Figure 1.
Calcium levels in PC12 cells exposed to SMF, the A2AR agonist CGS21680 or antagonist ZM241385.
(A) Extracellular Ca2+ was measured for cells maintained in calcium-free medium increased for time points up to 3.0 h in response to SMF exposure; p<0.05 for n = 3 independent experiments. (B) In a separate experiment cells were evaluated at the three hour time point when the largest difference between SMF-treated and untreated cells occurred but before cell integrity was compromised from the assay conditions (e.g., from using Ca2+ and Mg2+ free D-PBS). Cells treated with 1.0 µM CGS21680 experienced decreased Ca2+ release compared to control cells while co-treatment of the cells with this agonist and either 1.0 µM ZM241385 or SMF attenuated the CGS21680-induced decrease (p values for each comparison are shown on the chart for n = 3 independent experiments).
Figure 2.
Effect of Ca2+ flux and adenosine activators and blockers on A2AR mRNA and protein levels in PC12 cells.
(A) The A2AR agonist CGS21680 increased A2AR mRNA levels by over 5-fold while the antagonist ZM241385 as well as SMF decreased this agonist-enhanced A2AR transcription to close to control levels (p values for each comparison are shown on the chart for n≥3 independent experiments). (B) The A2AR agonist CGS21680 increased A2AR protein levels while the antagonist ZM241385 as well as SMF decreased A2AR in western blots; quantification of representative data is shown in (C); this experiment was repeated three times with similar results.
Figure 3.
Effect of L-type Ca2+ channel activators and blockers on A2AR mRNA and protein levels in PC12 cells.
(A) The L-type Ca2+ channel activator Bay K8644 increased A2AR mRNA levels in PC12 cells compared to untreated controls while the L-type Ca2+ blocker Nifedipine, as well as SMF exposure, decreased A2AR mRNA levels after 6.0 h of exposure (p<0.05 for each test condition compared to control for n≥3 independent experiments). (B) Increased A2AR mRNA resulting from exposure to Bay 8644 was reversed by concomitant exposure to SMF (p values are shown for n≥3 independent experiments).
Figure 4.
Cellular ATP and ADO levels in PC12 cells exposed to SMF, CGS21680 or ZM241385.
(A) Cells were incubated with 1.0 µM CGS21680, ZM241385, or exposed to SMF for 6.0 h. The cells were harvested and an equal number from each sample were used to prepare extracts and to measure intracellular ATP levels (p<0.05 for n≥3 independent experiments for each treatment condition compared to untreated control cells; a similar trend was observed for 3.0 h, but not all data points were statistically significant). (B) – (F) After 3.0 h incubation in D-PBS, the extracellular fluid was collected from PC12 cells and analyzed by HPLC to detect and quantify ADO. (B) ADO was not detected in samples from untreated control cells (elution of authentic ADO is shown in (C)) but was observed in samples from cells treated with (D) 1.0 µM CGS21680, (E) 1.0 µM CGS21680 plus 1.0 µM ZM241385, or (F) 1.0 µM CGS21680 plus exposure to SMF in (F). The HPLC assays were repeated three times with similar results; representative data is shown.
Figure 5.
cAMP levels in SMF, CGS21680, and ZM241385 treated PC12 cells.
Cells were exposed to each condition, harvested, lysed, and assayed for cAMP levels. Each test condition treatment condition varied from untreated control cells with p<0.05 for n = 3 independent experiments.
Figure 6.
Effect of CGS21680, ZM241385, and SMF on nitrite levels in PC12 cells.
Levels of nitrite were measured after 24 h of incubation with 1.0 µM of the A2AR agonist (CGS21680) or antagonist (ZM241385) or after exposure to SMF; p values are shown in comparison with untreated control cells for n = 3 independent experiments.
Figure 7.
Effect of CGS21680, ZM241385, and SMF on p44/42 MAPK phosphorylation and proliferation in PC12 cells.
(A) Western blots show the phosphorylation of p44/42 MAPK after exposure to SMF or 1.0 µM CGS21680 for 48 h, or after pretreatment with 1.0 µM ZM241385 or SMF for 30 min followed by the addition of 1.0 µM CGS21680 and an additional 48 h of incubation (total p44/42 MAPK is also shown); quantification by densitometry for a representative experiment (of n = 3 independent experiments) is shown in (B). (C) Proliferation of PC12 cells grown under the conditions indicated in (A), but for three days instead of 48 h, are given as measured by the MTT assay (p values for the various comparisons indicated on the figure are for n≥3 independent experiments).
Figure 8.
Effect of CGS21680, ZM241385, and SMF on neurite sprouting in PC12 cells.
Representative images of PC12 cells that were (A) untreated or treated with (B) CGS21680 (1.0 µM), (C) ZM241385 (1.0 µM), or (D) SMF for three days are shown. (E) Neurite outgrowth was quantified by counting the number of cells exhibiting neurites that were 1.5 times longer than the diameter of the cell and the proportion of cells with neurites was expressed as a percentage of the total number of cells; data is shown from counting at least 100 cells from each of five fields selected at random.
Figure 9.
Effect of CGS21680, ZM241385, and SMF on iron uptake in PC12 cells.
Intracellular iron was quantified using a colorimetric assay one hour after the addition of 50 µM FeSO4 to the medium (p values are shown comparing each test condition to controls for n≥3 independent experiments).
Figure 10.
Outline of the putative mechanism of SMF on lipid bilayers, Ca2+ flux, A2AR receptors, and downstream modulation of multiple effector systems.
(A) Phospholipid molecules possess diamagnetic anisotropy and align and reorient in the presence of moderate strength magnetic fields [60] thereby reducing the flexibility of the phospholipid acyl chains. The consequent stiffening of phospholipid molecules increases lateral compression and thickens the bilayer thereby altering the bulk biophysical properties of the membrane [79]. In turn, changes to membrane dynamics can affect the activity of embedded proteins ranging from signaling complexes (e.g., the toll like receptors [80]) to ion channels and membrane transporters [81]. (B) Specific examples of candidates for such SMF-mediated modulation include the sodium calcium exchanger (NCX) that transports Ca2+ out of a cell, the voltage gated L-type Ca2+ channel that transports Ca2+ into a cell, and the plasma membrane Ca2+ ATPase (PMCA) pump that hydrolyzes ATP to gain energy to remove Ca2+ from a cell (data relating to Ca2+ is given in Figure 1, concomitant changes to mRNA and proteins levels for A2AR in Figures 2 and 3, and ATP in Figure 4A). (C) ATP is linked to Ca2+ flux in another way, namely through metabolites such as adenosine (ADO, see Figure 4B-F). (D) ADO binds to adenosine receptors such as A2AR, which in turn can further modulate Ca2+ flux. (E) Calcium is a ubiquitous second messenger, leading to secondary responses that involve cAMP (Figure 5) or nitric oxide (Figure 6); in turn multiple effector systems (MES) can be engaged that affect additional endpoints including MAPK pathways (Figure 7), neurite outgrowth indicative of differentiation (Figure 8), and iron (Figure 9).