibitory protein phospholamban (PLB). We found that human ASM does not express PLB protein (although mRNA is detectable). Cytokine exposure did not induce PLB expression, raising the issue of how SERCA is regulated. We then found that direct SERCA phosphorylation (potentially via CaMKII) occurs in human ASM. In fura‐2 loaded human ASM cells, we examined [Ca 2+ ] i responses to ACh or bradykinin (zero extracellular Ca 2+ ) and found significant reduction in rates of fall of [Ca 2+ ] i transients in cells exposed to TNF‐α or IL‐13. We further found that CaMKII antagonist KN‐93 significantly slowed the fall of [Ca 2+ ] i transients in both controls and cytokine‐exposed human ASM cells. These data suggest that a) SERCA in human ASM is regulated by mechanisms such as CaMKII; and b) airway inflammation decreases SR refilling by blunting SERCA expression and function. Supported by NIH grants HL74309, the Flight Attendants Medical Research Institute (FAMRI), and the Department of Anesthesiology, Mayo Clinic.

Ovarian cancer is the eighth most common cancer and the fifth leading cause of cancer death among US women. Hepatoma‐derived growth factor (HDGF) is a heparin binding, secreted mitogen with prognostic value in multiple cancers. We previously reported that HDGF is overexpressed in ovarian cancer. Surprisingly, the mechanisms by which HDGF acts in any cell type remains to be fully elucidated. We hypothesized that overexpression of HDGF in ovarian cancer cells is functionally relevant and that HDGF confers pro‐survival and proliferative properties, promoting cancer growth. To elucidate intracellular and extracellular signaling mechanisms, we examined the effect of HDGF knockdown via siRNA, overexpression of HDGF and stimulation by full‐length recombinant protein. Immunofluorescence image analysis showed nuclear localization of HDGF in OV202 cell line. Additionally, HDGF was minimally secreted but passively released in greater amounts by necrotic cells. Extracellular HDGF stimulated phosphorylation of ERK and p38. In contrast, modulating intracellular HDGF did not affect MAPK pathways. In fact, intracellular HDGF functioned in a distinct manner by regulating cellular metabolism. This suggests that HDGF acts in ovarian cancer cells via two distinct mechanisms, both of which could be exploited for ovarian cancer therapy. Supported by the Mayo Graduate School (Giri) and the Department of Anesthesiology, Mayo Clinic (Prakash).

Introduction: Children with low Vitamin D levels are at increased risk of developing asthma. In the present study, we tested the hypothesis that the Vitamin D receptor (VDR) agonist, calcitriol, will reduce tumor necrosis factor‐α (TNF‐α)‐induced expression of chemokines in human fetal airway smooth muscle cells (fASM). Methods and Results Human fASM cells were dissociated from canalicular stage (18‐22 weeks gestation) lung tissue (de‐identified samples, in vitro use only and considered exempt by Mayo Institutional Review Board). Fetal ASM cells were pre‐treated with vehicle (0.001% DMSO) or 100 nM calcitriol for 1 h, and then exposed to 10 ng/mL TNFα for 6 or 24 h. Expression of CCL5 and CCL11 were measured by quantitative real time PCR and ELISA. Cells were transfected with VDR siRNA. Treatment with TNFα for 6 h increased mRNA expression and secretion of CCL5 and CCL11. While pre‐treatment with calcitriol reduced CCL5 and CCL11 expression in the presence of TNFα. Knockdown of the VDR expression demonstrated that calcitriol's effects are mediated through VDR. Conclusions These novel data demonstrate that calcitriol modulates cytokine enhancement of chemokines in developing ASM. Vitamin D may be a new therapeutic target in reducing inflammation in the immature airway. Supported by NIH F32 HL123075 (Britt), T32 HL105355 (Vogel), and R01s HL088029 and HL056470 (Prakash), and the Mayo Clinic Department of Anesthesiology (Pabelick, Prakash).