Calcium and the Man (from The MBL Newsletter, Vol. 8, No. 2, Winter 1983-4)

He was a stocky man who wore tattered tweed suits, the trousers sagging in heavy folds around his knees. Once, on the streets of Falmouth during World War II, some sailors took him for a bum - until he dug his pince-nez out of his pocket and ceremoniously fixed them to the bridge of his nose. He was a teacher whose every seminar "lit up the Lab," and a thinker whose theories echoed in the dark. Known to his students and colleagues as "The Boss," he was also regarded, in a curiously affectionate way, as a species of Fool - a man obsessed with the radical notion that the essential events of the living state were all governed by a single agent. According to one of his students, "he was a young rebel even when he was old." In the words of another, simply, he was "The Man".

From the late 1920's until his death in an auto accident in 1959, Lewis Victor Heilbrunn argued and gave evidence that the positively-charged ions of calcium played an ubiquitous role in cellular processes. Throughout that period his theories were regarded with skepticism. The last ten years, however, have seen a virtual explosion of research on intracellular calcium: over 2,000 papers were published on the subject in 1980 alone. Even more recently, calcium has come to be accepted by cell biologists as a "universal secondary messenger" - a thread linking such diverse cellular functions as muscle contraction, fertilization, sperm motility, nerve transmission, inflammation reactions, and even the neural modifications associated with learning.

"We really don't know if calcium acts as the universal messenger, as Heilbrunn believed," says Dr. Paul Gross, MBL Director and one of Heilbrunn's students. "But if by 'universal' you mean 'everywhere we've looked so far,' then it does seem to be true.

The importance of extracellular calcium in the formation of bone and teeth has been recognized for centuries, and as early as 1988 Sidney Ringer demonstrated that calcium was necessary for the contraction of skeletal muscle. Its role as a clotting agent in blood has also been known for some time. What Heilbrunn contributed was the profoundly radical proposal that the intracellular movement of calcium ions constituted the "trigger" for excitable systems. According to his "calcium release theory," in response to a primary stimulus - for example the electrical command of an "action potential," or nerve impulse - calcium ions are secreted inward from a region near the surface of the cell. Ca is normally found in very low concentrations in cells, and Heilbrunn thought that this quick burst of new ions somehow activated the cell's primary response: muscle fibers contract, cells divide, blood clots, and so on. In effect, Heilbrunn was arguing that the basic processes of existence all shared an undeniable physical kinship, and that their common relative was calcium.

That Heilbrunn's calcium release theory was not generally accepted really is no surprise. One important reason was the nature of the research itself. Heilbrunn's domain was colloidal chemistry; he understood calcium's action to be a regulation of the relative viscosity of living protoplasm. The rising fashions of biology during the 1940's favored biochemical studies of metabolic functions, and later, the limelight was occupied by the dramatic developments of molecular biology. Other than Heilbrunn's students and some of his colleagues at the MBL, few people seemed to be excited by shifts in the concentration of ions within cells.

Throughout the 30's and early 40's, Heilbrunn and his students, including Burr Steinbach and Daniel Mazia, concentrated efforts on calcium's role in protoplasmic clotting, and also found some initial evidence for calcium-mediated reactions in cell division, muscle contraction, and the stimulation of nerve fiber. No single experiment, however, gave Heilbrunn the irrefutable proof that he required to convince his critics - or more accurately, to interest them at all.

"It's really quite amazing that he knew what he did at the time," says Dr. Paul LeFevre, another of Heilbrunn's students and currently at the State University of New York, Stony Brook. "It was as if he had a direct pipeline to God."

Dr. LeFevre is quick to acknowledge, however, that celestial guidance is highly suspect, at least as an experimental method:

"Nobody could get that excited about calcium because Heilbrunn couldn't find the hard proof he needed to convince them. He seized on every bit of evidence he could find, but from the outside, it didn't look like he had it."

In 1947, Heilbrunn found new evidence for his theory that seemed to provide the requisite "hard proof." In experiments skillfully carried out with Dr. Floyd Wiercinski, minute amounts of calcium were microinjected directly into muscle cells, so that the membrane remained intact. The same painstaking procedure was used to test the intracellular effects of other ions. When the calcium was injected, the response was sharp and immediate: the muscle fibers pulled taut, shortening an average of 44%. None of the other ions naturally occurring in muscle tissue had any appreciable effect. Even potassium, which Albert Szent-Gyorgi had shown worked in conjunction with adnosine triphosphate (ATP) in contraction, only produced a shortening of 7%.

"It was fantastic to watch," Wiercinski remembers today. "We injected the calcium and the fiber instantly pulled up into a mass."

