Electromagnetic Fields - ACS Publications - American Chemical Society

Downloaded by UNIV MASSACHUSETTS AMHERST on July 31, 2012 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/ba-1995-0250.ch024

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Membrane Signal-Transduction Mechanisms and Biological Effects of Low-Energy Electromagnetic Fields Richard A. Luben Division of Biomedical Sciences and Department of Biochemistry, University of California, Riverside, CA 92521

The cell membrane represents a significant barrier between the ex tracellularenvironment and the interior of the cell, both in terms of electrical resistance and in terms of information transfer. Any at tempt to explain the effects of low-energy electromagnetic fields (EMFs) on cells requires explanation of how the very low energy produced by environmental EMFs can overcome the barrier of the cell membrane. One plausible postulate is that low-energyfieldsmay interact with already existing membrane signal-transduction mech anisms,which possess extremely high sensitivity and specificity for detecting and transducing low levels of signal in the extracellular environment. In this chapter, basic mechanisms of membrane signal transduction are reviewed, and specific examples of low-energy EMF effects on these processes are cited. A biochemical model is devel oped for possible interactions between EMFs and intracellular sig nal-transduction processes.

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(EMFs) have been implicated as a possible causal factor in observed correlations between development of childhood cancers and proximity to high-current power lines (i). A major difficulty in accepting such fields as the cause of these correlations has been the lack of convincing data to indicate that fields of such low strengths can interact with N V I R O N M E N T A L E L E C T R O M A G N E T I C FIELDS

0065-2393/95/0250-0437$12.00/0 ©1995 American Chemical Society In Electromagnetic Fields; Blank, M.; Advances in Chemistry; American Chemical Society: Washington, DC, 1995.

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cells in any way that might lead to development of cancer or other disorders (2). For example, the electric potential produced in tissues by exposure to power lines in the average household are in the range o f 1 V / m (5), whereas the membrane potential of most mammalian cells is about 10 V/m. Nevertheless, a number of biological activities such as prédation and navigation by animals have been shown to respond to changes in electric potential as small as (or even smaller than) the value of 1 V / m (4). This laboratory has studied the interaction of low-energy nonionizing E M F s with cells and tissues in a system in which low-energy E M F s are generally acknowledged to have biological effects: the stimulation of bone formation and fracture healing. Bone healing stimulated by electromagnetic energy has been a widely used and very successful therapeutic model for a number of years (5). In addition to fracture healing, a number of reports demonstrate that bone turnover rates can be modulated by EMFs, and this response suggests that EMFs may be a possible therapy for osteoporosis (6). A number o f laboratories have proposed (7) mechanisms by which EMFs can alter bone metabolism; most have focused on the interaction between osteoblasts and regulatory agents such as hormones and growth factors. Our laboratory seeks to examine the mechanisms by which E M F s interact with cells, especially those that may be involved in production of changes in cell growth or differentiation. Our results indicate that EMFs can have effects on bone cells by modifying the sensitivity of membrane signal-transduction systems. In this chapter the focus is on possible biochemical mechanisms that may be influenced by E M F exposure and not on the direct biophysical interaction between the field and membrane molecules themselves. The subject of biophysical models o f interaction has been reviewed elsewhere in this book and in several recent reviews (8). In this discussion it is assumed that exposure of cells to a field existing in the external cell medium (e.g., the extracellular fluid in an animal) changes the properties of a susceptible type (or types) of molecule on the external surface of the cell membrane and that this change in properties is reflected by a change in function of one or more signal-transduction systems existing in the membrane (4). Other hypothetical biophysical mechanisms such as direct magnetic interaction with intracellular metals (9) may also operate either in parallel with or alternatively to the models discussed here.


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Receptor-Mediated Signal Transduction The cell membrane presents a significant barrier to charged molecules (e.g., peptide hormones and neurotransmitters) that may carry a regulatory signal from one cell type to another. Similarly, the membrane, because of its high resistance, presents a major barrier to electric currents that are flowing in the medium outside the cell. The mechanisms developed by living cells to sense the presence of signaling molecules outside the cell have many of the properties that are required to receive an electrical signal as well: for example, sensitivity, amplification, rectification, and transduction can be accomplished by the enzyme systems residing in cell membranes. The receptors for hormones, neurotransmitters, and

In Electromagnetic Fields; Blank, M.; Advances in Chemistry; American Chemical Society: Washington, DC, 1995.


