2.1.

Cell Culture

Rodent neuroblastoma-glioma cells (NG108) were grown in Dulbecco’s modified Eagle’s medium without sodium pyruvate containing 10% fetal bovine serum, $1\mathrm{I.U.}/\mathrm{mL}$ penicillin, $0.1\mu \mathrm{g}/\mathrm{mL}$ streptomycin, 0.1 mM hypoxanthine, 400 nM aminopterin, and 0.016 mM thymidine. Chinese hamster ovarian cells ($\mathrm{CHO}\text{-}{\mathrm{hM}}_1$) stably expressing human muscarinic acetylcholine receptor type 1 (${\mathrm{hM}}_1$) were grown in F-12K medium containing 10% fetal bovine serum, $1\mathrm{I.U.}/\mathrm{mL}$ penicillin, and $0.1\mu \mathrm{g}/\mathrm{mL}$ streptomycin. Geneticin^® (G418) is used in the CHO medium to maintain the ${\mathrm{hM}}_1$ expressing phenotype. Both cell lines were cultured at 37°C, 5% ${\mathrm{CO}}_2$, and 95% humidity.

2.2.

Solutions

Solutions were exchanged through bath application using a Warner Instruments perfusion system at a flow rate of $2\mathrm{mL}/\min$. Unless otherwise noted, in most experiments, we used a standard external buffer solution (pH 7.4, 290 to 310 mOsm) that consisted of 2 mM magnesium chloride (${\mathrm{MgCl}}_2$), 5 mM potassium chloride (KCL), 10 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), 10 mM glucose, 2 mM calcium chloride (${\mathrm{CaCl}}_2$), and 135 mM sodium chloride (NaCl). In some experiments (which are noted in the text), the ${\mathrm{CaCl}}_2$ was replaced with 2 mM Na-ethylene glycol-bis($\beta$-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) to create ${\mathrm{Ca}}^{2+}$-free external buffer.

To investigate the sources of IR-induced intracellular ${\mathrm{Ca}}^{2+}$ rises and compare IR effects with well-known effects caused by endogenous PLC activation, some experiments were paired with ${\mathrm{G}}_{\mathrm{q}/11}$-coupled ${\mathrm{hM}}_1$ receptor agonist, oxotremorine (OxoM, $10\mu \mathrm{M}$), ${\mathrm{G}}_{\mathrm{q}/11}$-coupled ${\mathrm{B}}_1$ receptor agonist, bradykinin (BK, 100 nM), or RyR agonist caffeine (10 mM). Additionally, we used ${\mathrm{IP}}_3$ receptor (${\mathrm{IP}}_3\mathrm{R}$) blockers xestospongin C (XeC $20\mu \mathrm{M}$), 2-aminoethoxydiphenil borate (2-APB $50\mu \mathrm{M}$), and RyR blocker ryanodine ($10\mu \mathrm{M}$). Media, chemicals, and pharmaceuticals were obtained from Life Technologies, Tocris Bioscience, or Sigma-Aldrich. In initial experiments, propidium iodide (PI) (BD Bioscience) was added to the external solution to a concentration of $4\mu \mathrm{M}$ to verify cell viability⁴³ and safe IR fiber placement.

2.3.

Infrared Laser Stimulation

An Acculight Capella IR diode laser (Lockheed Martin) with a center wavelength of 1869 nm was used to stimulate the cells. As demonstrated in Fig. 1(a), the laser light was delivered to the sample by a $200\text{-}\mu \mathrm{m}$ core optical fiber. A $90\text{-}\mu \mathrm{m}$ region in the center of the fluorescent image was used to analyze the ${\mathrm{Ca}}^{2+}$ response, to ensure uniformity of exposure [Fig. 1(b), green circle]. The fiber tip (top edge) was positioned by a micromanipulator about $90\mu \mathrm{m}$ away from the center to avoid obstruction of the region of interest by the fiber. The laser pulse was synchronized with the microscope using the Olympus real-time controller. The rapid rise temperature during stimulation caused intensity fluctuations in the images, possibly from thermal lensing. This “spiking” artifact effect was manually removed from data sets for clear presentation of IR-induced intracellular ${\mathrm{Ca}}^{2+}$ changes.

