Effect of electromagnetic field on cyclic adenosine monophosphate (cAMP) in a human mu-opioid receptor cell model

Christina L. Rossa,b, Thaleia Telia, and Benjamin S. Harrisona

aWake Forest Institute for Regenerative Medicine, Wake Forest Baptist Health, Winston-Salem, NC, USA; bWake Forest Center for Integrative Medicine, Wake Forest Baptist Health, Winston-Salem, NC, USA

ABSTRACT

During the cell communication process, endogenous and exogenous signaling affect normal as well as pathological developmental conditions. Exogenous influences such as extra-low-frequency electromagnetic field (EMF) have been shown to effect pain and inflammation by modulating G-protein receptors, down-regulating cyclooxygenase-2 activity, and affecting the calcium/calmo-dulin/nitric oxide pathway. Investigators have reported changes in opioid receptors and second messengers, such as cyclic adenosine monophosphate (cAMP), in opiate tolerance and depen-dence by showing how repeated exposure to morphine decreases adenylate cyclase activity causing cAMP to return to control levels in the tolerant state, and increase above control levels during withdrawal. Resonance responses to biological systems using exogenous EMF signals suggest that frequency response characteristics of the target can determine the EMF biological response. In our past research we found significant down regulation of inflammatory markers tumor necrosis factor alpha (TNF-α) and nuclear factor kappa B (NFκB) using 5 Hz EMF frequency. In this study cAMP was stimulated in Chinese Hamster Ovary (CHO) cells transfected with human mu-opioid receptors, then exposed to 5 Hz EMF, and outcomes were compared with morphine treatment. Results showed a 23% greater inhibition of cAMP-treating cells with EMF than with morphine. In order to test our results for frequency specific effects, we ran identical experiments using 13 Hz EMF, which produced results similar to controls. This study suggests the use of EMF as a complementary or alternative treatment to morphine that could both reduce pain and enhance patient quality of life without the side-effects of opiates.

Introduction

Tissue injury results in the production of inflammatory mediators, several of which sensitize primary afferent nociceptors (Davis et al., 1993; Reuff and Dray, 1993), resulting in hyperalgesic pain (Ferreira, 1981; Fierreira et al., 1978). Hyperalgesic pain is often associated with inflammatory pain (Schultz et al., 2003). The largest family of receptors for pharmaceutical agents is the G-protein-coupled receptors (GPCRs), whose signal transduction pathway is well understood. For example, Gαs (stimulating) subunit increases adenylate cyclase (AC) activity, thereby stimulating the production of cyclic adenosine monophosphate (cAMP); whereas the Gαi (inhibitory) subunit decreases AC activity and inhi-bits the production of cAMP. The second messenger cAMP activates a specific number of tissue-specific cAMP-dependent protein kinases, ultimately affecting intracellular processes such as ion channel activity, release of neurotransmitters, regulation of transcription factors, and numerous other processes. GPCRs

determine ligand binding and selectivity (Karlsmark et al., 2003). A number of ligands inhibit the function of specific enzymes by competitive or non-competitive inhibition (Sen et al., 2009). A ligand that binds to the same active catalytic site as the endogenous substrate is a competitive inhibitor. Ligands that bind at different sites on the enzyme, alter the shape of the molecule, and reduce its catalytic activity, are called non-competitive inhibitors. Extracellular environments, and to some extent transmembrane regions, determine ligand bind-ing (Brenner and Steven, 2010). A substantial amount of literature suggest that hyperalgesia induced by tissue damage is initiated by the activation of AC – cAMP – protein kinase A (PKA) second messenger cascade, acti-vated at the mu-opioid receptor (MOR) site (England et al., 1996; Khasar et al., 1995; Malmberg et al., 1997; Taiwo and Levine, 1989, 1990, 1991, 1992). Agents that inhibit AC- and cAMP-dependent PKA prevent induc-tion of hyperalgesia by prostaglandin E2 (PGE2) and other inflammatory mediators.

CONTACT Christina L. Ross  chrross@wakehealth.edu  Wake Forest Institute for Regenerative Medicine, Wake Forest Baptist Health, Winston-Salem, NC 27109, USA.