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growth factors thus are logical targets for examination as the sites of E M F ef fects. It is necessary to understand the properties of such signal-transduction systems before we examine the possible ways in which EMFs may interact with the systems. One key feature of membrane signal-transduction systems is that the ac tivity of the overall pathway is largely under the control of a protein molecule known as a receptor. Receptors bind the signaling agent and pass on the signal to other components of the cell. Receptors may or may not have enzymatic activi ties of their own. Agents that bind to a receptor are called ligands. In general, specific ligands have a high affinity for their receptor, and the target tissue has a limited number of receptors; thus the specific binding sites tend to become satu rated at low concentrations of ligand. Only specific ligand binding generally results in a metabolically relevant response to the signal. The low concentrations of receptors in membranes is made up for by an extremely high affinity and specificity for the corresponding ligands. The signal-transducing activity of re ceptors is biochemically distinct from their ligand-binding properties; often the two activities reside in structurally distinct domains of the molecule. Receptors are susceptible in most cases to a variety of modulating agents, both intracellular and extracellular, such as antagonists, drugs, toxins, ionic composition of the medium, and, in the present case, electromagnetic energy. In many systems target cells are much less sensitive to a second dose of signaling ligand than to the first dose. This phenomenon is known as "desensitization" or "downregulation". These two terms are often employed to refer to two different mechanisms of decreasing tissue receptor responses. Downregulation usually refers to a process that produces a decreased number of binding sites for ligand in the tissue. The best established mechanism for this phenomenon is internalization and degradation of ligand-receptor complexes. Desensitization, on the other hand, refers to a diminished amplification of signal by membrane enzymes or other intracellular transduction processes. In several well-known systems (e.g., adrenergic receptors), this decrease in transduction capacity is mediated by phosphorylation of the receptor. Many different biochemical types of receptors exist, each of which util izes a different mechanism for transmitting the extracellular signal into the cell for further effects on biochemical pathways. Each receptor has, at a minimum, a portion of the molecule that binds ligands, a portion of the molecule that pierces the cell membrane, and a portion of the molecule that interacts with other intra cellular or membrane proteins to modulate their functions. Many receptors also have their own enzymatic activities that begin the cascade of events generating changes in cell function. Examples of different families of receptors are the ty rosine kinase family [e.g., the insulin receptor (10) and the epidermal growth factor receptor (11)]; the G-protein-linked receptor family (72), including recep tors for epinephrine and many other ligands; the ion-channel receptors (13), many of which interact with neurotransmitters; the nerve growth factor receptor family (14); the guanylate cyclase family of receptors (15); the tyrosine phos phatase family (16); and the Τ cell receptor and homologous receptors for lymphokines and cytokines (17). One benefit to the organism that is derived from


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having such complex mechanisms for membrane signal transduction is that the pathways can interact synergistically, a phenomenon known as "cross talk" (18). This cross talk increases the degree of amplification of signal and also allows for sophisticated control at a multitude of steps. Another benefit is that the multiple pathways can interact in feedback loops to help regulate each other. Thus the regulation of target tissue response can be at least partly handled by the tissue itself, without having to invoke systemwide feedback systems that may produce (possibly unwanted) side effects in a number of different tissues. This type of regulation is beneficial from both a heuristic and an energetic point of view. These receptor families present a significant but still limited variety of choices of possible mechanisms for transduction of the signal represented by the activated receptor into further intracellular actions. The detailed mechanisms of action of the various receptor types are well covered in other reviews (19). In this chapter we will focus on the ways in which bone cells in our laboratory have been found to respond to EMFs and the ways that these findings may illuminate the possible mechanisms of E M F effects on other cell types. The primary cell in which regulatory agents exert their effects on bone is the osteoblast; the osteoblast is in turn controlled primarily by the membrane receptors for parathyroid hormone (PTH). These receptors are members of the G-protein-linked family and carry out their actions by means of intracellular pathways involving adenosine 3',5'-cyclic monophosphate (cAMP), phospholipase C (PLC), protein kinase C (PKC), and C a (20). Evidence suggests that each of these pathways can be influenced by EMFs either directly or indirectly (7). Figure 1 depicts key points of osteoblast signal-transduction pathways. The key point in P T H action is activation of the receptor, which in turn causes activation of at least two GTPbinding membrane proteins (G proteins). These in turn modulate the activities of enzymes that produce modifications in cell activities. The following paragraphs describe the G-protein-linked activity of the P T H receptor in osteoblasts. Hormone binding induces a change in conformation of the P T H receptor. This change in conformation in turn increases the affinity of the receptor for membrane-resident G proteins. Many different types of G proteins exist, but they all share considerable sequence homology, and their functions are mediated in similar ways (21). The stimulatory G protein for adenylyl cyclase is designated G , and the inhibitory one is designated G\. Most evidence suggests that P T H primarily modulates the G protein in osteoblasts. The receptor-activated G protein binds G T P and activates adenylyl cyclase. The c A M P produced by adenylyl cyclase diffuses into the cytoplasm, where it binds to the c A M P dependent protein kinase (PKA). c A M P binds to the regulatory subunits of A kinase and causes dissociation of the protein and release of free catalytic subunits. These phosphorylate specific substrate proteins (their identities largely unknown in osteoblasts) and thereby lead to changes in their activities. In addition to activating G and adenylyl cyclase, the P T H receptor can also activate (by means of a different G protein having different specificities) P L C , which hydrolyzes the membrane phospholipid phosphatidylinositol bisphosphate (PIP2) to yield 1,4,5-trisphosphoinositol (IP ) and diacylglycerol 2 +