Fig. 1

(a) Diagram showing the position of the IR fiber in relation to the sample and (b) actual image of the cells and optical fiber. Cells in the green circle were used for measurements. The delivery fiber is outlined in red. PI is shown as the red fluorescence signal overlay. (From Olsovsky et al.⁴⁶)

All IR stimulation experiments were performed with the laser set to deliver 5 pulses (a 1-s, 5-Hz pulse train) with individual pulse durations from 2 (2.5 mJ) to 3 ms (3.8 mJ). Pulse energy was determined at the fiber and the absorption of water was not taken into account. To ensure that the IR laser pulse was not acutely damaging the cells, uptake of PI was monitored after IR pulse exposure. Uptake of PI can indicate damage to the plasma membrane and PI was seen in cells were directly beneath and in front of the fiber where the temperature rises were significantly higher [Fig. 1(b)]. Thus, the cells that were used for experiments [Fig. 1(b), green circle] were selected from a region that did not demonstrate any PI uptake after many minutes after the IR exposure.

2.4.

Measurement of Calcium

Cells were plated on poly-L-lysine coated glass coverslips and kept in a 37°C, humidified (5% ${\mathrm{CO}}_2$) incubator for 24 to 48 h before imaging. The cells were then loaded with Fura-2 ${\mathrm{Ca}}^{2+}$ probe in a standard external buffer solution containing $5\mu \mathrm{M}$ Fura-2 and 0.05% pluronic acid at 20°C for 30 min. The dye solution was then replaced with standard outside buffer solution for at least 15 min before imaging.

Fluorescent images were recorded using an Olympus epi-fluorescence microscope with a Lambda DG arc lamp and filter, a Hamamatsu Orca Flash 4.0 sCMOS camera, and an Olympus real-time controller. The real-time controller synchronizes the Lambda filter and camera so that an image using 340-nm excitation wavelength is captured immediately before another image using 380-nm excitation wavelength. The two images are compiled into a ratiometric image. The measured background and average autofluorescence for each cell line were subtracted before calculating the ratio. This ratio correlates to the concentration of calcium and is less vulnerable to artifact caused by variations in intensity due to, for example, defocus or sample thickness. The ratios were converted to ${\mathrm{Ca}}^{2+}$ concentrations using the following equation:

3.1.

Intracellular Ca²⁺ After Infrared Exposure

The resulting traces for the intracellular ${\mathrm{Ca}}^{2+}$ concentration increase after IR pulse exposures are shown in Fig. 2. A train of five IR pulses of 2, 2.5, or 3 ms duration started 660 ms after the beginning of image acquisition and lasted for 800 ms. In the ${\mathrm{Ca}}^{2+}$-containing standard outside buffer solution, noticeable intracellular ${\mathrm{Ca}}^{2+}$ increases appeared during IR pulses and peaked 260 ms after the train in both NG108 and $\mathrm{CHO}\text{-}{\mathrm{hM}}_1$. ${\mathrm{Ca}}^{2+}$ rise began immediately after the first IR pulse and increased with each subsequent pulse and has previously been shown to be evoked by each laser pulse.⁴⁵ The mean amplitudes at the peak of intracellular ${\mathrm{Ca}}^{2+}$ after 2 and 2.5 ms IR trains were significantly lower in $\mathrm{CHO}\text{-}{\mathrm{hM}}_1$ than in NG108 [Figs. 2(a) and 2(b)]. The delta changes were $12.3\pm 3.7\mathrm{nM}$ ($n=20$) versus $83.3\pm 21.1\mathrm{nM}$ ($n=9$) for 2 ms IR pulse trials and $26.4\pm 11.4\mathrm{nM}$ ($n=20$) versus $340.8\pm 106.8$ ($n=5$) for 2.5 ms pulses for $\mathrm{CHO}\text{-}{\mathrm{hM}}_1$ versus NG108, respectively ($p\le 0.0001$, unpaired two-tailed $t$-test). For the longer pulses [Fig. 2(c), 3 ms, 3.8 mJ], the intracellular ${\mathrm{Ca}}^{2+}$ increases were much higher than at lower IR pulses amplitudes, but increases observed between $\mathrm{CHO}\text{-}{\mathrm{hM}}_1$ and NG108 become statistically insignificant ($403.1\pm 58\mathrm{nM}$, $n=32$ versus $435.19\pm 105.4\mathrm{nM}$, $n=19$, respectively, $p\le 0.77$). Additionally, intracellular ${\mathrm{Ca}}^{2+}$ rise reaches a plateau 1.5 s after the end of the pulse train in CHO-hM1 but continues to rise in NG108 exposed cells. We then compared the increase in intracellular ${\mathrm{Ca}}^{2+}$ after a train of 3 ms pulses in ${\mathrm{Ca}}^{2+}$-chelated extracellular media [Fig. 2(d)]. We found these intracellular ${\mathrm{Ca}}^{2+}$ concentration increases to be much smaller than in experiments with ${\mathrm{Ca}}^{2+}$-containing external solution (postexposure $\mathrm{\Delta }{\mathrm{Ca}}^{2+}i18.4\pm 5.7\mathrm{nM}$, $n=21$ for $\mathrm{CHO}\text{-}{\mathrm{hM}}_1$ and $146.2\pm 59.4\mathrm{nM}$, $n=18$ for NG108), but still determined significant.

