Biology 466    Unsolved Problems Fall 2010

More on bone formation and degeneration

What is known and what is not known? What do we not know how to find out?
And sometimes don't know what alternative possible explanations there are!

It is known for sure that bone is made stronger where larger forces are imposed on it
(unless maybe it's a response to frequent exertion of forces, instead of magnitude: e.g. whether tennis players' arm bones get stronger than weight-lifters' arm bones)

But it is not known how forces are detected, or by which cells.
Detection could be by means of piezoelectric voltages resulting from crystal distortions.

Or detection might be by electro-osmotic voltages caused by compression or stretching of extracellular matrices. Maybe the reported measurements of piezoelectric voltages of bone were really caused by electro-osmosis, but the scientists doing the work perhaps didn't realize that there is such a thing as electro-osmosis, and believed that any voltages that they detected being produced by compressing or stretching bone must be piezoelectric. It's not so easy to invent experimental criteria to distinguish between electro-osmosis voltages versus piezoelectric voltages. Also, how could bone cells distinguish one voltage from the other? Has it ever really been proven that voltages stimulate increased bone formation? If so, does voltage stimulate osteoblasts to deposit more bone, or does voltage inhibit osteoclasts from dissolving bone? Also it is not known whether the maximum size of the voltage is what matters, versus how often, or how quickly this voltage changes.

The degeneration of bone in astronauts might be caused by the same mechanism as the weakening of bone that occurs when a leg or arm is in a cast, i.e. reduced forces acting on, or exerted by the bone. Or the causes might be different! Weightlessness might affect tissues in some important way different than taking the load off bones and muscles. For example, gravity could cause some (lighter) materials to rise relative to other (heavier) materials in body fluids, and the failure of this tendency to redistribution might be the true cause of the degeneration of bones and muscles in astronauts. Also, someone should persuade NASA to quit using the term "microgravity" as a supposedly more accurate term that weightlessness. In fact, weightlessness is the correct term. It shouldn't take a Rocket Scientist to get that right!

Osteoporosis in older people, especially women, might result from reduced loads on bones, or it might result from reduced sensitivity to loads, or neither. It might result from increased destruction of bone by osteoclasts, or it might result from decreased deposition of bone by osteocytes, or reduced differentiation of new osteocytes from osteoblasts; or it could result from increased differentiation of osteoclasts, either from stem cells in the bone marrow, or by fusion of macrophages (that had already been differentiated, except into a slightly different cell type). These processes might be controlled by amounts of particular cytokines (such a #6), or by changes in cell sensitivity to those cytokines. More of a signal protein, versus more sensitivity to that protein, are not the same thing, but these two possibilities are often difficult to distinguish, and many people don't bother.

It is known for sure that osteoclasts dissolve calcium phosphate by secreting acid (but don't ask me whether it's acetic acid or hydrochloric acid [as in the stomach], or why researchers don't consider the possibility of absorbing bases, even OH-, as a means of increasing acidity outside a given cell. It is also known for sure that osteoclasts secrete proteolytic enzymes that digest the (1/3 !) of bone which is collagen. The evidence is also strong that one side of each osteoclast secretes the acid and the proteolytic enzymes. Apparently (and I believe it, but wish I knew more evidence), each osteoclast sticks tightly to adjacent bone around a ring-like shape, and secretes the acid and the collagen-digesting enzymes into the enclosed space, where it remains concentrated and from which they cannot freely leak out. This directional polarization of secretion by osteoclasts, plus differences in leakage of acid and enzymes out of the enclosed space, are two more important variables. Either or both might change in response to mechanical loads, nearby voltages, age, weightlessness or concentrations of cytokine number 6.

The true answers to these questions are still available to be discovered. Although you were born too late to figure out how DNA encodes genes, you have arrived just in time to design and/or carry out whatever experiments can decide conclusively between the different possibilities listed above. You would do a lot of good for medicine by answering even one of these questions. Just inventing experimental criteria could be a big advance; so could noticing relevant implications in existing data, which could include the human genome base sequence. I don't see how; but that is exactly the way of puzzles and riddles; they jump quickly from being impossible to being obvious.

The existence and cause of resting potentials is well established for the case of nerve cells and muscle cells. A plasma membrane enzyme called the sodium pump uses energy from hydrolysis of ATP to pump sodium ions out of cells, and (somewhat less directly) pump potassium ions into the cytoplasm. In addition, the plasma membrane is highly permeable to potassium ions, so that enough of them leak out (maybe one in a million), each potassium ion carrying its positive charge, so that an approximately 70 millivolt voltage difference is produced, with the outside of each cell being that much positive relative to the (more negative) cytoplasm. Notice the slight paradox of a positive voltage being produced outside each cell as a result or a higher concentration of a positive ion inside each cell. How can more positive ions inside cause a positive voltage outside?! As biological paradoxes go, this is not especially difficult. Scientists solved it in the late 1800s. But it's too difficult for the writers of more than half of introductory biology textbooks; they fall back on two favorite pseudo-explanations: that the cytoplasm contains an excess of negatively-charged proteins; and that the greater concentration of sodium ions (positive+) outside the cell causes the resting potential. If you ever find yourself a member of a committee choosing between alternative textbooks on introductory biology, you can weed out those that are most inaccurate about resting potentials, osmotic pressure, and auto-immunity. Many of them have also have delusions about the maximum size of cells being limited by surface to volume ratios. If that were true, then tetraploid cells would be stressed, skeletal muscle cells a thousand time the volume of average cells would be impossible, not to mention oocytes. It is a tragedy that elementary textbooks are no longer checked for accuracy, as advanced textbooks still are.

