Life at the Cell and Below-Cell Level. The Hidden History of a Fundamental Revolution in Biology
Gilbert N. Ling, Ph.D.
Pacific Press
ISBN 0-9707322-0-1

"Dr. Ling is one of the most inventive biochemist I have ever met."
Prof. Albert Szent-Györgyi, Nobel Laureate

Chapter 4.

Evidence for a Cell Membrane Covering All Living Cells
(p. 14-25)

The following is a summary of the evidence for the existence of a cell membrane covering all living cells.

4.1 From studies of cell volume changes and solute permeability

Cell volume change, the first subject matter of cell physiological experi­mentations and observations, reflects largely the movement of water in and out of cells. In the membrane theory, water movement in turn reflects the permeability of the cell membrane to water, and the permeability or imper­meability to substances dissolved in water.

(1) A semipermeable diffusion barrier at the cell surface

Chambers and Chambers showed that non-injurious dyes, which could not penetrate the cell from the outside, diffused rapidly through the cytoplasm when micro-injected into the cell interior. They stopped on reaching the cell surface, unable to escape from the cell.22

(2) Plasmolysis

When a mature plant cell is exposed to a concentrated solution of table salt (sodium chloride, NaCl) or cane sugar (sucrose), the inside of the plant cell or protoplast (a name coined by von Hanstein,27 Figure 1B) shrinks away from the containing cell wall (Figure 1). The degrees of shrinkage varies with the strength of the NaCl or sucrose solution used. This phenomenon was extensively studied by Hugo de Vries28 and is known as plasmolysis.

 In 1871 de Vries observed that the protoplast of beet roots remained shrunken for days in a concentrated solution of sodium chloride (NaCl).24 This observation led him to conclude that the plasma (cell) membranes of these cells are (absolutely) impermeable to NaCl. In the context of the membrane theory, this observation is of life-death importance. For it substantiates a basic tenet of the membrane theory: a substance, which at high concentration causes sustained shrinkage, must be absolutely and perma­nently impermeant.

To explain the need for this tenet, consider the following. Many verte­brate cells spend their entire life-spans—which may be as long as 100 years—in tissue fluids containing NaCl as its main osmotically-active in­gredient. If NaCl can enter the cell, even slowly, the cell would sooner or later be chock-full of NaCl, lose its osmotic equilibrium and probably die. Therefore, the cell membrane must be impermeable to NaCl, permanently and absolutely.

A high point in the experimental study on plasmolysis occurred in the year 1918, when K. Höfler invented a method for measuring quantitatively the volume of the (irregularly-shaped) shrunken protoplast29 (Figure 1 Â). Applying this method, he showed that in the mature cells of the plant, Tradescansia elongata, the product of the volume of the protoplast, V, and the concentration of sucrose in the bathing solution, C, is close to being a constant. This invariance of the product, VC, was hailed as a decisive quantitative confirmation of the membrane theory. It shows that the living cell indeed behaves like a perfect osmometer in solutions containing differ­ent concentrations of an impermeant solute like sucrose.30

Further investigations, however, led Höfler to reverse his earlier stand.31 Thus in later studies, he discovered that it is not the whole cell en­closed by the cell membrane that behaves like a perfect osmometer. Rather, it was only the central vacuole enclosed by the vesicular membrane (or tonoplast), which obeys the constant VC relationship: This new discovery poses a serious difficulty for the membrane theory.

At high concentrations, sucrose causes the shrinkage of the central vacuole. This is a fact. This shrinkage could not have happened without su­crose first entering into the cell's cytoplasm, which completely surrounds the central vacuole. Sucrose could not have entered the cytoplasm without first penetrating the plasma or cell membrane, which in turn completely surrounds the cytoplasm. Therefore, the cell membrane is not impermeable to sucrose—in contradiction to the basic tenet that a solute that causes sus­tained plasmolysis like sucrose is impermeant to the cell membrane.

Other studies showed that in concentrated solutions of an electrolyte (e.g., a salt of potassium ion, K+), the surrounding cytoplasm may actually swell even as the central vacuole is shrinking.32 This observation too can­not be explained by the membrane theory.

 (3) Transient and sustained volume changes

As I have just pointed out above, sodium chloride (NaCl) is the major ubiq­uitous component of the tissue fluids of all vertebrates including frogs and humans.

Charles E. Overton—a distant cousin of Charles Darwin—showed in 1902 that an isolated frog sartorius muscles—a thin and flat muscle on the inside surface of the thigh, often highly developed in tailors of early days (sartor, Latin)—retained their normal weights in a 0.7% NaCl solution.25 Such a weight-preserving 0.7% NaCl solution was described as isotonic by H. J. Hamburger; higher and lower concentrations of NaCl solutions were respectively called hypertonic and hypotonic26 When 5% methyl alcohol (methanol) was included in an isotonic 0.7% NaCl solution, the muscle im­mersed in the solution showed no change from its previous weight in the 0.7% NaCl solution alone. However, in a solution containing 3% ethylene glycol and 0.35% NaCl solution, the muscle shrank first followed by a slow return to a higher weight, close to that produced by the unadulterated 0.35% NaCl solution alone.