© 2015 Taylor & Francis

Downloaded by [Christina Ross] at 11:21 30 December 2015

2     C. L. ROSS ET AL.

Cyclic AMP is required for cell communication in the hypothalamus/pituitary gland axis and for the feed-back control of hormones of the sympathetic nervous system (SNS) (Vargas et al., 2001). It is synthesized from adenosine triphosphate (ATP) by adenylyl cyclase (AC) located on the inside of the cell membrane, and is an important signal carrier necessary for the proper biological response of cells to hormones and other extracellular signals that initiate inflammatory pain through the cAMP response element binding (CREB) protein cycle. Cyclic AMP is activated by a range of signaling molecules via AC stimulatory G-protein (Gs)-coupled receptors and inhibited by agonists of AC inhibitory GPCRs (Gi) (Billington and Penn, 2003; Khoury et al., 2014). GPCRs also form homo- and/or hetero-dimers, and their impact on receptor physiology and pharmacology has attracted a lot of interest (CRR, 2013). Data suggest that the MOR is a heterodimer that heterodimerizes only at the cell surface, and the oligo-mers of opioid receptors and heterotimeric G-proteins are the bases for MOR heterodimer phenotypes (Markova and Mostow, 2012). There is accumulating evidence that oligomerization of GPCRs can alter the selectivity and affinity of ligand binding, and this has been reported for opioid receptor heterodimers (Andreadis and Geer, 2006; Gurtner et al., 2008). Due to this phenomenon an alteration in binding properties could sensitize receptors to ligands, thereby allowing responses to lower agonist concentrations. Receptor dimerization can also result in the formation of novel binding sites. There are many documented examples of interactions between GPCRs coupling preferentially to different signaling pathways, which result in a potentia-tion of Ca2+ signaling (Fiorotto and Klish, 1991; Gourevitch et al., 2014). GPCRs are components of multi-protein signaling complexes that regulate the localization and function of receptors which form com-plexes with a wide variety of signaling and regulatory proteins. For these experiments we have chosen the mu-opioid receptor as our target, because it is most associated with potent analgesics currently used to con-trol pain (Tzschentke et al., 2014).

Opioid dependence has been reported to be asso-ciated with changes in the cAMP systems in experi-ments in vitro. For example, in neuroblastoma cells, acute treatments with morphine and other opiates inhi-bit adenylate cyclase (AC) activity resulting in a decrease of cAMP levels (Sharma et al., 1975; Traber et al., 1975); however, after repeated exposure to mor-phine, the AC activity and cAMP levels return to con-trol levels in the tolerant state and increased above control levels during withdrawal (Benalal and Bachrach, 1985; Sharma et al., 1975; Traber et al.,

1975). These findings are the basis for cAMP as the mechanism of action for the development of morphine dependence (Mamiya et al., 2001). An increase in AC activity and cAMP levels in the brain represent bio-chemical associations of morphine dependence (Collier, 1980; Kuriyama et al., 1978), whereby cAMP levels are regulated by AC and phosphodiesterases (PDEs) (Thompson, 1991). It has been substantiated that 3-isobutyl-1-methylxanthine (IBMX)-modulated forsko-lin induces behavior that resembles morphine withdrawal syndrome in naïve rats and increases nalox-one-precipitated morphine withdrawal syndrome in morphine dependent rats (Collier and Francis, 1975; Rasmussen et al., 1990).

In humans, electromagnetic field (EMF) therapy has proven to be a safe, non-invasive, easy-to-use method to treat the source of pain and inflammation (Markov, 2007; Ross and Harrison, 2013). Research has shown that therapeutic applications at extra-low frequency (ELF) EMF (1–100 Hz) levels stimulate the immune system by suppressing inflammatory responses at the cell-membrane level (O’Connor et al., 1990). Double-blind, placebo-controlled clinical trials (Stiller et al., 1992) report EMF passes through the skin into the body’s conductive tissue (Hannan et al., 1994; Stiller et al., 1992; Traina et al., 1998), reducing pain and the onset of edema shortly after trauma (Chalidis et al., 2011; Rohde et al., 2009). In one such study low-frequency pulsed EMF (PEMF) therapy at 0.1 to 64 Hz was reported to improve mobility, and reduce pain and fatigue in fibromyalgia patients (Sutbeyaz et al., 2009). Both human and in vitro studies report EMF to be effective in the treatment of pain and inflammation in osteoarthritis (OA) (Li et al., 2013; Sadoghi et al., 2013), without the addictive side-effects of opiates. It has been proposed that charge receptors or other kinds of sensors at the extracellular membrane could recog-nize EMF by their ability to resonate with varying frequencies (Funk and Monsees, 2006).

