Assisi Loop targets

Assisi Animal Health markets a pulsed radio frequency electromagnetic emitting device that has been specifically designed to facilitate the binding of Ca²⁺ to small and very well conserved protein called calmodulin. This device has been through numerous clinical trials with promising results. Much of the marketing seems to be focused on CaCaM activating nitric oxide synthase as a mechanism of action. This post concludes with another possible mechanism: CaCaM inactivating TRPV1, the target of capsaicin creams and CBD.

Featured Image: Calmodulin introduction

Calmodulins bind calcium ions at both the N- and C- termini. the featured image from RCSB shows a variety from the single cell organism called Paramecium. Calmodulins have positive (blue) and negative charges (red). Ca²⁺ binding sites are found on both flexible sides. The binding pockets have flexible amino acid side chains also with charged residues that are subject to thermal motion. The thermal motion figured into mathematical predictions by Arthur Pilla and coworkers.

Colacicco and Pilla earlier work [1,2]

Some of the earlier published work was using a device based on US patent 4105017.

Each positive pulse signal portion is composed of at least three segments, of which the peak amplitude of the final segment is no less than about 25 percent of the peak amplitude of the first segment;

• positive pulse signal duration 0.2-1msec
• < ~ 1/9 the duration of the negative pulse signal
• repetition rate of the pulses 10-100 Hz;
• the frequency of the pulse portions within each pulse group 2000 and 10000 Hz.,

The patented electromagnetic generator, provided by Electro-Biology, Inc., seems to be different from the Assiss. The older device consisted of

• asymmetric pulses trains of 5 ms duration
• repetition rate of 15 Hz.
• rate of change of the magnetic field was 0.1 G /|is for the main polarity pulse.

Tibiae from chicken embryos.

Much of the discussion in this paper involved Na⁺/K⁺ and Ca²⁺ ATPase in biological function and the thinking of the day that PEMF affected these functions.

treatment	bicarb	phosphate	acetate	phosphate	bicarb +	phosphate	pH5
buffer detail	20	20	10	5	140	20	50
Control	24.1	25	1.2	11.5	20.4	34.1	33.6
Field	28.9	29.1	1.3	11.7	28.1	46.8	38.2

A focus on ATP

These authors switched to a cancer cell line for focus on the Na⁺/K⁺-ATPase, or sodium pump. Ouabain inhibits this Na Pump. Mg²⁺ is the preferred counter ion for negatively charged ATP.

	cont	1mM oabain	2.5mM NaATP	2.5mM MgATP
Control	33000	16000	22000	16000
Field	33000(100)	16000 (100)	26 000 (118)	29 000 (181)

Huehsam & Pilla: the Lorentz Model and Thermal Noise [3,4]

These authors addressed some criticisms of the ion cyclotron resonance ICR model:

• It does not address the viscosity of the biological fluid.
• the diameter of the cyclotron orbit would be much larger than the total size of the biological target itself
• The rotation of the target protein and the shape of the active sites were discussed.
• Citing previous work: “the induced electric field from an AC magnetic field in the order of 100 mT with frequency below 100 Hz is not expected to produce significant bioeffects”
  

Some of the differential equations are hard to follow. One of the 1988 papers on energetics of Ca²⁺ binding to calmodulin. Ca2+ was given two sites on each end (C- and N-terminus). Ca²⁺ is continuously coming on and off each site. The occupancy of one site may influence the binding to other sites.

[4] attempted to fine tune the equations and assumptions regarding CaCaM and PC-`12 neurite outgrowth studies of Blackman covered on this site in a previous post. Panel B of Figure 6 attempted to fit the model to the data. It is beyond the scope of this post to evaluate whether or not the authors performed a good job based on what we know 15 years later. Instead a PDF file of this publication was keyword searched for “Hz” and “q/m” to gain a better idea of the thought processes that went into the development of the Assissi loop. “24 Hz in previous studies Larmor frequency, suggests a charge –mass ratio equal to that of unhydrated Mg²⁺ as a potential target for the magnetic field effect.”

We have a huge disconnect with some very fancy mathematics, data from other publications concerned with unhydrated Mg²⁺ resonance AND previous experimental data collected with a patented device that seems to be concerned with Mg²⁺ ATP and the pumps it powers. One may suppose that a good portion of the cellular Mg²⁺ is stabilizing ATP.

A transition to CaCaM and eNOS [5]

The logic behind this publication is a little bit obscure. CaCaM has so many targets including myosin light chain kinase reported in earlier literature. Endothelial nitric oxide synthase has so many modes of regulation. Without question CaCaM is one of them.

The premise that one can use a radio frequency PEMF device to selectively enhance the binding of Ca²⁺ to calmodulin and only activate eNOS is unrealistic. Other factors may be regulating eNOS. The above image illustrates phosphorylation on serine amino acid side chains by: protean kinase A (PKA), Ca²⁺CaM kinase II (CAMKII), AMP regulated kinase, and so on. Glutationylation is the attachment of glutathione to cysteine side chains. Nitrosylation is the attachment of nitric oxides to cyteine side chains. ,

A neuronal cell line was used in this study. The EMF signal consisted of a 27.12 MHz radio frequency (RF) carrier, pulse-modulated with a 2 ms burst repeating at 2 Hz, known commonly as a pulsed radio frequency (PRF) signal configured on a theoretical model of Ca²⁺ binding to CaM as the target pathway. CaCaM dependent signaling, increased immediate NO release from MN9D cells in response to an acute non-toxic LPS challenge by approximately three-fold vs. that from control samples (P < 0.001)

Fig. 1. Effect of a non-thermal pulse-modulated RF signal on peak NO release from
MN9D dopaminergic cells during challenge with LPS (100 ng/mL). NO was measured in real-time with a NO selective electrode. Results show EMF produced an immediate (<5 s) three-fold increase in peak NO. Fig. 2. Effect of 15 min exposure to the identical RF signal on NO in conditioned medium from human fibroblasts. The RF signal increased NO (via Griess determination of nitrite) about two-fold. The CaM antagonist W-7 blocked the EMF effect, but had no effect on baseline NO already present from the serum depletion
challenge

• Peak nitric oxide production was increased ~4x over the control
• Adding a CaM blocker prevented the increase in nitrite, a degradation product of nitric oxide.

