Getting The Message Across In their 1957 paper, Theodore W. Rall, Earl W. Sutherland, and Jacques Berthet announced the landmark discovery of the biological activity of cy- clic AMP (CAMP) and its role as a second messen- ger, They were studying the activation of liver phosphorylase (the key initial enzyme in the breakdown of glycogen) by epinephrine and gluca- gon when they found CAMP as a "heat stable fac- tor." The second messenger system began as a "two-staged process" in which the hormone pro- duced the heat stable factor in the membranous fraction, and the membranous fraction activated phosphorylase in the supernatant fraction. Rall, Sutherland, and Berthet's success in study- ing hormone action in cell-free homogenates opened a new era in biochemistry. Sutherland, who won the Nobel Prize in 1972 for this research, wrote in the mid-1950s what seems now to be re- markably obvious: " . there is reason to feel that the hormones may act at the molecular level" [Earl W. Sutherland, "Introduction," in Cyclic AMP, G.A. Robison, R.W. Butcher, and E.W. Suth- erland, Eds. (Academic Press, New York, 1971) p. 2.1 Sutherland died in 1974. Although the initial re- search on CAMP was not funded by NIH, it became the basis for Sutherland's first NIH grant. In an interview with THE JOURNAL OF NIH RE- SEARCH, Henry Bourne, chairman of the depart- ment of pharmacology at the University of Califor- nia at San Francisco, spoke of the elegance and value of this paper. Rall, who is now professor of pharmacology at the University of Virginia Medi- cal School in Charlottesville, recalled some of the circumstances that surrounded the work. Henry Bourne "I tell my students that this paper is the fountainhead from which all that we pres- ently know about hormone action comes," says Bourne. "It's a genuine landmark pa- per that's lovely to read and fun to teach." Bourne uses the paper to demonstrate sev- eral aspects of science in gen- eral and signalling in particular. "It is the first instance of taking a cell apart to show different components mediating the action of a hormone. Strange as it may seem, at that time there were many researchers who considered the cell to be such a complex and vital object that one would never be able to tease it apart. This paper showed that you could use the fundamental method of biological science, which is to take things apart and put them back together, to under- stand the cell. That is really the nub of it." "They knew the beginning of the signalling pathway, that is, glucagon and epinephrine stimu- lation and they had an endpoint [the phosphory- lase reaction] that was clear and measurable that they could attribute to the action of the hormone. They dissected what was in between. "Even the footnotes tell us something important. about the signalling pathway. In Footnote 3 they gave the composition of cyclic AMP And they said that there was something in their extracts that de- grades it; that, we know now, is the enzyme phos- phodiesterase. Footnote 4 tells us about the gen- erality of their findings because homogenates of dog heart membranes behaved similarly. Finally, at the end of the paper they stated that the heat- stable factor [cyclic AMP] did not have a reproduc- ible effect on purified phosphorylase. Now we know that it does not work on purified phosphory lase because there is a cyclic AMP-dependent ki- nase in between. "Several elements of luck went into this thing, too. One is that cyclic AMP is itself stable to boil- ing. If they had taken on some of the other second messengers that we know now, they would have flunked. Secondly, they picked the right incuba- tion time. Under their conditions the phosphodi- esterase was chewing up the cyclic AMP almost as fast as it was made. Had they waited 20 or 30 min- utes instead of 10, the membranes might have got- ten a little soggy and sick and unable to make enough CAMP to let them see their effect in the boiled stuff. "Finally, there is another point that was not ap- preciated until the '70s. The ATP they used in the reactions was contaminated with GTP [guanosine triphosphate-required by adenylyl cyclase for the synthesis of cyclic AMP]. If you really wash mem- branes well and get rid of endogenous GTP and if you have pure ATP the cyclase does not. work very well. Marty Rodbell, Lutz Birnbaumer, and their colleagues discovered that at the NIH about 15 years later." Theodore Ball "I have lost. count of how many wrong ideas got us to do the right experiments," Rall says about the experi- ments in which he and his colleagues identified the bio- logical activity of CAMP and from which the second-mes- senger concept arose. "There were several incorrect ideas that got us going; in retrospect, some of the no- tions were downright stupid. But the fact that those mistaken working hypotheses were formu- lated and acted upon allowed the experiments to be done. "The principal stupid hypothesis is my personal claim to fame. Notice that we made sucrose THE.OURNALOF NIH ?ESEAXH J4NUAR9EWJA?Y 1390 'WL ? 