In this study we hypothesize that an EMF at certain frequencies can be therapeutic, therefore we are looking for a similar down regulatory effect of EMF on cAMP as would be seen in morphine treatment. EMF has a number of well-documented therapeutic effects on cells and tissues afflicted with inflammatory pain (Ross and Harrison, 2013). Reports of resonance frequency responses of biological systems to exogenous EMF sig-nals suggest the frequency response characteristics of the tissue can determine the EMF response (Bawin et al., 1975; Markov, 1981). EMF parameters such as frequency, field strength, and time of exposure, all account for the mechanistic pathway affecting inflam-matory pain mediators. Oscillating EMF exerts forces on free ions present on both sides of the plasma mem-brane which move across the cell surface through the transmembrane proteins creating a forced intracellular vibration. This force is responsible for phenomena such as the influx of extracellular calcium and the binding affinity of calmodulin (CaM) – the primary transduc-tion pathway to second messengers such as cAMP. Because of the important ramifications for treating inflammatory pain, we investigated the effect of EMF on the cAMP second messenger pathway, which con-tributes to maintenance as well as the initiation of hyperalgesia.

Materials and methods

A Chinese Hamster Ovary (CHO-K1) cell line trans-fected with human mu(µ)-opioid receptors (catalog #6605, ChanTest Corp, Cleveland, OH) was cultured in flasks using Ham’s F-12 1X modified media with L-glutamine (Mediatech, Inc., Manassas, VA); 10% FBS, 1% non-essential amino acids (100X, #11140-050, Gibco, Grand Island, NY); 0.4 mg/ml Geneticin (G418, #10131-027, Gibco, Grand Island, NY); and 1% penicillin-streptomycin (Cellgro#30-002-CL, Fisher Scientific, Pittsburg, PA), then incubated in 5% CO2 at 37 °C and 100% humidity until ∼80% confluent. Cells were detached from flasks using trypsin, then centri-fuged at 1500 rpm for 5 min and reseeded in two sepa-rate 6-well culture plates (treatment and control) at a density of 2.5 × 105 cells/mL.

Stimulation of cAMP

Cyclic AMP was stimulated by exposing µ-CHO cells to 10µM forskolin (Sigma #F6886, St. Louis, MO). Forskolin is a labdane diterpene produced by the Indian coleus plant, and is commonly used in medical research to raise cAMP levels (Gris et al., 2010). Forskolin re-sensitizes cell receptors by activating ade-nylyl cyclase (AC) to catalyze the levels of cAMP. Cells were also stimulated with 0.5 mM 3-isobutyl-1-methylxanthin (IBMX, Sigma #17018, St. Louis, MO), which is a phosphodiesterase inhibitor that will prevent the degradation of the phosphodiester bond in the second messenger molecule cAMP. All stimulation times were 15 min as is standard for this protocol (Xu et al., 2003). Duplicate experiments were performed on CHO-K1 cells used as negative control. Outcomes were measured based on EC50 or half maximal effec-tive concentration of the morphine (CatchPoint Cyclic-AMP Fluorescent Assay Kit #R8088, Molecular Devices, Sunnyvale, CA).

ELECTROMAGNETIC BIOLOGY AND MEDICINE 3

Experimental groups

Six individual groups were used in this experiment: (1) µ-CHO cells only (baseline) [positive control]; (2) µ-CHO cells + forskolin/IBMX stimulant [F only]; (3) µ-CHO cells + forskolin/IBMX stimulant + 0.1 mM morphine [F + M]; (4) µ-CHO cells + forskolin/IBMX stimulant + EMF treatment [F + E]; (5) µ-CHO cells + forskolin/IBMX stimulant + 0.1 mM morphine + EMF treatment [F + M + E]; and (6) CHO-K1 [negative con-trol]. In the F + M + EMF group, morphine was added immediately before cells were exposed to EMF.