Fig. 2

Comparison of intracellular ${\mathrm{Ca}}^{2+}$ increases after train of IR pulses of different duration between NG108 and $\mathrm{CHO}\text{-}{\mathrm{hM}}_1$ cell lines. (a–c) Exposures were performed in ${\mathrm{Ca}}^{2+}$ containing extracellular media. (d) Experiments performed in ${\mathrm{Ca}}^{2+}$ chelated outside media. Error bars (gray area) represent the standard error (SE) of the mean of 5 to 32 cells per group. Vertical ticks above $x$-axis indicate the IR pulses train.

From our previous work suggesting that ${\mathrm{Ca}}^{2+}$ rises from IR pulse exposure were due to ${\mathrm{Ca}}^{2+}$ influx from extracellular media,^21,46 we hypothesized that the exposure may create small pores in the plasma membrane. However, the presence of an intracellular ${\mathrm{Ca}}^{2+}$ rise in the absence of external ${\mathrm{Ca}}^{2+}$ suggests that ${\mathrm{Ca}}^{2+}$ increases after IR exposure is not the result of simple passive diffusion through a permeabilized plasma membrane, but rather, a complex and regulated process, possibly through the involvement of ${\mathrm{IP}}_3$-sensitive endoplasmic reticulum (ER) stores and ${\mathrm{Ca}}^{2+}$-induced-${\mathrm{Ca}}^{2+}$-release (CICR) from ryanodine-sensitive ${\mathrm{Ca}}^{2+}$ stores. Additionally, in both ${\mathrm{Ca}}^{2+}$-containing and ${\mathrm{Ca}}^{2+}$-chelated solution, $\mathrm{CHO}\text{-}{\mathrm{hM}}_1$ did not exhibit as high a ${\mathrm{Ca}}^{2+}$ increase as NG108. The composition and distribution of plasma membrane ion channels responsible for normal cellular homeostasis and function are markedly different between mammalian excitable and nonexcitable cells. Similar differences are also present in the membranes of major intracellular ${\mathrm{Ca}}^{2+}$ stores. For example, muscular, neuronal, and cardiomyocyte cells widely express both RyR and ${\mathrm{IP}}_3\mathrm{R}$ in the sarco-ER, but nonexcitable cells express mostly intracellular ${\mathrm{IP}}_3\mathrm{R}$.⁴⁷ The differences in expression in the ER of RyR and ${\mathrm{IP}}_3\mathrm{R}$ in excitable and nonexcitable cells could be one of the main regulatory mechanisms responsible for the sensitivity of these cells to external stressors, such as IR stimulation.

Furthermore, the ${\mathrm{Ca}}^{2+}$ rise can be blocked in IR-exposed NG108 and $\mathrm{CHO}\text{-}{\mathrm{hM}}_1$ in ${\mathrm{Ca}}^{2+}$-chelated external media supplemented with thapsigargin.⁴⁶ Thapsigargin blocks SERCA, which normally pumps ${\mathrm{Ca}}^{2+}$ from the cytosol into the lumen of the sarco-ER,^48–50 thereby resulting in the depletion of intracellular stores. Remaining ${\mathrm{Ca}}^{2+}$ is eventually cleared by plasma membrane ${\mathrm{Ca}}^{2+}$ pumps.⁵¹ Thus, the lack of a ${\mathrm{Ca}}^{2+}$ increase after IR stimulation seen in these depleted cells suggests that increase in ${\mathrm{Ca}}^{2+}$-free solution may originate from ER or ryanodine-sensitive ${\mathrm{Ca}}^{2+}$ stores.