Before his death Dr Pilla published a review of nitric oxide / vascular related studies with his radio frequency PEMF device. [6] RF PEMF reduced knee pain in osteoarthritis as well as post-operative pain in breast reduction patients. IL-1b in wound exudates was concomitantly 2.5-fold higher in the control “Results suggest that PRF acted through the CaM/NO signaling pathway.” This review presented data from a rat model of myocardio infarction and subsequent blood vessel regrowth. Inhibition of nitric oxide synthase inhibitor L-NAME reversed protection via RF PEMF. In 2005 the human proteome was screened for CaCaM binging proteins revealing many known and previously unknown targets. [7]

An Assisi canine post operative pain clinical trial

In 2019 a (mostly) Dachshund clinical trial of the Pilla rfPEMF device was used to handle post operative pain for surgery to correct acute intervertebral disc extrusions (IVDEs) and the resulting spinal cord injury. The potential for neuroprotective effects was also examined. Treated dogs in this randomized trial received PEMF (15 min every 2 h for 2 weeks then twice-daily for 4 weeks) or placebo starting immediately after diagnosis.

• Plasma glial fibrillary acidic protein (GFAP) concentration was measured as a spinal cord injury marker. significantly lower at 2 weeks in the PEMF group.
• Post-operative pain was quantified by measuring mechanical sensory thresholds (MSTs) at control and surgical sites and was significantly improved by PEMF
• There was no difference in primary outcome or in secondary measures of gait, but proprioceptive placing was significantly better at 6 weeks

Somehow the discussion kept to the company’s origins. The mechanism of action was attributed to the the calcium-calmodulin (CaM) nitric oxide (NO) cascade. Activation of the CaM/NO/ cGMP pathway downregulates IL-1b, limiting inflammation and reducing inducible NOS (iNOS) activity as well as increasing production of fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor. The authors cited literature regarding these pathways. [8]

Assissi Loop target: Transient Receptor Potential channels

Many transient potential receptor Ca²⁺ channel family members have sites that bind Ca²⁺ CaM. TRPV1, the capsaicin (chili pepper) receptor is one example with two CaM binding sites..

Excellent cartoon of TRP family member domains

Click on the above link for an image that has got to be copy right protected. The point is that CaC²⁺ -calmodulin has targets that go far beyond nitric oxide synthase.

The green pore is where Ca²⁺ enters the cell from the outside. NOTE: Only two of the four subunits of TRPV1 are shown here. One is gray and the other is color coded. Two more subunits are behind the plan of view.

The Ca²⁺-calmodulin tuned Assisi Loop might be targeted more than just nitric oxide synthase isoforms. It might also be turning off TRPV1 and other family members of this super family. There are many ways to activate eNOS and inactivate TRPV1. Perhaps some specificity may be enabled with the Assisi Loop by use of these factors.

References

1. Colacicco G, Pilla AA. Electromagnetic modulation of biological processes: bicarbonate effect and mechanistic considerations in the Ca-uptake by embryonal chick tibia in vitro. Z Naturforsch C Biosci. 1983 May-Jun;38(5-6):465-7. Sci-Hub free article
2. Colacicco G, Pilla AA. Electromagnetic modulation of biological processes: ATPase function and DNA production by Raji cancer cells in vitro. Z Naturforsch C Biosci. 1983 May-Jun;38(5-6):468-70 Sci-Hub free article
3. Muehsam DJ, Pilla AA. A Lorentz model for weak magnetic field bioeffects: part I–thermal noise is an essential component of AC/DC effects on bound ion trajectory. Bioelectromagnetics. 2009 Sep;30(6):462-75 Sci-Hub free article
4. Muehsam DJ, Pilla AA. A Lorentz model for weak magnetic field bioeffects: part II–secondary transduction mechanisms and measures of reactivity. Bioelectromagnetics. 2009 Sep;30(6):476-88. free article
5. Pilla AA. Electromagnetic fields instantaneously modulate nitric oxide signaling in challenged biological systems. Biochem Biophys Res Commun. 2012 Sep 28;426(3):330-3. Sci-Hub free paper
6. Pilla AA. Pilla AA. Nonthermal electromagnetic fields: from first messenger to therapeutic applications. Electromagn Biol Med. 2013 Jun;32(2):123-36. Electromagn Biol Med. 2013 Jun;32(2):123-36. Sci Hub free paper
7. Shen X, Valencia CA, Szostak JW, Dong B, Liu R. Scanning the human proteome for calmodulin-binding proteins. Proc Natl Acad Sci U S A. 2005 Apr 26;102(17):5969-74. PMC free paper
8. Zidan N, Fenn J, Griffith E, Early PJ, Mariani CL, Muñana KR, Guevar J, Olby NJ. The Effect of Electromagnetic Fields on Post-Operative Pain and Locomotor Recovery in Dogs with Acute, Severe Thoracolumbar Intervertebral Disc Extrusion: A Randomized Placebo-Controlled, Prospective Clinical Trial. J Neurotrauma. 2018 Aug 1;35(15):1726-1736. free paper