PEMF exposure

Using a commercially manufactured 11-inch diameter Helmholtz Coil (SpinCoil-11-X, Micro Magnetics, Fall River, MA), both µ-CHO cells and CHO-K1 cell groups underwent the same experimental conditions. The [F + E] and [F + M + E] groups were exposed to a 5 Hz EMF for 15 min to determine treatment effect on cAMP. The time point of 15 min was selected as it is the amount of time needed for cAMP accumulation levels to be main-tained fully until the end of the incubation period. The EMF coils were driven by an alternating current power supply with adjustable frequency and amplitude. From the coil center the uniform field strength was measured to be approximately 1.5 µT (see flux density schematic in Figure 1a). Each coil carried a 50% duty sine wave in the same direction (Figure 1b). The EMF was charac-terized using a Gauss meter (Sypris Model 5180; Pacific Scientific-OECO, Milwaukie, OR). The frequency was quantified using an oscilloscope (Model TDS 20248; Tektronix Inc, Beaverton, OR). Experiments were per-formed under ambient conditions. After exposure to EMF, a live/dead cell assay was performed using calcein acetomethoxy (catalog # C697959; Life Technologies, Carlsbad, CA), showing no significant change in cell viability (data not shown). Measurements were taken using a Spectramax M5 plate reader (Molecular Devices LLC, Sunnyvale, CA), at 530 nm excitation level and 590 nm emission level. Control samples were kept in the same conditions without exposure to EMF. The EMF exposed cells were kept in a warm bath to ensure a consistent 37 °C temperature during these experi-ments. Background EMF was measured and averaged the same as Earth’s (∼0.5 Gauss).

Statistical analysis

All data measured are presented as mean ± standard error of the mean (S.E.M.), for n = 4 samples. For all assays, a one-way analysis of variance (ANOVA) with

4     C. L. ROSS ET AL.

Tukey’s post hoc test was used to assess the differences between the controls and the cells exposed to the EMF, with p < 0.05 considered statistically significant for all tests.

Results

The aim of this study was to investigate the effect of a low-frequency EMF on cAMP, which is a second mes-senger transcription factor used for intracellular signal transduction on the AC-cAMP-PKA-dependent path-way. This pathway contributes to the initiation and maintenance of hyperalgesia. In this experiment we have chosen to study the human mu (µ) opioid receptor in particular because it belongs to the GPCR super family of receptors whose activation leads to a cascade of events that inhibit AC and decrease cAMP levels, and also has a strong binding affinity with morphine (Osugi et al., 1996). Using n = 4 samples for three trials, we compared the results of an EMF treatment with that of morphine. Since the opiate receptor ligand morphine has a considerably higher binding affinity for mu-opioid receptors (MORs) than for other opioid recep-tors, and its occupation can inhibit AC and cAMP formation, we hypothesized the EMF would affect the binding affinity of the MORs. What we found was that a statistically significant inhibition of cAMP did indeed occur in the EMF-exposed cells. EMF exposure on the MOR transfected cells reduced the amount of cAMP by 23% more as compared with the morphine-treated sample. CHO-K1 cell line produced results similar to basal (baseline) (Figure 2).

In the experimental group where MORs stimulated with forskolin increased cAMP levels, which were exposed to 5 Hz EMF [F + EMF], there was a

statistically significant (p < 0.05 for n = 4 samples) decrease in cAMP levels (Figure 3). After finding an inhibitory effect of EMF on cAMP at 5 Hz, we wanted to compare it with a different frequency in order to determine if the effect was frequency dependent. Therefore, we duplicated the experiment using 13 Hz to compare with the outcome of the 5 Hz treatment. This number was chosen due to the lack of published evidence of any therapeutic value related to its fre-quency. As shown in Figure 2, the effect of EMF on mu-cells was more inhibitive at 5 Hz than at 13 Hz (Figure 2a and c). While we hypothesized that EMF would improve the efficacy of morphine, this did not appear to be the case.

Data represent standard error of mean (S.E.M.). A statistically significant difference (p < 0.05) between morphine and EMF [F + M vs. F + EMF and F + M vs. F + M + EMF] was observed in cells exposed to 5 Hz frequency, but not between EMF and morphine com-bined [EMF + F vs. EMF + F + M]. EMF does not appear to counteract the inhibition potential of morphine.

Discussion

Pain killers target membrane transport proteins, including ligand- and voltage-gated ion channels. At ligand-gated ion channels, drugs can bind at the same site as the endogenous ligand and directly compete for the receptor site. Although membrane ion channels and protein phosphorylation can be indirectly affected by GPCRs through effector pro-teins such as AC and second messengers such as cAMP, they can short circuit the second messenger pathway and gate the ion channels directly (Brown et al., 1988). This bypassing of the second messenger pathway is observed in mammalian cardiac myocytes where Ca2+ channels are able to survive and function in the absence of cAMP, ATP, or protein kinase C (PKC), when in the presence of the activated α-subunit of the G protein (Yatani et al., 1987 ). Here

Gα, which is stimulatory to AC, acts on the Ca2+ channel directly as an effector. This short circuit is membrane-delimited, allowing direct gating of Ca2+ channels by G-proteins to produce effects more quickly than the cAMP cascade could (Brown et al., 1988). Some drugs directly bind and inactivate vol-tage-gated ion channels; these are ion channel pro-teins that do not have endogenous ligands, but open or close as a function of the membrane voltage potential. This membrane voltage potential has been suggested as the mechanism of action for the biolo-gical effect of EMF through the plasma membrane (Adey, 1974; Liboff, 1985; McLeod et al., 1987) – across which the EMF signal induces a voltage change (Figure 4).