77 homogenates of liver cells. As a matter of fact, the experiment will work as well or better if other ho- mogenizing media are used. But at the time, I thought it was crucial, and it convinced me to do the experiment. I had just done my Ph.D. thesis with Albert Lehninger, and I had learned to grind up liver tissue to prepare and study these magic things called mitochondria. The use of isotonic su- crose was very important for getting happy mito- chondria. I thought that. if sucrose keeps mlto- chondria happy, we could perhaps keep some other part of the cell-we didn't know what part-happy. So that's what I did. "You have to put yourselves into the minds of the people who did research in the 195Os, includ- ing Earl Sutherland, who had come out of the [Carl and Gerty] Cori lab [at Washington Univer- sity, St. Louis]. They thought there was something magic about the structure of the intact cell that was necessary for hormones to act. Sutherland seemed reticent to undertake this broken-cell ex- periment. But I said, `Give me a couple of months' and talked him into it. "Then, of course, at the start I used the wrong animal. I reasoned that since so much of mamma- lian biochemistry had been discovered using rat liver, this tissue would be the best to use initially. We now know that this type of experiment does not work using any kind of rodent liver; in fact, it probably would work only when using the livers of carnivores, such as dogs or cats. "After a couple of really disappointing months messing around with rat liver, I was getting a little desperate. Sutherland% foot was tapping an the floor, we had some other experiments to do, and I was under the gun. All the work an phasphoryla- tian in Sutherland's lab had used dog liver, and I knew the hormones would produce a large and rapid activation of the enzymes in slices of dog liver. So I decided to incubate slices as if I was to reproduce this observation, but I would try to `fool' the system by adding the hormones to farti- fied homogenates instead of to the slices. It was pretty wasteful, because I needed only 10 or 20 grams from a 400-gram dog liver, but I figured it was my last shot. "I had not seen Sutherland for a day and a half. I do not know how he knew, but for some reason, right at the time the incubation was over, he ap- peared to watch the outcome. The reaction was so obvious that we just about dropped all the tubes. That first experiment showed nearly a doubling of the rate of phosphorylase activation. Sutherland was not usually a demonstrative person, but you could tell he was absolutely ecstatic. We were scheduled to go away the following weekend (even before we had any confirmation), and we spent the whole weekend fantasizing about what this meant. "Then there was a Belgian postdoc in the lab, Jacques Berthet, who had just gotten his degree with Christian de Duve. He had spent his six or so years in a cold room making sucrose homogenates of liver and performing very precise fractionations by differential centrifugation. Berthet was very upset with me about the way I did those experi- ments. Looking over my shoulder, he watched me centrifuge the homogenates in an angle rotor far brief periods timed with a wrist watch, just to get something reasonably smooth that could be pipetted. Then I did the `Lehninger Hard Pour' where the supernatant material was decanted with a smooth and continuous motion that allowed you to see the pellet string out along the side of the tube. As soon as the hunks and chunks reach the top, you quit. I had done it many times before, and the supernatant material worked. "Berthet was so offended by this procedure that he wrote me a `proper' protocol for centrifugation I must use a horizontal yoke, not an angle rotor. and I must centrifuge a prescribed height of sus- pension at a certain rpm for a defined time. More- over, the supernatant must be harvested by care- ful aspiration, not by pouring. So I did it, but none of the supernatant fractions obtained by this pro- cedure responded to hormone. "I was furious with him. But as it turned out, that was the way we found out that the superna- tant would not respond unless you add back a lit- tle bit of the particulate fraction [the hunks and chunks]. Then we did the experiment in two stages, incubating the particulate fraction with the hormones, heating the mixture, and adding the `cooked stuff' [`Kochsaft' in Figure 41 to the super- natant. Fortunately, for no good reason, we in- cluded MgATP in the first stage, and such experi- ments reproduced the effect of the hormone in the whole homogenate. Voil$, second messenger! You'll notice a much less sexy term was used in the pa- per, something like "intracellular mediator." "Things went like fury in the next few months. As I recall, that first experiment was November 5, 1955. By the time the paper was submitted in July 1956, cyclic AMP had been crystallized-it went from a gleam in somebody's eye to crystals in roughly seven months. Some chemistry was done in the next couple months, and a footnote describ- ing the stoichiometric content of adenine, ribose, and phosphate was slipped into the galleys before publication in January 1957. "Even before the chemistry was done we had a bioassay for the heat stable factor, so we looked in other tissues with other hormones. We found, thanks to our friend and colleague down the hall, Robert Haynes, that ACTH [adrenocorticotropic hormone] stimulated the formation of the `heat stable factor' in the adrenal cortex. That started the notion that we were doing business with a general phenomenon." -GAYLLOHSEGALLAGHER 70 THE JOURNALOF NIH RESEARCH JANUARY-FEBRUARY 7990 VOL 2