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Regulation of smooth muscle contraction/relaxation: paradigm shifts and quantifying arrows

Perhaps the best indication that a paradigm shift in physiological thinking has occurred is when we are compelled to change the content in our Medical Physiology texts. Over the last 40 years, our teaching of smooth muscle contractile function has been subjected to several such abrupt shifts. In the 1970s, our teaching schematic diagrams were dominated by major arrows pointing primarily to systems that regulate intracellular Ca2+ concentration ([Ca2+]i), with another minor arrow pointing from [Ca2+]i to ‘contractile proteins’. Since smooth muscle studies lagged behind skeletal muscle, it was assumed that this meant a thin-filament regulatory system based on troponin–tropomyosin. A major shift occurred when it was shown that there is no troponin in smooth muscle, but rather a thick-filament regulation based on phosphorylation of the myosin regulatory light chain (MRLC) by myosin light chain kinase (MLCK). Thus in the 1980s, the primary arrow from calcium now pointed to Ca2+–calmodulin, then to MLCK, eventually leading to phosphorylated MRLC (p-MRLC) and activation of the contractile system. This mechanism worked well for smooth muscle actomyosin preparations, but another shift occurred when it was discovered that the mechanism could not be so readily applied to intact preparations. Studies in the mid 1980s showed that while isometric force increases monotonically to a steady state value, [Ca2+]i, contractile velocity, ATPase activity and p-MRLC rapidly reach a peak but then decrease to a steady state, some one-third to one-fifth of their peak values. This steady state was termed the ‘latch’ state, by analogy to the invertebrate smooth muscle state of ‘catch’. The appearance of the ‘latch state’ is now a staple of our current texts. The discovery of actin binding proteins, calponin and caldesmon, ushered the next shift, which was characterized by renewed interest in thin-filament regulation and the possible modulatory role of these proteins in the latch state. In our current era, beginning in the late 1990s, the discovery of the major role played by regulation of myosin light chain phosphatase ushered in the latest paradigm shift. The evidence suggests that regulation of the response to a given [Ca2+]i, that is, regulation of the ‘Ca2+ sensitivity’ via modulating phosphatase activity, is as important as regulation of [Ca2+]i in the control of contractility. 
 
There are currently two major themes in this paradigm (Fig. 1). One is related to activation of Rho kinase by the small G protein RhoA. Rho kinase in turn phosphorylates the myosin binding subunit, MYPT1, of myosin regulatory light chain phosphatase (MRLCP), reducing its binding to myosin and concomitantly its phosphatase activity. Lower phosphatase activity in turn results in greater p-MRLC and increased activation of actin-myosin interaction for a given [Ca2+]i. Inhibition of Rho kinase, then, is a powerful strategy for relaxing smooth muscle, resulting from increased phosphatase activity. A second theme is based on a protein called CPI-17 and its inhibitory effect after phosphorylation by protein kinase C on MRLCP. Thus for both contraction and relaxation, of which the latter may not simply be a reversal of the former, control of [Ca2+]i as well as that of Ca2+ sensitivity must be considered. 
 
 
 
Figure 1 
 
A schematic of current understanding of the pathways regulating contractile activity in smooth muscle 
 
 
 
Given all these arrows and regulatory pathways, the goal of physiology is ultimately to quantify the relations amongst the pathways. To do this requires time course measurements of a wide variety of variables, including force, [Ca2+]i, p-CPI-17, p-MRLC, p-MYPT1 and p-RhoA. This is a formidable task, given that many of these proteins have multiple phosphorylation sites. In this issue of The Journal of Physiology, Kitazawa and colleagues (2009) tackle this challenge for the important case of the relaxation of vascular smooth muscle elicited by the NO–cGMP–PKG signalling pathway. This study and a previous study on contraction in response to phenylephrine (Dimopoulos et al. 2007) provide our most detailed information on the interactions and temporal significance of these pathways. 
 
The results of this study support the hypothesis that the vasorelaxation response to NO includes rapid and slow phases which correspond to distinct signalling cascades. The rapid phase includes inhibition of Ca2+ release from the sarcoplasmic reticulum, and thus decreased MLCK and PKC activity resulting in decreased p-CPI-17 and de-inhibition of MRLCP. The slow phase continues the drop in [Ca2+]i, as well as dephosphorylation of p-MYPT1, now ascribable to Rho kinase inactivation due to RhoA phosphorylation and inactivation. These results for NO relaxation are similar to those associated with receptor-mediated contraction, with dephosphorylation rather than phosphorylation associated with a rapid phase involving CPI-17 and a steady state modulated via MYPT1. 
 
It would be of interest to investigate whether these results from rabbit femoral artery are representative of arterial smooth muscle in general, and resistance vessels in particular. Studies of the latter, of course, are limited by the available tissue mass, but some indication of the validity of extrapolating these studies would be very important. One might ask about the advantages of such a dual regulatory system, in that one might not suspect that fine control of rapid transients would be critical to vascular function of resistance vessels. However, increasing our knowledge of regulation of smooth muscle at this level is critical for development of strategies for relaxation of smooth muscle, for example, during vasospasm of cerebral or coronary arteries.