Oscillating EMF exerts forces on free ions present on both sides of the plasma membrane that move across the cell surface through transmembrane proteins, creat-ing a forced intracellular vibration. This vibration is responsible for phenomena such as the influx of extra-cellular Ca2+, as well as efflux of intracellular Ca2+. A rise in cytosolic Ca2+ concentration is used as a signaling messenger in nearly all eukaryotic cells (Berridge et al., 2000; Clapham, 2008). Oscillations in cytosolic Ca2+ concentration are observed in all cell types, suggesting they represent a universal signaling mode (Thomas et al., 1996). Changes in the plasma membrane potential can alter the activity of voltage-gated Ca2+ channels leading to bursts of Ca2+ entry (Parekh, 2011), which can result directly in Ca2+ oscil-lations which can trigger second messengers through the activation of cell-surface receptors that activate enzymes such as AC, modulator of cAMP. Regardless of the mechanism, oscillations in cytosolic Ca2+ con-centration can be supported for a few minutes in the absence of external Ca2+, showing that Ca2+ recycling across the intracellular stores is the primary mechanism for driving them (Parekh, 2011). Oscillation triggered in Ca2+-free solution does decrease with time, because a fraction of the Ca2+ released during each cycle is trans-ported out of the cell, causing less Ca2+ to be available to support the next oscillation. To sustain oscillation requires Ca2+ entry, and this is accomplished through Ca2+ channels in the plasma membrane (Parekh, 2010; Parekh and Putney, 2005).

EMF has also been reported to be instrumental in the binding affinity of calmodulin (CaM) – the primary transduction pathway to second messengers cAMP, and cyclic guanosine monophosphate (cGMP), found to influence inflammatory pain (Pilla et al., 2011). Since EMF decreased the amount of cAMP by 23% compared with morphine, it is either causing a change in the conformation of the GPCRs, or it is affecting the sec-ond messenger cAMP via the voltage-gated calcium channels (VGCCs). This action could be achieved through several high thresholds, slowly activating Ca2+ channels in neurons which are regulated by G-proteins (Morris and Malbon, 1999). The activation of α-subunits of G-proteins has been shown to cause rapid closing of voltage-dependent Ca2+ channels, which causes difficulties in the firing of action poten-tials (Stryer et al., 2007). This inhibition of voltage-gated Ca2+ channels by GPCRs has been demonstrated in the dorsal root ganglion of a chick and other cells lines (Morris and Malbon, 1999). Other studies have indicated roles for Gα subunits in the inhibition of Ca2+ channels (Jeon et al., 2013; Zhang et al., 2008). Since receptors normally pick up signals in the cells’ environ-ment, and G-proteins couple these signals to the effec-tors (sending the signal to the cytoplasm), it is feasible that the EMF was able to short circuit the second messenger pathway and gate the ion channels directly. This would account for a moderately stronger down-regulation of cAMP with EMF treatment than with morphine.

Conclusion

The therapeutic effects of low-frequency EMF have been reported for years; however, a mechanism of action has yet to be elucidated. Here we compared the effects of EMF on cAMP at both 5 Hz and 13 Hz. The 5 Hz frequency showed a stronger inhibitory effect in cAMP expression on [F + EMF] than the 13 Hz fre-quency. EMF appears to competitively inhibit cAMP as morphine does; however, EMF exposure does not have the addictive side-effects of morphine. If EMF changes the conformation of the MOR receptor, along with stabilizing Ca2+ flux, then it would explain the outcomes we observed. If EMF is able to induce homeostasis via the stabilization of Ca2+, then it appears to be frequency specific as our study suggests.

Declaration of interest

The authors wish to thank the Guth Endowment Fund for providing financial support for this project (120-330-740196).

References

Adey, W. (1974). Dynamic patterns of brain cell assemblies.

