Table Of ContentTransmembrane Potentials
and
Characteristics
of
Immune and Tumor Cells
Editor
Richard C. Niemtzow, M.D., Ph.D.
Major, USAF, MC
Department of Radiology
Radiation Oncology Section
Malcolm Grow USAF Medical Center
Andrews AFB, Maryland
Boca Raton London New York
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PREFACE
Historically, interest in the investigation of cell membranes has fallen somewhere among
the hematologist interested in the surface properties of the membranes of erythrocytes; the
physiologist interested in excitability, impulse generation, and synaptic transmission; the
histologist interested in the characteristics of the fixed preparation; the biochemist interested
primarily in cellular metabolic events; and the cellular physiologist interested in cellular
function. Sometimes overlooked by these investigators has been the contribution of the cell
membrane in regulating total organism homeostatic balance.
In particular, the importance of transmembrane potentials as a measure of cell function
and activity has not been recognized by the medical profession. While there has been
considerable work on the evaluation of the role of the cell membrane in the generation and
transmission of electrical impulses in tissues such as muscles and neurons, it has only been
within the past decade that the cell membrane has been recognized as an important regulator
in many critical cellular functions.
With knowledge that is now available, it seems appropriate to turn our attention to these
areas of membrane function and apply our knowledge of membrane properties in an effort
to understand the role that membrane events play in normal cellular function and in the
integration of various cells into major tissue and organ systems. Once these questions are
answered, we should then turn to the obvious problem of perhaps being able to intervene
so that abnormal cell membrane functions can be corrected. It may well be that the resolution
of these issues could provide the solution to the problems of dysplastic growth that is seen
in cancer and to the secrets of the entire germination process related to the initiation of the
growth and development of the organisms.
As emphasized by the various authors of this timely text, changes in the cell membrane
associated with responses of the immune system display specific characteristics which can
be associated with various states of rest and stimulation. The activated macrophage and, to
a lesser extent, the other mononuclear cells of the immune system have characteristic mem-
brane properties which can serve as markers of cellular\activity. These properties are both
reproducible and indicative of the initiation of important cellular and immunologic functions.
These characteristics have been well substantiated enough that it seems reasonable to think
in terms of the classification of cells according to their “fingerprint” as characterized by
their electrophysiological qualities.
The cell membrane acts as the interface between the intracellular environment and the
external milieu. Therefore, the membrane must act as the primary interactive site for external
agents, To do this, cells of the immune system have developed specialized surface receptor
molecules which recognize molecules acting as chemical messengers for the immune system.
That lymphokines from a human T cell supernatant can activate macrophages and lympho-
cytes while ions are otherwise controlled suggests one or more of the surface membrane
receptors may interact with this hormone-like material to mediate substantial alteration of
intracellular processes. Besides specific, receptor-mediated responses, there is fairly con-
vincing information that cellular protein synthesis can be initiated thrpugh altered concen-
trations of the common physiological ions and that control of cellular division can similarly
be controlled in vitro.
The sources of the different transmembrane potentials of various specialized cells, the
role of specific ions in initiating cellular events, the function of various transport mechanisms
designed to maintain homeostasis, and the role of hormones, enzymes, and other organic
molecules remains to be completely explained. Recent improvements in technology, in-
cluding the ability to measure various cellular events with various types of microelectrode
measurements and the ability to grow various cells in culture offer an opportunity to extend
our knowledge in these areas.
The series of chapters of this book represent a major step in synthesizing our understanding
of the events that occur in the body defense mechanism that we now know as the immune
system. That we can reproduce desirable changes in the basic cells of the immune system
that represent changes observed in vivo suggests that we are getting closer to being able to
manipulate events that can control various cellular activities and that we might soon be able
to alter the extracellular environment to produce various beneficial cellular changes. It is
becoming progressively more clear that the events which occur at the membrane level,
recorded as transmembrane potentials, represent a mystery that is deserving of complete
definition. This area of research will indeed be exciting to watch.
Ralph D. Reynolds, M.D.
INTRODUCTION
This book is a unique effort to review published works and to encourage investigation
into the young field of immunological and neoplastic mechanisms through the measurement
of transmembrane potentials. This is an exciting application of a biological measurement to
the field of immunology, because classically, interest in the measurement of transmembrane
potentials has generally centered only on “excitable” cells such as neurons and muscles.