The explanations offered are as follows: Methyl alcohol is, like water, highly permeable to the cell membrane. Hence, its addition to an isotonic NaCl solution caused no change in the weight of the muscle (if one as­sumes that the dilution of NaCl thus brought about is trivial). Ethylene glycol is also permeant, but less so than methanol. Therefore the inclusion of ethylene glycol caused first a shrinkage followed by a return to normal and above normal weight as more and more ethylene glycol enters the cells. In NaCl solutions at a concentration higher than 0.7% or in a 0.7% NaCl solu­tion containing also 3% glucose, the muscle shrank and stayed shrunken. These observations were seen as once more confirming the basic tenet of the membrane theory that only concentrated solutions containing solutes like NaCl or glucose, which are absolutely and permanently impermeant to the cell membrane can cause sustained cell shrinkage. Then something un­expected came up again.

In 1937 and 1938, D. Nasonov, E.I. Aizenberg33 and I. Ye. Kamnev34 from the former Soviet Union made a simple discovery. They first showed that in a normal isotonic Ringer's solution containing in addition 4% su­crose, frog muscle shrank until it reached and then sustained a smaller vol­ume (Figure 2)—as one would have expected on the basis of the membrane theory. However, what one did not expect was that as the muscle was shrinking, sucrose was actually entering into and accumulating inside the shrinking cells. This inward permeation of sucrose continued until a steady level—lower than that in the external medium—was reached and then maintained (For additional details of their experiments, see [8.2].)


 Figure 2. Time course of shrinkage of frog muscle cells in a Ringer's solution containing 4% sucrose (A). Muscle cell volume is given as percentage of the initial cell volume (left ordinate). Concentration of concomitantly-accumulated sucrose in the cells (B) is expressed as weight percent per 100 g. of tissue water (right ordinate). (Nasonov and Aizenberg33 and Kamnev34)

 These simple but highly important findings showed that sucrose, which causes sustained shrinkage, is in fact permeant. Thus, like Höfler's later observations, this discovery also refutes the tenet—vital to the membrane theory—that only impermeant solutes can cause sustained shrinkage. Permeant solutes can cause sustained shrinkage also.

With the advent of radioisotopes and associated technology in the late 1930's and early 1940's, cell physiology entered a new age of enlighten­ment. Indeed with the radioactive tracer technique, (true) permeabil­ity—the sine que ïîï of the membrane theory—can now be studied with precision and without ambiguity, often for the first time {[13.3]; [15.2(1)]}.

Thus the data shown in Table 1 once more confirm the existence of a diffusion barrier to labeled sucrose and NaCl as the surgical amputation of the frog muscle cells doubled the rate of penetration of sucrose into the muscle cells.23 However, the data also demonstrate that before the surgical amputation of the cell membrane, labeled sucrose could and did enter into (intact) muscle cells. Other studies have repeatedly and unequivocally es­tablished that solutes like sucrose23,35 and NaCl36 are permeant to the cell's membrane, confirming that permeant solutes can cause sustained cell shrinkage, a truth already suggested in Höfler's later discovery, and estab­lished by Nasonov, Aizenberg and Kamnev. And, in consequence, the elementary fact that living cell maintain steady and unchanging volumes in isotonic NaCl or sucrose solution can no longer be explained by the membrane theory.

To be continued

Ðàçäåëû êíèãè
"Life at the Cell and Below-Cell Level.
The Hidden History of a Fundamental Revolution in Biology":

Contents (PDF 218 Kb)
Preface (
PDF 155 Kb)
Answers to Reader's Queries (Read First!) (
PDF 120 Kb)

1. How It Began on the Wrong Foot---Perhaps Inescapably
2. The Same Mistake Repeated in Cell Physiology
3. How the Membrane Theory Began
4. Evidence for a Cell Membrane Covering All Living Cells
5. Evidence for the Cell Content as a Dilute Solution
6. Colloid, the Brain Child of a Chemist
7. Legacy of the Nearly Forgotten Pioneers
8. Aftermath of the Rout
9. Troshin's Sorption Theory for Solute Distribution
10. Ling's Fixed Charge Hypothesis (LFCH)
11. The Polarized Multilayer Theory of Cell Water
12. The Membrane-Pump Theory and Grave Contradictions
13. The Physico-chemical Makeup of the Cell Membrane
14. The Living State: Electronic Mechanisms for its Maintenance and Control
15. Physiological Activities: Electronic Mechanisms and Their Control by ATP, Drugs, Hormones and Other Cardinal Adsorbents
16. Summary Plus
17. Epilogue 

A Super-Glossary
List of Abbreviations
List of Figures, Tables and Equations
References (
PDF 193 Kb)
Subject Index
About the Author

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