You may be surprised to learn (because 90+% of real biologists don't seem to be aware of it) that all the cells of the body have resting potentials. (All? Well, almost all. And if you know of exceptions, please tell me.) Fibroblasts, liver cells, leucocytes, oocytes? You name it, and it has a sodium pump, an excess of potassium ions in the cytoplasm, leakage of potassium ions out through the plasma membrane, and a resulting trans-membrane voltage in the -50 to -80 millivolt range, inside versus outside. In the particular case of oocytes, this voltage depolarizes during fertilization, and is a key part of two sets of mechanisms for preventing any more sperm for getting into the oocyte, after the first one has gotten in.

But for what purposes do all those other differentiated cell types expend all that energy (as much as a third or half of all their ATP) bailing out sodium and letting potassium leak out (assuming I am correctly informed that they do!). It could be part of some sym-port or anti-port mechanisms for getting sugars, or something like that. Most of what I know, or think I know, about the existence of these voltages has come from conversations with scientists at national and international conferences (and not from refereed journal articles, unfortunately). They were very good scientists, and they seemed absolutely confident on the subject. On the other hand, it isn't easy to measure trans-membrane voltages, except in giant cells like oocytes, skeletal muscle cells, and squid giant axons. Rapid changes in voltages are much easier to detect, and had been studied in nerves long before giant axons made intracellular electrodes practical. For example, imagine if chemotaxis by leucocytes was coordinated by local differences in their "resting" potential: how could you detect it. For a long time there was a very trendy hypothesis that invasiveness of non-cancerous cells is controlled by "electrotonic coupling" through "gap junctions”, which got their name from an early misunderstanding of their structure. That hypothesis has gone out of fashion, but the evidence for it remains unchanged. It wasn't disproved: scientists just got bored with it. This boredom resulted from lack of progress, not from disproof. Another reason that major facts sometimes get ignored is that they don't fit into anybody's theory. "Who ordered that?" as a certain physicist once said about an unexpected discovery.

Another thing no one knows is whether these "resting potentials" are causally related to how tissue culture cells respond to "DC" electric fields. For about five years, that was a very popular subject, and then went out of fashion. No one explanatory theory got proven, so most biologists lost interest. Two other factors may have been: first, the danger of electrocution inherent in these kinds of experiments; and second, realization that piezoelectric effects can only produce "AC" electric fields, when the surroundings conduct electricity. Body fluids and tissue culture media both conduct electricity well enough that current will quickly drain away any voltage that's not being continually renewed by a power source. We used an electrophoresis power supply whose safety device had been disconnected! I don't recommend that, & if I ever do more such experiments will use batteries.

Any cell in a voltage gradient will feel (at least!) two effects. Small electrophoretic forces will pull on any positive or negative proteins or lipids in the plasma membrane. The charges, and the forces, may or may not be strong enough to displace the charged molecules; but there will be some force. Little or no electrophoretic force will be exerted on cytoplasmic molecules, because the plasma membrane conducts electric currents much less than either the cytoplasm itself, or the surrounding medium. Current will go around cells. For this same region, the voltage difference between the inside and the outside of cells will be very different along the cells, in the axis of the voltage gradient. Some areas will be depolarized and others will be hyperpolarized (I think!). Some very good scientists have told me three different conclusions about trans-membrane voltages of living cells subjected to voltage gradients. If a cell is 50 microns long, and is in a one volt per millimeter voltage gradient, that will produce a 50 millivolt voltage difference between one end of the cell and the other end. (That was one reason we used one volt per millimeter: to make the math easier.)

If a cell's internal potassium concentration is enough to give it a 70 millivolt "resting" potential, and you subject it to a 50 millivolt difference between one end of the cell and the other, then is one end going to have a 120 millivolt drop across its plasma membrane? Or is the other end going to have a 20 millivolt drop? Or what? And is the potassium going to get drained out through any membranes whose transmembrane voltage is dropped below 70 millivolts?

Why do epithelial cells crawl toward the negative electrode, and macrophages crawl toward the positive electrode, and fibroblasts line up perpendicular to the field? Are these responses to electrophoretic displacement of membrane molecules; an if so, which ones? And do these molecules change cell-substratum adhesion, or change contractility, or what? I don't encourage people to do research on this topic; but it's an illustration of how many loose ends there are in cell biology.




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