  1. Mixed systems. Influence of exogenous low-level cur-rents. Are weak oscillating fields detected by the brain?. Neurosci. Res. Prog. Bull. 12:140–143.

Andreadis, S. G., Geer, D. J. (2006). Biomimetic approaches to protein and gene delivery for tissue regeneration. Trends Biothechnol. 24:331–337.

Bawin, S., Kaczmarek, L., Adey, W. (1975). Effects of modu-lated VHF fields on the central nervous system. Ann. N.Y. Acad. Sci. 247:74–81.

Benalal, D., Bachrach, U. (1985). Opiates and cultured neu-roblastoma x glioma cells. Effect on cyclic AMP and poly-amine levels and on ornithine decarboxylase and protein kinase activities. Biochem. J. 227:389–395.

Berridge, M., Lipp, P., Bootman, M. D. (2000). The versatility and universality of calcium signalling. Nat. Rev. Mol. Cell Biol. 1:11–21.

Billington, C., Penn, R. B. (2003). Signaling and regulation of G protein-coupled receptors in airway smooth muscle. Respir. Res. 4:2.

Brenner G., Steven, C. (2010). Pharmacodynamics. In: Pharmocology. 4th Ed, Chapter 3. Philadelphia: Elsevier. pp. 28–35.

Brown, A., Yatani, A., Imoto, Y., et al. (1988). Direct coupling of G proteins to ionic channels. Cold Spring Harb. Symp. Quant. Biol. 53:365–373.

CRR. (2013). Optimal care of chronic, non-healing, lower extremity wounds: A review of clinical evidence and guide-lines [Internet]. Report. Ottawa (ON): Canadian Agency for Drugs and Technologies in Health; 2013 Dec. Available from: http://www.ncbi.nlm.nih.gov/pubmedhealth/ PMH0064398/ (accessed 19 Sep 2014).

Chalidis, B., Sachinis, N., Assiotis, A., Maccauro, G. (2011). Stimulation of bone formation and fracture healing with pulsed electromagnetic fields: Biologic responses and clin-ical implications. Int. J. Immunopathol. Pharmacol. 24:17–20.

Clapham, D. (2008). Calcium signaling. Cell. 131:1047–1058. Collier, H. (1980). Cellular site of opiate dependence. Nature.

283:625–629.

Collier, H., Francis, D. (1975). Morphine abstinence is asso-ciated with increased brain cyclic AMP. Nature. 255:159–162.

Davis, K., Meyer, R. A., Campbell, J. N. (1993). Chemosensitivity and sensitization of nociceptive afferents that innervate the hairy skin on monkey. J. Neurophysiol. 69:1071–1081.

England, S., Bevan, S., Docherty, R. J. (1996). PGE2 modu-lates the tetrodotoxin-resistant sodium current in neonatal rat dorsal root ganglion neurones via the cyclic AMP-protein kinase A cascade. J. Physiol. 495:429–440.

ELECTROMAGNETIC BIOLOGY AND MEDICINE 7

Ferreira, S. (1981). Inflammatory pain, prostaglandin hyper-algesia and the development of peripheral analgesics. Trends Pharmacol. Sci. 2:183–186.

Fierreira, S., Nakamura, M., Castro, m. S. A. (1978). The hyperalgesic effects of prostacycline and prostaglandin E2. Prostaglandins. 16:31–37.

Fiorotto, M., Klish, W. J. (1991). Total body electrical con-ductivity measurements in the neonate. Clin. Perinatol. 18:611–627.

Funk, R., Monsees, T. (2006). Effects of electromagnetic fields on cells: Physiological and therapeutical approaches and molecular mechanisms of interaction. Cells Tissues Organs. 182:59–78.

Gourevitch, D., Kossenkov, A. V., Zhang, Y., et al. (2014). Inflammation and its correlates in regenerative wound healing: An alternative perspective. Adv. Wound Care (New Rochelle). 3:592–603.

Gris, P., Cheng, P., Pierson, J., et al. (2010). Molecular assays for characterization of alternatively spliced isoforms of the mu opioid receptor (MOR). Methods Mol. Biol. 617:421–435.

Gurtner, G., Werner, S., Barrandon, Y., Longaker, M. T. (2008). Wound repair and regeneration. Nature. 453:314–321.

Hannan, C., Liang, J., Allison, J., Pantazis, C., et al. (1994). Chemotherapy of human carcinoma xenografts during pulsed magnetic field exposure. Anticancer Res. 14:1521–1524.