3.2.

Role of Intracellular Ca²⁺ Stores in Ca²⁺ Rises After Infrared Exposure

To investigate the role that these ${\mathrm{Ca}}^{2+}$ stores may be playing in the INM response, we then performed a series of experiments with agonists of the RyR and ${\mathrm{IP}}_3\mathrm{R}$ in NG108 and $\mathrm{CHO}\text{-}{\mathrm{hM}}_1$. First, to demonstrate the functional expression of intracellular ER receptors and capability of NG108 to adjust to changes in extracellular ${\mathrm{Ca}}^{2+}$, a series of solution changes were conducted during ${\mathrm{Ca}}^{2+}$ imaging (Fig. 3). NG108 were bathed in ${\mathrm{Ca}}^{2+}$-chelated buffer for 30 min before beginning ratiometric ${\mathrm{Ca}}^{2+}$ imaging to partially deplete intracellular ${\mathrm{Ca}}^{2+}$ out of the unstimulated cells.⁵² The normal resting intracellular ${\mathrm{Ca}}^{2+}$ concentration is between 50 and 100 nM,^53,54 but this exposure depleted it to $15\pm 3\mathrm{nM}$ [Fig 3(a)]. Shortly after beginning perfusion of cells with ${\mathrm{Ca}}^{2+}$-containing solution, the resting intracellular ${\mathrm{Ca}}^{2+}$ concentration reached a normal $52\pm 3\mathrm{nM}$ due to a capacitive ${\mathrm{Ca}}^{2+}$ entry mechanism. Treatment of NG108 with 100 nM BK peptide caused activation of the ${\mathrm{G}}_{\mathrm{q}/11}$-coupled ${\mathrm{B}}_1$ receptors, consequentially initiating ${\mathrm{PIP}}_2$ signaling and production of the ${\mathrm{IP}}_3$. After the ${\mathrm{IP}}_3$-induced ${\mathrm{Ca}}^{2+}$ spike, we applied 2-APB ($50\mu \mathrm{M}$) in ${\mathrm{Ca}}^{2+}$-chelated buffer to block ${\mathrm{IP}}_3\mathrm{R}$ and slightly deplete intracellular ${\mathrm{Ca}}^{2+}$ stores.⁵⁵ This manipulation prevented the ${\mathrm{IP}}_3$-induced ${\mathrm{Ca}}^{2+}$ spike after secondary application of the BK and confirmed the functional role of ${\mathrm{IP}}_3\mathrm{R}$ in NG108. Similarly, $\mathrm{CHO}\text{-}{\mathrm{hM}}_1$ stably expresses the ${\mathrm{G}}_{\mathrm{q}/11}$-coupled ${\mathrm{hM}}_1$ receptors, so application of a high concentration of ${\mathrm{hM}}_1$ agonist OxoM ($10\mu \mathrm{M}$) resulted in a strong ${\mathrm{IP}}_3$-dependent ${\mathrm{Ca}}^{2+}$ response [Fig. 3(b)].²⁹ To demonstrate CICR from ryanodine stores, we applied caffeine (10 mM) to sensitize RyR and allowed basal cytosolic calcium levels to actuate CICR.^56,57 A cytoplasmic ${\mathrm{Ca}}^{2+}$ rise can be seen in the NG108, but $\mathrm{CHO}\text{-}{\mathrm{hM}}_1$, which do not contain ryanodine stores, shows no response [Figs. 3(c) and 3(d)].

To investigate the role of RyR and ${\mathrm{IP}}_3\mathrm{R}$ in the IR-induced changes in intracellular ${\mathrm{Ca}}^{2+}$ dynamics, we exposed both NG108 and $\mathrm{CHO}\text{-}{\mathrm{hM}}_1$ to a 3-ms IR pulses train in the presence of several receptor antagonists. Antagonists of RyR and ${\mathrm{IP}}_3\mathrm{R}$ dramatically reduced IR-induced intracellular ${\mathrm{Ca}}^{2+}$ response in both cell lines [Fig. 4(a)], suggesting that physiological ${\mathrm{Ca}}^{2+}$ regulatory mechanisms are predominate in the cellular response to IR stimulation.