The orientation of this book primarily focuses on human and murine mononuclear cells
and the neuroblastomas. It is assumed that the reader desires to understand the theoretical
knowledge not only of techniques, but of the latest concepts of cellular physiology as it
relates to electrokinetic events at the membrane level. It is hoped that this field of interest
will develop and mature as has already been noted in the well-established area of excitable
cell electrophysiology.
All living cells exhibit small electrical voltages across their membrane. These electrical
voltages, called transmembrane potentials, can be correlated directly with the state of the
membrane and the metabolic processes that alter the movements of molecules across the
membrane and the inherent membrane permeability. Immune cells exhibit transmembrane
potentials which are characteristic of their immunological role. These electrophysiological
events offer a useful means to better understand and appreciate immunological mechanisms
at the cellular level.
Tumor cells also exhibit certain electrokinetic activity which may be specific to neoplastic
development. The permeability of these membranes to specific ionic movements may be
necessary to assure cellular division. The magnitude of the voltages and the configuration
of the membrane to accommodate the channeling of specific ions may be significant to
cellular division and development.
The classical technique of measuring transmembrane potentials requires consideration of
three principal elements: (1) the glass microelectrode and its properties, (2) the microman-
ipulator with optical cellular identification, and (3) the electronic equipment to amplify and
discriminate useful information. The literature abounds with detailed information, and for
this reason, the subject is covered briefly along with a technique to suspend cells for the
purpose of achieving multiple microglass electrode penetrations with little physiological and
electrical artifacts in the chapter dealing with “Transmembrane Potentials of Human Lym-
phocytes.” Intracellular fluorescent dyes as voltage indicators will be considered as alter-
native means of measurements as these techniques are becoming increasingly more popular.
Cellular membrane physiology is developed to include the theory of transmembrane po-
tentials, the membrane model, the Hodgkin-Huxley model of specific ionic channels, the
ionic properties of resting and active membranes, and the independent effects of sodium,
potassium, and other ions on the generated potentials.
A modem and brief presentation of optical and electronic microscopic description will
delineate the basic morphology of human and murine mononuclear cells. Neoplastic cell
criteria and morphology will be briefly summarized. The immunology and role of the
lymphocytic and phagocytic cells will be described as well as their separation techniques.
The text then turns from the fundamental concepts and techniques to focus in detail on
the transmembrane potentials of the lymphocyte, macrophage and the neuroblastoma cell.
It is hoped that these basic expositions will encourage further creative application of elec-
trophysiological techniques into the area of immunology and cancer.
In 1971, Dr. Clarence D. Cone, Jr. published a paper in the Journal of Theoretical Biology
entitled “Unified Theory on the Basic Mechanism of Normal Mitotic Control and Onco-
genesis”, a paper which has since become a classic in the field of mitogenesis regulation.
Since Cone’s theory of mitogenesis regulation aptly ties together in functional relationship
many of the primary immunological and oncological mitogenic phenomena covered in this
present volume, the paper is being reproduced here in original form with the permission of
Academic Press, Inc. (London). A brief discussion by Dr. Cone of subsequent work based
on the “sodium theory of mitogenesis control” along with additional potential implications
is presented following the reprint.
Finally, the last chapter is a clinical approach of extracting skeletal transmembrane po-
tential information from the patient. The transfer of information from the human organism
to the electronic circuit should neither be accepted as a simplistic approach nor ignored
because of its possible lack of explanation due to complex organic events. Transmembrane
potentials measured directly from skeletal muscle as a convenient means of biological status
in the cancer patient being treated by both radiation and chemical therapies remains to be
developed. This proposal has a certain appeal, especially when neoplastic and immunological
mechanisms can be explored electronically in vivo.
Opinions and viewpoints expressed herein by the writers and publisher are their own and
not to be considered an official expression of the Department of the Air Force.
Richard C. Niemtzow, M.D., Ph.D.