Jeon, J., Roh, S. E., Wie, J., et al. (2013). Activation of TRPC4β by Gαi subunit increases Ca2+ selectivity and controls neurite morphogenesis in cultured hippocampal neuron. Cell Calcium. 54:307–319.

Karlsmark, T., Agersley, R. H., Bendz, S. H., et al. (2003). Clinical performance of a new silver dressing, Contreet Foam, for chronic exuding venous leg ulcers. J. Wound Care. 12:351–354.

Khasar, S. G., Ouseph, A. K., Chou, B., et al. (1995). Is there more than one prostaglandin E receptor subtype mediating hyperalgesia in the rat hindpaw? Neuroscience. 64:1161–1165.

Khoury, E., Clément, S., Laporte, S. A. (2014). Allosteric and biased g protein-coupled receptor signaling regulation: Potentials for new therapeutics. Front Endocrinol (Lausanne). 8:68.

Kuriyama, K., Nakagawa, K., Naito, K., Muramatsu, M. (1978). Morphine-induced changes in cyclic AMP meta-bolism and protein kinase activity in the brain. Jpn. J. Pharmacol. 28:73–84.

Li, S., Yu, B., Zhou, D., et al. (2013). Electromagnetic fields for treating osteoarthritis. Cochrane Database Syst. Rev. 12: CD003523.

Liboff, A. (1985). Geomagnetic cyclotron resonance in living cells. J. Biol. Phys. 13:99–104.

Malmberg, A., Brandon, E., Idzerda, R., et al. (1997). Diminished inflammation and nociceptive pain with pre-servation of neuropathic pain in mice with a targeted mutation of the type I regulatory subunit of cAMP-depen-dent protein kinase. J. Neurosci. 17:7462–7470.

Mamiya, T., Noda, Y., Ren, X., et al. (2001). Involvement of cyclic AMP systems in morphine physical dependence in mice: Prevention of development of morphine dependence by rolipram, a phosphodiesterase 4 inhibitor. Br. J. Pharmacol. 132:1111–1117.

Markov, M. (2007). Expanding use of pulsed electromagnetic field therapies. Electromagn. Biol. Med. 26:257–274.

Markov, M. (1981). Biological mechanisms of the magnetic-field action. IEEE Trans. Magn. 17:2334–2337.

Markova, A., Mostow, E. N. (2012). US skin disease assess-ment: Ulcer and wound care. Dermatol. Clin. 30:107–111.

McLeod, B., Smith, S. D., Cooksey, K. E., Liboff, A. R. (1987). Ion cyclotron resonance frequencies Ca++-dependent motility in diatoms. Bioelectromagnetics. 6:1–12.

Morris, A., Malbon, C. C. (1999). Physiological regulation of G protein-linked signaling. Physiol. Rev. 79:1372–1430.

O’Connor, M., Bentall, R., Monahan, J. (1990). Emerging Electromagnetic Medicine Conference Proceedings. New York: Springer-Verlag.

Osugi, T., Ding, Y., Miki, N. (1996). Characterization of single-stranded cAMP response element binding protein (ssCRE-BP) from mouse cerebellum. Ann. N. Y. Acad. Sci. 801:39–50.

Parekh, A. (2011). Decoding cyctosolic Ca2+ oscillations. Trends Biochem. Sci. 36:78–87.

Parekh, A. (2010). Store-operated CRAC channels: Function in health and disease. Nat. Rev. Drug Discov. 9:399–410.

Parekh, A., Putney, J. W. J. (2005). Store-operatied calcium channels. Physiol. Rev. 85:757–810.

Pilla, A., Fitzsimmons, R., Muehsam, D., et al. (2011). Electromagnetic fields as first messenger in biological sig-naling: Application to calmodulin-dependent signaling in tissue repair. Biochim. Biophys. Acta. 1810:1236–1245.

Rasmussen, K., Beitner-Johnson, D., Krystal, J., et al. (1990). Opiate withdrawal and the rat locus coeruleus: Behavioral, electrophysiological, and biochemical correlates. J. Neurosci. 10:2308–2317.

Reuff, A., Dray, A. (1993). Sensitization of peripheral afferent fibers in the in vitro neonatal rat spinal cord tail by bradykinin and prostaglandins. Neuroscience. 54:527–535.