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Membrane Signal-Transduction Mechanisms of ELF EMFs 441

Figure 1. Major signal-transduction pathways in osteoblasts showing sites of action of extremely lowfrequency(ELF)fieldsfsee text for description). PTE is parathyroid hormone; PTHR is PTE receptor; PTH-LP is PTE-like peptide; 1,250E D is 1,25 dihydroxyvitamin D ; PLC is phospholipase C; PKC is protein kinase C; DAG is diacylglycerol; PIP is phosphatidylinositol bisphosphate; IP3 is 1,4,5-trisphosphoinositol; IP is 1,3,4,5-tetrakisphosphoinositol; AC is adenylyl cyclase; ALPase is alkaline phosphatase; VDR is vitamin D receptor; PDE is phosphodiesterase. (Reproduced with permissionfromreference 47. Copyright 1991.) 2

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(DAG). Both the IP and the D A G from this reaction can serve as second messengers (22). D A G activates a cellular protein kinase known as P K C (25). P K C phosphorylates a different set of proteins from protein kinase A (PKA), and this phosphorylation leads to a separate pattern of activities in the cell. The phorbol ester family of tumor promoter molecules, which are structural analogs of D A G , activates P K C independently of ligand-receptor signal transduction (24) and thereby produces inappropriate growth regulation in cells. Phorbol esters have been shown (25) to mimic some of the actions of P T H in bone cells by activating the P K C enzyme pathway. IP also acts as a second messenger in bone cells (19) by interacting with receptors on intracellular membranes to release free C a from intracellular stores (probably endoplasmic reticulum or a related vesicular organelle). This release can raise intracellular free C a by as much as tenfold 3

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(i.e., from around 10~ M to about ΚΓ M ) . This increase in turn can activate a variety of Ca -dependent processes such as calmodulin-dependent protein kinases and/or phosphodiesterases. IP also can be both phosphorylated and dephosphorylated to form other active metabolites, for example, 1,3,4,5tetrakisphosphoinositol (IP ), which seems to stimulate C a influx and efflux across the plasma membrane by opening ligand-gated and possibly also voltagegated ion channels. 2+

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Importance ofPhosphorylation in Signal Transduction Regulation of the phosphorylation status of intracellular proteins is a key feature of the vast majority of signal-transduction pathways (26). The structures and functions of receptors, enzymes, structural proteins (e.g., microtubules), and intracellular modifiers (e.g., transcription factors) can be altered dramatically by phosphorylation. Changes in activity also can be brought about by removal of phosphate from previously phosphorylated proteins (phosphatase activity). Pro teins may possess multiple possible phosphorylation sites, each of which may be phosphorylated or dephosphorylated by a different panel of enzymes. This prop erty leads to a multiplicity of different possible phosphorylation states for most proteins regulated by phosphorylation. A variety of different activities may in turn be associated with the different possible phosphorylation states of each protein. Thus different signal-transduction pathways can modulate the same proteins in different ways, by activating different patterns of protein kinases and protein phosphatases. Because of the multiplicity of possible regulation levels, signal-transduction pathways have great flexibility in responding to diverse needs of the organism. Protein kinases regulated by signal-transduction pathways are ubiquitous both in their number and in their substrate specificity; moreover, protein phos phatases may be nearly as universal (27). Signal-transduction pathways can modulate these kinases and/or phosphatases directly, as a result of processes set in motion by ligand binding to receptors, or indirectly, for example by stimulat ing the transcription of genes for kinase or phosphatase enzymes. Moreover, the response to signals may be determined by the preexisting phosphorylation state of receptors or other molecules participating in the pathway. Many of the inter actions between pathways referred to already are commonly manifested at the level of phosphorylation or dephosphorylation of intermediate modifiers com mon to the interacting pathways (e.g., the kinase and phosphatase enzymes themselves). Thus, phosphorylation and dephosphorylation may be said to be central regulatory mechanisms by which many i f not all signal-transduction pathways function and interact in the cell. Actual identities, however, are known for only a few of the multiple intracellular proteins phosphorylated or dephos phorylated in response to regulatory ligands. This area of research is active and expanding.