THE EDITOR
Richard C. Niemtzow, M.D., Ph.D., did his undergraduate work at Emory University,
Atlanta, Georgia and Goddard College, Plainfield, Vermont, where he received a Bachelor
of Arts degree. He obtained his M.D. degree at the Faculty of Medicine of Montpellier
University, Montpellier, France at the end of 1977. Dr. Niemtzow’s research interests were
fostered and developed at the Department of Chemotherapy and Immunotherapy under the
direction of Dr. Bernard Serrou and at the National Institute of Biomechanics under the
direction of Professor Pierre Rabischong while completing his training in France. Dr. Niem-
tzow completed his residency in radiation oncology at the University of Texas Medical
Branch, Galveston, Texas, in 1981. During his residency, he published numerous papers
and a book chapter dealing with transmembrane potentials of mononuclear cells. He was
the recipient of several grants dealing with this research and helped organize an International
Symposium on the Fundamental Mechanisms in Human Cancer Immunology at Galveston,
Texas. In 1981, Dr. Niemtzow served as a radiation oncologist at David Grant USAF Medical
Center, Travis AFB, California. In April 1983, he was transferred to Malcolm Grow USAF
Medical Center, Andrews AFB, Maryland as chief clinical investigator to run a FDA-
approved clinical trial dealing with cancer therapy. He is currently continuing development
in new cancer therapeutic modalities and is also practicing radiation oncology at Malcolm
Grow USAF Medical Center where he is a staff physician in the United States Air Force.
Dr. Niemtzow received his Ph.D. degree in Biological Sciences from Pacific Western
University, California, in February 1985.
CONTRIBUTORS
Clarence D. Cone, Jr., Ph.D. Richard C. Niemtzow, M.D., Ph.D.
President Major, USAF, MC
Therapeutical Systems Corporation Department of Radiology
Yorktown, Virginia Radiation Oncology Section
Malcolm Grow USAF Medical Center
Andrews AFB, Maryland
Douglas C. Eaton, Ph.D.
Professor of Physiology and Biophysics J. Regino Perez-Polo, Ph.D.
University of Texas Medical Branch Professor of Biochemistry
Galveston, Texas Department of Human Biological Chem-
istry and Genetics
University of Texas Medical Branch
Hardin Jones, Jr., Ph.D. Galveston, Texas
Research Associate, Physiology
Clinical Investigation Center Ralph D. Reynolds, M.D.
Naval Regional Medical Center Chairman
Oakland, California Department of Medicine
Ellis Fischel State Cancer Center
Columbia, Missouri
J. Hamilton Licht
Commander, Medical Corps, USNR Cynthia Robbins-Roth, Ph.D.
Head, Nephrology Division Technology Associate
Assistant Director, Clinical Investigation Business Development
Center California Biotechnology, Inc.
Naval Regional Medical Center Mountain View, California
Oakland, California and
Assistant Clinical Professor of Medicine Jeffrey L. Rossio, Ph.D.
University of California Program Resources, Inc.
San Francisco, California NCI — Frederick Cancer Research Facility
Frederick, Maryland
Richard G. Moulton, Ph.D. Joseph A. Sisson, M.D.
Associate Professor Chairman
School of Basic Life Sciences Department of Pathology
University of Missouri—Kansas City Franklin Square Hospital
Kansas City, Missouri Baltimore, Maryland
TABLE OF CONTENTS
Chapter 1
The Electrical Properties of Cells............................................................................................... 1
Douglas C. Eaton
Chapter 2
Use of Fluorescence as a Voltage Indicator in Mononuclear Cells ....................................21
Jeffrey L. Rossio and Richard C. Niemtzow
Chapter 3
Lymphocytes and the Immune Response...............................................................................33
Cynthia Robbins-Roth
Chapter 4
The Role of the Macrophage in Immunity..............................................................................43
Richard G. Moulton
Chapter 5
Morphology of Mononuclear and Malignant Cells..................................................................61
J. A. Sisson
Chapter 6
Transmembrane Potentials of Human Lymphocytes..............................................................69
Richard C. Niemtzow
Chapter 7
Membrane Properties of Peritoneal Macrophage.....................................................................87
Douglas C. Eaton
Chapter 8
Neuroblastoma: Maturation and Differentiation.....................................................................103
J. Regino Perez-Polo
Chapter 9
Unified Theory on the Basic Mechanism of Normal Mitotic Control and
Oncogenesis.................................................................................................................................117
Clarence D. Cone, Jr.
Chapter 10
Measurement of Transmembrane Potential in the Study of Systemic Disease...............143
J. Hamilton Licht and Hardin Jones
Index 155