Rohde, C., Chiang, A., Adipoju, O., et al. (2009). Effects of pulsed electromagnetic fields on IL-1b and post operative pain: A double-blind, placebo-controlled pilot study in breast reduction patients. Plast. Reconstr. Surg. 125:1620–1629.

Ross, C., Harrison, B. S. (2013). The use of magnetic field for the reduction of inflammation: A review of the history and therapeutic results. Altern. Ther. Health Med. 19:47–54.

Sadoghi, P., Leithner, A., Dorotka, R., Vavken, P. (2013). Effect of pulsed electromagnetic fields on the bioactivity of human osteoarthritic chondrocytes. Orthopedics. 36:e360–365.

Schultz, G., Sibbald, G. R., Falanga, V., et al. (2003). Wound bed preparation: A systematic approach to wound man-agement. Wound Repair Regen. 11:1–28.

Sen, C., Gordillo, G. M., Roy, S., et al. (2009). Human skin wounds: A major and snowballing threat to public health and the economy. Wound Repair Regen. 17:763–771.

Sharma, S., Nirenberg, M., Klee, W. (1975). Morphine recep-tors as regulators of adenylate cyclase activity. Proc. Natl. Acad. Sci. USA. 72:590–594.

Stiller, M., Pak, G. H., Shupack, J. L., et al. (1992). A portable pulsed electromagnetic field (PEMF) device to enhance healing of recalcitrant venous ulcers: A double-blind, placebo-controlled clinical trial. Br. J. Dermatol. 127:147–154.

Stryer, L., Berg, J., Tymoczko, M., John, L. (2007). Biochemistry (6th ed.). San Francisco: WH Freeman.

Sutbeyaz, S., Sezer, N., Koseoglu, F., Kibar, S. (2009). Low-frequency pulsed electromagnetic field therapy in fibro-myalgia: A randomized, double-blind, sham-controlled clinical study. Clin. J. Pain 25:722–728.

Taiwo, Y., Levine, J. D. (1989). Prostaglandin effects after elimination of indirect hyperalgesic mechanisms in the skin of the rat. Brain Res. 492:397–399.

Taiwo, Y., Levine, J. (1990). Direct cutaneous hyperalgesia induced by adenosine. Neuroscience. 38:757–762.

Taiwo, Y., Levine, J. (1992). Serotonin is a directly-acting hyperalgesic agent in the rat. Neuroscience. 48:485–490.

Taiwo, Y., Levine, J. (1991). Further confirmation of the role of adenyl cyclase and of cAMP-dependent protein kinase in primary afferent hyperalgesia. Neuroscience. 44:131–135.

Thomas, A., Bird, G. S., Hajnóczky, G., et al. (1996). Spatial and temporal spects of cellular calcium signaling. FASEB J. 10:1505–1517.

Thompson, W. (1991). Cyclic nucleotide phosphodiesterases: Pharmacology, biochemistry and function. Pharmacol. Therap. 51:13–33.

Traber, J., Gullis, R., Hamprecht, B. (1975). Influence of opiates on the levels of adenosine 3′:5′-cyclic monopho-sphate in neuroblastoma×glioma hybrid cells. Life Sci. 16:1863–1868.

Traina, G., Romanini, L., Benazoo, F., Cadossi, V. (1998). Use of electric and magnetic stimulation in orthopaedics and traumatology: Consensus conference. J. Ortho. Trauma. 24:1–31.

Tzschentke, T., Christoph, T., Kögel, B. Y. (2014). The mu-opioid receptor agonist/noradrenaline reuptake inhibition (MOR-NRI) concept in analgesia: The case of tapentadol. CNS Drugs. 28:319–329.

Vargas, M., Abella, C., Hernandez, J. (2001). Diazepam increases the hypothalamic-pituitary-adrenocortical (HPA) axis activity by a cyclic AMP-dependent mechan-ism. Br. J. Pharmacol. 133:1355–1361.

Xu, H., Lu, Y. F., Rothman, R. B. (2003). Opioid peptide receptor studies. 16. Chronic morphine alters G-protein function in cells expressing the cloned mu opioid receptor. Synapse. 47:1–9.

Yatani, A., Codina, J., Imoto, Y., et al. (1987). A G protein directly regulates mammalian cardiac calcium channels. Science. 238:1288–1292.

Zhang, Y., Chen, Y. H., Bangaru, S. D., et al. (2008). Origin of the voltage dependence of G-protein regulation of P/Q-type Ca2+ channels. J Neurosci. 28:14176–14188.