We will take a brief look at what cells are, and the stuff that is inside them, as our mental constructions that guide our modeling can often bring an unseen bias to bear upon our understanding of living systems. We will follow the historical development of concepts in cell physiology as it may be that historical inertia has held back our questioning of the most basic principles that underlie the modern conception of cell structure and function.
I. Opposing concepts in Cell Physiology: History & Background
1. The early history of cell physiology
Our initial understanding of the cellular nature of life came from the development of the microscope. In 1665, Robert Hooke described similarities between the fine structure of cork and a honeycomb under a magnifying glass. He called these small units cella, a Latin word for "small room", and later extended this name to describe similar units he saw in living plant tissues.
Anton Von Leeuwenhoek, the first person to observe "animalcules" through a microscope, didn’t at all like what he saw.
In the 1830's, the cell theory of living systems was refined by Schleiden & Schwann, stating that cells are the basic units of life. The internal contents of cells was called protoplasm and described as a jelly-like substance, sometimes called living jelly. At about the same time, colloidal chemistry began its development, and the concepts of bound water emerged. A colloid being something between a solution and a suspension, where brownian motion is sufficient to prevent sedimentation.
The idea of a semipermeable membrane, a barrier that is permeable to solvent but impermeable to solute molecules was developed at about the same time. The term osmosis originated in 1827 and its importance to physiological phenomena realized, but it wasn’t until 1877, when the botanist Pfeffer proposed the membrane theory of cell physiology. In this view, the cell was seen to be enclosed by a thin surface, the plasma membrane, and cell water and solutes such as K+ existed in a physical state like that of a dilute solution. In 1889 Hamburger used hemolysis of erythrocytes to determine the permeability of various solutes. By measuring the time required for the cells to swell past their elastic limit, the rate at which solutes entered the cells could be estimated by the accompanying change in cell volume. He also found that there was an apparent nonsolvent volume of about 50% in red blood cells and later showed that this includes water of hydration in addition to the protein and other nonsolvent components of the cells.
2. Evolution of the membrane and bulk phase theories
Two opposing concepts developed within the context of studies on osmosis, permeability, and electrical properties of cells. The first held that these properties all belonged to the plasma membrane whereas the other predominant view was that the protoplasm was responsible for these properties.
The membrane theory developed as a succession of ad hoc additions and changes to the theory to overcome experimental hurdles. Overton (a distant cousin of Charles Darwin) first proposed the concept of a lipid (oil) plasma membrane in 1899. The major weakness of the lipid membrane was the lack of an explanation of the high permeability to water, so Nathansohn (1904) proposed the mosaic theory. In this view, the membrane is not a pure lipid layer, but a mosaic of areas with lipid and areas with semipermeable gel. Ruhland refined the mosaic theory to include pores to allow additional passage of small molecules. Since membranes are generally less permeable to anions, Michaelis concluded that ions are adsorbed to the walls of the pores, changing the permeability of the pores to ions by electrostatic repulsion. Michaelis demonstrated the membrane potential (1926) and proposed that it was related to the distribution of ions across the membrane. Harvey and Danielli (1939) proposed a lipid bilayer membrane covered on each side with a layer of protein to account for measurements of surface tension. In 1941 Boyle & Conway showed that the membrane of frog muscle was permeable to both K+ and Cl-, but apparently not to Na+, so the idea of electrical charges in the pores was unnecessary since a single critical pore size would explain the permeability to K+ , H+, and Cl- as well as the impermeability to Na+, Ca+, and Mg++.
Over the same time period, it was shown (Procter & Wilson, 1916) that gels, which do not have a semipermeable membrane, would swell in dilute solutions. Loeb (1920) also studied gelatin extensively, with and without a membrane, showing that more of the properties attributed to the plasma membrane could be duplicted in gels without a membrane. In particular, he found that an electrical potential difference between the gelatin and the outside medium could be developed, based on the H+ concentration. Some criticisms of the membrane theory developed in the 1930's, based on observations such as the ability of some cells to swell and increase their surface area by a factor of 1000. A lipid layer cannot stretch to that extent without becoming a patchwork (thereby losing its barrier properties. Such criticisms stimulated continued studies on protoplasm as the principle agent determining cell permeability properties. In 1938, Fischer proposed that water in the protoplasm is not free but in a chemically combined form—the protoplasm represents a combination of protein, salt and water—and demonstrated the basic similarity between swelling in living tissues and the swelling of gelatin and fibrin gels. Nasonov (1944) viewed proteins as the central components responsible for many properties of the cell, including electrical properties.
