Dick Murphy: three decades as the touchstone of smooth muscle physiology. Focus on "Cooperative attachment of cross bridges predicts regulation of smooth muscle force by myosin phosphorylation"

Patrick F. Dillon

Department of Physiology, Michigan State University, East Lansing, Michigan 48824

IT IS APPROPRIATE that the paper corresponding to the retirement of Richard A. Murphy as a physiology professor should again raise our level of understanding of smooth muscle contractile regulation. As one of the dominant figures in smooth muscle physiology for the past 30 years, Dick Murphy has led a cadre of students and fellows in discovering, verifying, and disseminating novel, sophisticated, and, in his own quiet manner, radical ideas. Bracketed by the demonstration of the smooth muscle length-tension relationship (7) and the current article in focus (Ref. 10, see page C594 in this issue) echoing the striated muscle exposure of multiple actin-myosin binding sites following a shift in tropomyosin, the observations from Murphy's laboratory set new standards and challenged established paradigms.

The mechanism of latch, the slow detachment of dephosphorylated cross bridges discovered in Murphy's laboratory, has been a central focus of smooth muscle research for 25 years. It is ironic that Murphy has been criticized for not embracing thin filament smooth muscle regulation as a radical alternative to latch, since latch itself was discovered because Murphy made an important, initially radical observation: different stimuli could produce different mechanics in smooth muscle (8). In 1974, Herlihy and Murphy (8) reported that although potassium stimulation produced a greater force in smooth muscle than electrical stimulation, electrical stimulation produced a higher velocity of shortening. The radical nature of this finding, not appreciated at the time, showed that smooth muscle mechanics did not depend directly on the number of attached cross bridges, as was expected by analogy to striated muscle. In finding that smooth muscle mechanics were, at least in part, a product of the mode of stimulation, the groundwork for exploiting the complexities of smooth muscle contractile regulation was laid.

The basis of the latch hypothesis was the observation that myosin phosphorylation in smooth muscle was not correlated with force but with shortening velocity (4). In the absence of any odd but anticipated mechanism derived from striated muscle, the biochemical discoveries of the direct correlation between myosin ATPase activity and myosin light chain phosphorylation (2, 5) predicted that an increase in phosphorylation should always be accompanied by an increase in force. It was here that Murphy's ability to move forward in multiple directions paid off. Working in Murphy's laboratory, Aksoy, using methods developed by Driska, found that prolonged potassium stimulation of vascular smooth muscle produced steady, maintained tension, but that light chain phosphorylation rose and then fell, despite the tension maintenance. Dillon, following up the Herlihy work, discovered that shortening velocity correlated with the rise and fall of phosphorylation, not the force. This novel observation, made because Murphy's laboratory moved in multiple directions, forced the development of the latch hypothesis and a decisive break with the mechanics of striated muscle.

Of the many principles students learned under Murphy, two stand out. First, understand the technical limitations of your methods so well that if you make an unusual observation, you can believe it is a consequence of the tissue, not an artifact of the experiment. Second, follow developments in parallel fields, because these can be a guidepost for new discoveries in your own. If the first principle let Murphy have confidence in the phosphorylation/velocity findings in his own laboratory, it is the second principle that placed those findings in a larger context and, appropriately, allowed their extension. The linkage between myosin light chain phosphorylation and velocity has been verified many times, in many laboratories, and forms a basic element in our understanding of smooth muscle. Just as Murphy drew insight from biochemical work in the laboratories of Hartshorne (5) and Adelstein (2), so for the past 25 years have workers in smooth muscle, and even a few in striated muscle, tested ideas that have come from Murphy.

Within four years of the first report of latch, more than a dozen laboratories were working, at least in part, on aspects of the latch hypothesis. Murphy's second principle, following important findings in parallel fields, had echoed throughout the field of muscle physiology. In what may have been a first, striated muscle researchers now looked to a discovery in smooth muscle as a new paradigm for work in their own field (1, 3, 9). With the formation of the latch hypothesis, workers, especially within the smooth muscle community, were devising experiments to confirm, critique, expand, or reject this new idea. Extending far beyond the dozens of scientists trained by Murphy himself, the testing of these ideas provided funding and training for a generation of smooth muscle research, even in those laboratories most critical of Murphy. During innumerable symposia throughout the 1980s, Murphy defended the ideas his laboratory had produced and continued to generate.

It is significant to note that, even in the presence of the most severe criticism, Murphy remained a true gentleman throughout those years. Always soft-spoken, he let the data speak for itself. If the many people who worked in the Murphy laboratory provided the data that led to the ideas that are now such a part of smooth muscle, in talk after talk, Murphy acknowledged his staff in a manner few others emulate. It is common for a speaker to thank various staff members at the conclusion of a presentation, but Murphy went a step further. During those years when the latch debate was most robust, as each slide came up during a talk, Murphy would cite the student or fellow who done that experiment, creating a point of pride in each of us. Scientists are paid not so much in money or power but in the prestige their work generates. Murphy never took the prestige alone but, instead, spread it to the people he inspired to work late into the night. That he bore any criticism alone is a testimonial to the respect he continues to receive.