Fig. 3

Intracellular RyR and ${\mathrm{IP}}_3\mathrm{Rs}$ in the NG108 and $\mathrm{CHO}\text{-}{\mathrm{hM}}_1$ cells. (a) Demonstration of the NG108 cells ($n=9$) capability to adjust to changes in extracellular ${\mathrm{Ca}}^{2+}$ concentration and respond to 100 nM BK-induced ${\mathrm{IP}}_3\mathrm{Rs}$ activation. (b) OxoM ($10\mu \mathrm{M}$)-induced intracellular ${\mathrm{Ca}}^{2+}$ rise in $\mathrm{CHO}\text{-}{\mathrm{hM}}_1$ cells ($n=15$) due to ER ${\mathrm{IP}}_3\mathrm{Rs}$ activation. (c) Caffeine (10 mM)-induced ${\mathrm{Ca}}^{2+}$ increase due to intracellular RyR receptors activation in the NG108 cells ($n=17$). (d) Lack of response to 10 mM caffeine in CHO-hM1 cells ($n=18$). Error bars (black outline with gray area fill) represent the SE of the mean.

Fig. 4

NG108 and $\mathrm{CHO}\text{-}{\mathrm{hM}}_1$ ${\mathrm{Ca}}^{2+}$ responses in ${\mathrm{Ca}}^{2+}$-containing outside buffer after trains of 3-ms duration IR pulses with and without intracellular RyR and ${\mathrm{IP}}_3\mathrm{R}$ blockers. (a) IR-induced changes in intracellular ${\mathrm{Ca}}^{2+}$ dynamics. The traces without antagonists are the same as in Fig. 2 and presented here for comparison. Vertical ticks above $x$-axis indicate the IR pulses train. (b) Magnification of the ${\mathrm{Ca}}^{2+}$ responses with RyR and ${\mathrm{IP}}_3\mathrm{R}$ antagonists. Error bars (black outline with gray or black/gray checked pattern fill areas) represent the SE of the mean ($n=11$ to 16).

The ${\mathrm{IP}}_3$ stores, as shown above (Fig. 4), are present in both NG108 and $\mathrm{CHO}\text{-}{\mathrm{hM}}_1$. We pretreated $\mathrm{CHO}\text{-}{\mathrm{hM}}_1$ cells for 20 min with XeC ($20\mu \mathrm{M}$), a specific inhibitor of the ${\mathrm{IP}}_3$-dependent ${\mathrm{Ca}}^{2+}$ release.⁵⁸ In $\mathrm{CHO}\text{-}{\mathrm{hM}}_1$, XeC nearly completely blocked the rise in intracellular ${\mathrm{Ca}}^{2+}$ levels (postexposure $\mathrm{\Delta }{\mathrm{Ca}}^{2+}i3.3\pm 0.5\mathrm{nM}$, $n=13$), even in ${\mathrm{Ca}}^{2+}$-containing outside buffer. Despite the fact that such a small response is within normal intracellular ${\mathrm{Ca}}^{2+}$ physiological fluctuations, the rise correlates temporally with IR exposure [Fig. 4(b)]. This small increase could be due to capacitive entry of extracellular ${\mathrm{Ca}}^{2+}$ through diacylglycerol (DAG)-sensitive TRP/SOC channels or from incomplete block of the ${\mathrm{IP}}_3\mathrm{R}$.^34,35,59,60 In NG108, a similar small intracellular ${\mathrm{Ca}}^{2+}$ response could lead to CICR from RyR ${\mathrm{Ca}}^{2+}$ stores. Indeed, IR stimulation of NG108 cells in ${\mathrm{Ca}}^{2+}$-chelated outside buffer and treated with XeC ($20\mu \mathrm{M}$) resulted in a small, but significant ${\mathrm{Ca}}^{2+}$ rise (postexposure $\mathrm{\Delta }{\mathrm{Ca}}^{2+}i38.2\pm 6.4\mathrm{nM}$, $n=11$). Additionally, NG108 pretreated with RyR antagonist ryanodine⁶¹ ($10\mu \mathrm{M}$) in ${\mathrm{Ca}}^{2+}$-containing buffer, showed a large reduction in ${\mathrm{Ca}}^{2+}$ rise after IR stimulation (postexposure $\mathrm{\Delta }{\mathrm{Ca}}^{2+}i16.1\pm 6.2\mathrm{nM}$, $n=16$), with the small rise in ${\mathrm{Ca}}^{2+}$ possibly resulting from ${\mathrm{IP}}_3$ stores or capacitive entry^37,40 without CICR [Fig. 4(b)]. These results show that ${\mathrm{IP}}_3$ stores are involved in ${\mathrm{Ca}}^{2+}$ signaling from INM in both cell lines but are not the sole ${\mathrm{Ca}}^{2+}$ source in NG108.