By the 1940’s, the bulk phase theories were not as well developed as the membrane theories. In 1941, Brooks & Brooks published a monograph The Permeability of Living Cells, which rejects the bulk phase theories
3. The emergence of the steady-state membrane pump concept
With the development of radioactive tracers, it was shown that cells are not impermeable to Na+. This was difficult to explain with the membrane barrier theory, so the sodium pump was proposed to continually remove Na+ as it permeates cells. This drove the concept that cells are in a state of dynamic equilibrium, constantly using energy to maintain ion gradients. In 1935, Lohmann discovered ATP and its role as a source of energy for cells, so the concept of a metabolically-driven sodium pump was proposed.
The tremendous uccess of Hodgkin, Huxley, and Katz in the development of the membrane theory of cellular potentials, with differential equations that modeled the phenomena correctly, provided even more support for the membrane pump hypothesis.
The modern view of the plasma membrane is of a fluid lipid bilayer that has protein components embedded within it. The structure of the membrane is now known in great detail, including 3D models of many of the hundreds of different proteins that are bound to the membrane.
These major developments in cell physiology placed the membrane theory in a position of dominance and stimulated the imagination of most physiologists, who now apparently accept the theory as fact—there are, however, a few dissenters.
4. The reemergence of the bulk phase theories
In 1956, Troshin published a book, The Problems of Cell Permeability, in Russian (1958 in German, 1961 in Chinese, 1966 in English) in which he found that permeability was of secondary importance in determination of the patterns of equilibrium between the cell and its environment. Troshin showed that cell water decreased in solutions of galactose or urea although these compounds did slowly permeate cells. Since the membrane theory requires an impermeant solute to sustain cell shrinkage, these experiments cast doubt on the theory. Others questioned whether the cell has enough energy to sustain the sodium/potassium pump. Such questions became even more urgent as dozens of new metabolic pumps were added as new chemical gradients were discovered.
In 1952, Gilbert Ling became the champion of the bulk phase theories (almost completely isolated from the rest of biology for his cheek) and proposed his association-induction hypothesis of living cells.
II. The Association-Induction Hypothesis
The association-induction (AI) hypothesis is based on the concept that water in cells is in a unique state and that the living state does not exist without such water. This state is called polarized multilayers and relies on the spacing of polarized groups on the protein backbone to increase the effective distance over which they affect water molecules. Water in polarized multilayers is in a higher energy state than in bulk water and can differentially exclude solutes. The AI hypothesis holds that the majority of intracellular ions are adsorbed onto charged sites on macromolecules; the preferential adsorption of K+ being due to the inductive effect of the neighboring molecular groups affecting the electron densities of carboxyl groups on proteins. Surprisingly, this apparently radical theory yields almost identical experimental predictions as the membrane pump theory for most questions. Ling and others have doggedly pursued the areas where there seem to be differences in the predictions, and have claimed success in showing that the AI hypothesis is the better.
Biologists, unlike physicists, seems to show very little inclination to question the fundamental building blocks of the modern conceptual model. Thus Ling and his followers have been largely ignored or dismissed as crackpots. Interestingly, Raymond Damadian, the inventor of MRI based the idea on the AI hypothesis. At worst, they have exposed many weaknesses involved with treating cells as bags of salty water.
In cryobiology, we depend on models of the behavior of cells with respect to mass transfer at various temperatures. It is convenient to treat the cell like a sac of ordinary water with ions dissolved in it, with the permeability barrier existing at the plasma membrane, but one should always remind themselves that this is a model of convenience. The evidence is now clear that water and ions in cells behave differently than in bulk solution (at least some compartments do). A re-evaluation of the assumptions in ideal solution theory are now overdue; perhaps the next decade will see the development of basic hypotheses describing the state of water in cells.
The outside of a cell is a complicated structure.
And the inside of a cell is even worse. The contents of a cell are now called 'cytoplasm' rather than 'protoplasm' to reflect the fact that it is considered to be a normal aqueous solution.
Consider these images when modeling the interior of the cell as a dilute aqueous solution.
Document last updated Oct. 12, 1998.
Copyright © 1998, Ken Muldrew.