Heilbrunn's and Wiercinski's findings met with some initial interest, followed by a seemingly fatal rebuttal. The eminent English physiologist A.V. Hill, after first supporting the work, abruptly reversed himself and argued convincingly that the rate of diffusion of calcium ions from the exterior region of a cell to the interior was much too slow to account for th enearly instantaneous contraction of myofibrils deep within the cell. According to Dr. Marian LeFevre, another of Heilbrunn's students, "In those days, whatever A.V. Hill said was instant law."

Nothing followed the Hill paper but the familiar hush of neglect. Heilbrunn's "hard proof" had fallen prety to a fundamental law of scientific practice: an hypothesis must be explicable in terms of existing knowledge. Truth is relative to what we already know.

Ironically, the knowledge Heilbrunn needed to counter Hill's objection was already in the literature; the problem was that no one had paid any attention to it for fifty years. In 1902 Emilio Veratti, an investigator in the laboratory of Camillo Golgi, had noticed and described an elaborate filigree of tubules surrounding each sarcomere (the contractile unit of muscle) in striated, skeletal muscle tissue. Veratti could ascribe no function to this network, and his discovery was more or less swallowed up by the more aggressive attentions given to his famous mentor. Not until 1955 did Stanley Bennett and Keith Porter (long an MBL investigator and later to become the institution's director) rediscover Veratti's system of tubules with the aid of the electron microscope.

The network was soon found to consist of two separate but functionally related systems: the sarcoplasmic reticulum, a cuff-like network surrounding each sarcomere, and the T-system, a web of hollow, deeply-defined crevices in the cell membrane. The T-system lies against the reticulum where it forms a series of sacs called "terminal cisternae." Later research proved that large stores of calcium are sequestered in these sacs. an action potential delivered to the muscle is instantly conveyed via the T-system to the sarcoplasmic reticulum, which releases its store of calcium simultaneously in all parts of the cell. Bioelectricity - not diffusion - is the mechanism of delivery, and thus Hill's objections to a calcium release theory of contraction were no longer relevant.

All this became clear, of course, only after Heilbrunn's death. The process was confirmed in the early 1970's by injecting muscle fibers with aequorin, a remarkable, bioluminescent substance first isolated in 1963 by Osamu Shimomura, now a year-round MBL investigator. With perfect consistency, aequorin obliges physiologists by glowing a deep blue - and only in the presence of free calcium. Aequorin is, in effect, a natural witness for Heilbrunn's calcium-release theory.

How calcium activates skeletal muscle contraction is now fairly well understood, at least in broad outline. Briefly, according to the "sliding filament theory" of contraction, thick filaments of the protein myosin are dovetailed in each muscle sarcomere with thin filaments of the protein actin. When the muscle is relaxed, a calcium-binding protein called troponin acts as a sort of clutch on the actomyosin complex, physically interfering with the interaction of the two contractile proteins. The deluge of calcium ions released by the stimulus of an action potential is quickly bound to the troponin, subtly altering its shape. the clutch disengages, and in an ATP-fueled reaction, the myosin filaments grip and pull upon the actin filaments. When the action potential ceases, the calcium is pumped back into the sarcoplasmic reticulum, the troponin once again nestles between the actin and myosin, and the muscle relaxes.

In his time, Heilbrunn was considered to be overinsistent about the role of Ca in the cell - or as Paul LeFevre puts it, "he was thought of as a calcium maniac." But the final irony of Heilbrunn's radical notions is that they did not go far enough. In addition to the processes already mentioned, cellular transport, photreception, the formation and disassembly of microtubules, and the movement of chromosomes are all processes mediated by calcium. At the MBL on a given summer day, one could walk through the halls of Lillie and Whitman to find Andrew Szent-Gyorgi studying the role of calcium in muscle contraction, or Rodolfo Llinas examining its parts in the release of neurotransmitters at nerve synapses. Gerald Weissman, Leonard Nelson and Daniel Alkon might be investigating its function in, respectively, cell aggregation, sperm motility, and the cellular mechanisms of learning. But any such list would be incomplete - it is safe to say that among physiologists, at least, there isn't anyone working at the MBL whose research is not in some way concerned with the role calcium plays as an intracellular messenger.

Such research - Heilbrunn's legacy - has already been of direct clinical benefit. "Calcium channel blockers" are now used to intercept the action of Ca in heart muscle contraction, thus to relieve muscle spasms in coronary arteries and to lower high blood pressure. Other "calcium antagonists" have been proposed in the treatment of muscular dystrophy, cancer, cystic fibrosis, arthritis, and diabetes. The theory behind the action of such drugs is that if we can learn to "regulate the regulator," our powers to keep the whole living system in balance will be immeasurably enhanced.

For this, some thanks are due to The Man.