Our observations further suggest that differences between excitable and nonexcitable cells in IR-induced ${\mathrm{Ca}}^{2+}$ responses could be due to distinct expression of intracellular RyR and ${\mathrm{IP}}_3\mathrm{R}$ in the ER of these cells. RyR and ${\mathrm{IP}}_3\mathrm{R}$ have been shown to be activated in parallel with store-operated ${\mathrm{Ca}}^{2+}$ entry (SOCE) and strongly contribute to the global ${\mathrm{Ca}}^{2+}$ response.⁶² Depletion of these intracellular ${\mathrm{Ca}}^{2+}$ stores can initiate SOCE through plasma membrane SOC channels. Thus, much of the observed ${\mathrm{Ca}}^{2+}$ increase in the NG108 could be due to direct or indirect activation of RyR and additional to SOCE intracellular ${\mathrm{Ca}}^{2+}$ regulatory mechanism. In NG108, it has been demonstrated that depolarization-induced ${\mathrm{Ca}}^{2+}$ entry evoked CICR only from the ryanodine-sensitive stores,⁶³ which greatly contribute to general ${\mathrm{Ca}}^{2+}$ response.

Previous experiments on HeLa cells, cardiomyocytes, and neurons have demonstrated the critical role of intracellular ${\mathrm{Ca}}^{2+}$ regulation in thermal gradient stimulation mechanisms. In HeLa cells, during second-long heating of $<1\mathrm{deg}$, a decrease in ${\mathrm{Ca}}^{2+}$ was observed, theorized to be due to an increase of SERCA activity along with a decrease in the open probability of the ER ${\mathrm{IP}}_3\mathrm{R}$ and RyR. After the exposure, the rapid cooling was hypothesized to increase the open probability of these ER ${\mathrm{Ca}}^{2+}$ conducting channels, leading to an overshoot of cytoplasmic ${\mathrm{Ca}}^{2+}$. This IR-induced ${\mathrm{Ca}}^{2+}$ uptake by SERCAs and its asymmetrical outflow via intracellular ER ${\mathrm{IP}}_3\mathrm{R}$ were proposed as a general mechanism of the temperature-dependent changes in ${\mathrm{Ca}}^{2+}$ dynamics.²⁴ While we did not observe a decrease in ${\mathrm{Ca}}^{2+}$ in these experiments, due to the brevity of our pulses and experimental parameters, this hypothesized sensitivity of the ER ${\mathrm{IP}}_3\mathrm{R}$ could contributed the ${\mathrm{Ca}}^{2+}$ overshoot observed from IR pulses. IR rapid heating/cooling of water also creates capacitive photothermal currents, which results in plasma membrane depolarization/repolarization¹⁹ and thus possible activation of the voltage sensitive phosphatase (Ci-VSP). Recently, Ci-VSP was shown to regulate ${\mathrm{PIP}}_2$ signaling in the plasma membrane^64–66 and could be accounted for the initial depletion during IR-induced cellular response.

Previous IR pulse experiments in cardiomyocytes and spiral and vestibular ganglion neurons indicated that the calcium signaling originated from the mitochondria. However, three (ryanodine, cyclopiazonic acid, and ruthenium red) of the pharmaceutical compounds used in these studies have direct severe inhibitory effect on the RyR and ER, indicating a likely critical importance of internal ${\mathrm{Ca}}^{2+}$ ER pools/receptors in IR-induced INM in addition to alteration of mitochondrial function.²⁶ By using two cell lines with innate differences in ER receptors, we demonstrate the role that the interplay between these two receptors has on the response the IR exposure.