In the current article in focus (10), Richard Murphy ends his research career following the same principles that have been a hallmark for three decades. The underlying observations leading to this new work were the technical improvements in the measurement of the basal levels of myosin regulatory light chain phosphorylation. With the original latch work performed using swine carotid artery media having significant basal tone, elevated basal phosphorylation levels were not a surprise. But the use of other tissue having lower or zero tone, but with significant basal phosphorylation, created a need to revisit the latch hypothesis, particularly because it had moved to a more sophisticated level with the work of Hai and Murphy (6).

The strongest element in the work of Rembold et al. (10) is the confirmation that even in the presence of cooperativity, latch bridges have to exist to explain the force-phosphorylation data: the absence of latch would lead to a rapid fall in force that does not occur following dephosphorylation. The absence of force at significant basal phosphorylation, not present in the Hai model, does not negate the requirement that latch must exist following prolonged activation. In proposing the cooperativity model, the article invokes a shift in tropomyosin whose magnitude fits that of the shift produced by calcium-bound troponin in striated muscle. Given the helical nature of tropomyosin polymers around the thin filament, it is energetically unlikely that any model requiring extensive uncoiling of tropomyosin is accurate. Still, a cooperative model requires that any tropomyosin shift cannot be due to random thermal energy but that a molecular mechanism occurring during activation must be involved. The leading candidates for such a mechanism, caldesmon and calponin, are cited, but the lack of a clearly established regulatory role for these proteins makes experimental confirmation problematical. Conjecture in the article that a second regulatory system beyond light chain phosphorylation may not be needed requires that this system alone would be able to explain the cooperativity with a molecular mechanism. Given the limited number of possibilities, the myosin head complex would have to be a major candidate for contact and shift of tropomyosin.

It is in the best traditions of science that even as Dick Murphy leaves the world of research, he leaves it with unanswered questions. The next generation of smooth muscle physiologists would do well to follow up the ideas and possibilities he leaves behind. Like any good target, he has continued to move. It is our good fortune that he has graced us with his presence and our better fortune that he will still be around to hear our ideas and give us his thoughts. We should all listen closely.

FOOTNOTES


Address for reprint requests and other correspondence: P. F. Dillon, Dept. of Physiology, Michigan State Univ., East Lansing, Michigan 48824 (E-mail: dillon{at}msu.edu).

REFERENCES

1. Barany K, Sayers ST, DiSalvo J, and Barany M. Two-dimensional electrophoretic analysis of myosin light chain phosphorylation in heart. Electrophoresis 4: 138–142, 1983.[ISI]

2. Chacko S, Conti MA, and Adelstein RS. The effect of the phosphorylation of smooth muscle myosin on actin activation and Ca2+ regulation. Proc Natl Acad Sci USA 74: 129–133, 1977.[Abstract]

3. Crow MT and Kushmerick MJ. Correlated reduction of velocity of shortening and the rate of energy-utilization in mouse fast-twitch muscle during a continuous tetanus. J Gen Physiol 82: 703–720, 1983.[Abstract]

4. Dillon PF, Aksoy MO, Driska SP, and Murphy RA. Myosin phosphorylation and the crossbridge cycle in arterial smooth muscle. Science 211: 495–497, 1981.[ISI][Medline]

5. Gorecka A, Aksoy MO, and Hartshorne DJ. The effect of phosphorylation of gizzard myosin on actin activation. Biochem Biophys Res Commun 71: 325–331, 1976.[ISI][Medline]

6. Hai CM and Murphy RA. Cross-bridge phosphorylation and the regulation of latch state in smooth muscle. Am J Physiol Cell Physiol 254: C99–C106, 1988.[Abstract/Free Full Text]

7. Herlihy JT and Murphy RA. Length-tension relationship of smooth muscle of the hog carotid artery. Circ Res 33: 275–283, 1973.[ISI][Medline]

8. Herlihy JT and Murphy RA. Force-velocity and series elastic characteristics of smooth muscle from the hog carotid artery. Circ Res 34: 461–466, 1974.[ISI][Medline]

9. Perry SV. Phosphorylation of the myofibrillar proteins and the regulation of contractile activity in muscle. Philos Trans R Soc Lond B Biol Sci 302: 59–71, 1983.[ISI][Medline]

10. Rembold CM, Wardle RL, Wingard CJ, Batts TW, Etter EF, and Murphy RA. Cooperative attachment of cross bridges predict regulation of smooth muscle force by myosin phosphorylation. Am J Physiol Cell Physiol 287: C594–C602, 2004.[Abstract/Free Full Text]





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