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Regulation of Cell Growth The relationships between signal-transduction processes, cell growth, differentiation, and neoplastic transformation of cells are far too complex for thorough discussion here. However, in the current context, two points should be made: first, many genes known to be oncogenes are clearly analogous to either hormones/growth factors or hormone/growth factor receptors. Second, intracellular regulatory pathways such as the cell division cycle discussed already and the promotion of differentiation and gene expression are very likely to be modulated by a multitude of signal-transduction pathways both in normal cells and in neoplastically transformed cells. For example, the well-known "tumor suppressor" R B gene product (28) not only acts as a cell cycle regulatory element but also undergoes a cell-cycle-dependent phosphorylation reminiscent of the cdc2 kinase (29). Moreover, the R B gene product is phosphorylated (inactive) in cells in which proliferation is induced by tumor promoters or differentiation agents that activate signal-transduction pathways, including P K C (30, 31). The R B gene product in turn appears to regulate the expression of c-fos, a transcriptional regulatory factor whose expression is tumorigenic, whose activity is regulated by phosphorylation, and which is the cellular homolog of one of the most potent viral oncogenes (32). These and many other examples suggest an intimate connection between signal-transduction pathways and the induction of tumorigenesis (33). Clearly, any environmental influence (e.g., E M F ) that modifies signaltransduction pathways in normal cells could also influence the potentially tumorigenic pathways in susceptible cells, either by enhancing the likelihood of transformation by other tumorigenic stimuli or by acting in a directly tumorigenic manner.

Potential Interactions of EMFs with Signal-Transduction Pathways Figure 2 summarizes several hypothetical ways in which low-energy E M F s could interact with membrane molecules to modulate or disrupt signaltransduction processes, by using the activation of adenylyl cyclase as a model (34). Each of the potential interactions with this signaling system could also be involved in interactions of extremely low frequency (ELF) EMFs with similar steps in other mechanisms of signal transduction. The model is based on the assumption that low-energy E M F effects are initially localized to the cell membrane. Possible interactions include the following. 1.

Ligand binding could be modified because of interactions of the field either with free ligand or with the receptor. Possible charge flows due to field oscillations or cyclotron resonance might produce changes in the conformations of protein domains critical for binding. Alternatively, changes in the membrane-extracellular fluid interface could produce less specific alterations in the binding constants of the reaction.



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Figure 2. Possible interactions between ELFfieldsand signal transduction by the G-protein-linked adenylyl cyclase activation system. See text for description. (Reproduced with permissionfromreference 47. Copyright 1991.)

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The conformational changes associated with activation of the receptor might be disrupted by E L F fields due to changes in membrane charge or phospholipid composition. Alternatively, the field might induce a specific change in receptor conformation, which would prevent both binding and activation by ligand. In some cases the receptor might be induced to undergo premature desensitization (or desensitization might be postponed and thus lead to enhanced activity of the receptor).

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Membrane fluidity might be changed either directly (e.g., by fieldinduced changes in charge distribution of phospholipid groups) or indirectly (e.g., by changes in intracellular phospholipid synthesis). This fluidity change in turn would lead to changes in the association kinetics of the separate protein components needed to form the ternary complexes required for activation of adenylyl cyclase.

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The properties, kinetics, and/or phosphorylation state of the G protein or its subunits could be altered. For example, even small changes in the GTPase activity of the

Electromagnetic Fields - ACS Publications - American Chemical Society

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