Previously, we found that IR pulses initiated the intracellular phosphoinositide ${\mathrm{PIP}}_2$ signaling cascade in $\mathrm{CHO}\text{-}{\mathrm{hM}}_1$.²¹ This response appeared similar to one initiated by activation of ${\mathrm{G}}_{\mathrm{q}/11}$-coupled receptors and resulted in ${\mathrm{IP}}_3$ production with possible consequential depletion of the intracellular ER ${\mathrm{Ca}}^{2+}$ stores. ${\mathrm{IP}}_3$ is a main component of the intracellular calcium signaling and provides a direct link between cellular plasma membrane and prime intracellular ${\mathrm{Ca}}^{2+}$ store, the ER.^30,67–70 The exact mechanism of IR-induced activation of ${\mathrm{PIP}}_2$ signaling is unknown, but hypothetical schematics of the IR-induced ${\mathrm{Ca}}^{2+}$ responses are presented in Fig. 5.

Fig. 5

Simplified hypothetical schematic of IR-induced ${\mathrm{Ca}}^{2+}$ response between (a) nonexcitable and (b) excitable cells.

In nonexcitable cells [Fig. 5(a)], IR-induced PLC-dependent ${\mathrm{PIP}}_2$ hydrolysis or depletion leads to production of ${\mathrm{IP}}_3$ and DAG (green arrows). DAG and its derivative, arachidonic acid, activate ${\mathrm{Ca}}^{2+}$-conducting TRP SOC channels^34,35,40,60 and ${\mathrm{IP}}_3$ initiates intracellular ${\mathrm{Ca}}^{2+}$ release through activation of ${\mathrm{IP}}_3\mathrm{R}$ on the ER (blue arrows out of ER). Intracellular ${\mathrm{Ca}}^{2+}$ activates cytoplasmic PKC, which has a high affinity to DAG.^71,72 Active PKC translocates toward DAG (purple arrows) and phosphorylates TRP channels, keeping them in the open state longer.⁷³ Extracellular ${\mathrm{Ca}}^{2+}$ started to influx into cytosol through TRP/SOC channels due to the SOCE mechanism (red arrows). High levels of intracellular ${\mathrm{Ca}}^{2+}$ catalyze PLC activity, leading to stronger ${\mathrm{PIP}}_2$ hydrolysis, and potentiating the reaction described above.^29,74 High intracellular ${\mathrm{Ca}}^{2+}$ is eventually pumped out of the cell by plasma membrane ${\mathrm{Ca}}^{2+}$-ATPase and into ER stores by SERCA.^75,76 In excitable, specifically neuronal cells [Fig. 5(b)], in addition to the reactions described above and SOCE, the intracellular ${\mathrm{Ca}}^{2+}$ increase is achieved through additional mechanisms, including strong sensitization of neurons by ${\mathrm{IP}}_3$ and ${\mathrm{Ca}}^{2+}$-activated ryanodine-sensitive ${\mathrm{Ca}}^{2+}$ release.^63,77–79 The interplay of these two intracellular ${\mathrm{Ca}}^{2+}$ pools is critically important, since it leads to much stronger phenotypic ${\mathrm{Ca}}^{2+}$ response. ${\mathrm{Ca}}^{2+}$- and ${\mathrm{PIP}}_2$-dependent modulation of the neuronal potassium channels leads to changes in membrane potential and depolarization.^30,80 As a consequence of depolarization and activation of the VGCC, ${\mathrm{Ca}}^{2+}$ influx could also evoke CICR through RyR receptors.^63,79 Last, the overall neuronal activity induces ${\mathrm{Ca}}^{2+}$ influx through excitatory neurotransmitters and receptor-operated ${\mathrm{Ca}}^{2+}$ channels.^81–84 Therefore, IR-induced changes of intracellular ${\mathrm{Ca}}^{2+}$ signaling and dynamics in neurons can explain both stimulation and modulation mechanisms. While additional studies are needed, our experiments presented here indicate that intracellular ${\mathrm{IP}}_3\mathrm{R}$ in the ER play an important role in both excitable and nonexcitable cell lines, with the IR-induced ${\mathrm{Ca}}^{2+}$ response augmented by RyR in excitable cells, thus strongly reinforcing our hypothesis.