Pretreatment of alpha-toxin-permeabilized smooth muscle with ATP gamma S (adenosine 5'-O-(thiotriphosphate)) under conditions resulting in minimal (< 1%) thiophosphorylation of the myosin light chain increases the subsequent calcium sensitivity of force output and myosin light chain phosphorylation. The change in calcium sensitivity results at least in part from a 5-fold decrease in myosin light chain phosphatase activity. One of the few proteins thiophosphorylated under these conditions is the 130-kDa subunit of myosin light chain phosphatase. These results suggest that thiophosphorylation of this subunit leads to a decrease in the activity of the phosphatase, and that phosphorylation and dephosphorylation of the subunit may play a role in regulating myosin light chain phosphatase activity.

ABSTRACT''Catch'' is a condition of prolonged, highforce maintenance at resting intracellular Ca 2؉ concentration ([Ca 2؉ ]) and very low energy usage, occurring in invertebrate smooth muscles, including the anterior byssus retractor muscle (ABRM) of Mytilus edulis. Relaxation from catch is rapid on serotonergic nerve stimulation in intact muscles and application of cAMP in permeabilized muscles. This release of catch occurs by protein kinase A-mediated phosphorylation of a high (Ϸ600 kDa) molecular mass protein, the regulator of catch. Here, we identify the catch-regulating protein as a homologue of the mini-titin, twitchin, based on (i) a partial cDNA of the purified isolated protein showing 77% amino acid sequence identity to the kinase domain of Aplysia californica twitchin; (ii) a polyclonal antibody to a synthetic peptide in this sequence reacting with the phosphorylated catchregulating protein band from permeabilized ABRM; and (iii) the similarity of the amino acid composition and molecular weight of the protein to twitchin. In permeabilized ABRM, at all but maximum [Ca 2؉ ], phosphorylation of twitchin results in a decreased calcium sensitivity of force production (halfmaximum at 2.5 vs. 1.3 M calcium). At a given submaximal force, with equal numbers of force generators, twitchin phosphorylation increased unloaded shortening velocity Ϸ2-fold. These data suggest that aspects of the catch state exist not only at resting [Ca 2؉ ], but also at higher submaximal [Ca 2؉ ]. The mechanism that gives rise to force maintenance in catch probably operates together, to some extent, with that of cycling myosin crossbridges.Isometric force production in the anterior byssus retractor muscle (ABRM) of Mytilus edulis, initiated by cholinergic nerve stimulation, is maintained for a prolonged period of time after cessation of stimulation. This condition, during which relaxation of force occurs at an extremely slow rate, lasting minutes, or even hours, has been termed ''catch.'' When catch occurs, the intracellular Ca 2ϩ concentration ([Ca 2ϩ ]), which was transiently elevated as a result of the stimulus, has declined to near-resting levels (1, 2). In this invertebrate smooth muscle, calcium activates contraction by direct binding to myosin (3, 4), and its subsequent removal establishes the catch state (5). The catch state is characterized by a marked slowing of crossbridge cycling rate, measured as energy usage (6-8) and mechanical behavior such as force-velocity relations and force redevelopment after a quick release (9-12). Catch exemplifies the high economy of smooth muscle: the ability to maintain force with a low expenditure of energy. In the ABRM, rapid relaxation, or release of catch, occurs on stimulation of serotonergic nerves, a response that is mediated by an increase in cellular cAMP and the activation of protein kinase A (13, 14).We have shown that the catch state is regulated by the cAMP-dependent phosphorylation of a high molecular mass (Ϸ600 kDa) protein in the intact and permeabilized ABRM (15). Seve...

Recent experiments on permeabilized anterior byssus retractor muscle (ABRM) of Mytilus edulis have shown that phosphorylation of twitchin releases catch force at pCa > 8 and decreases force at suprabasal but submaximum [Ca2+]. Twitchin phosphorylation decreases force with no detectable change in ATPase activity, and thus increases the energy cost of force maintenance at subsaturating [Ca2+]. Similarly, twitchin phosphorylation causes no change in unloaded shortening velocity (Vo) at any [Ca2+], but when compared at equal submaximum forces, there is a higher Vo when twitchin is phosphorylated. During calcium activation, the force-maintaining structure controlled by twitchin phosphorylation adjusts to a 30% Lo release to maintain force at the shorter length. The data suggest that during both catch and calcium-mediated submaximum contractions, twitchin phosphorylation removes a structure that maintains force with a very low ATPase, but which can slowly cycle during submaximum calcium activation. A quantitative cross-bridge model of catch is presented that is based on modifications of the Hai and Murphy (1988. Am. J. Physiol. 254:C99-C106) latch bridge model for regulation of mammalian smooth muscle.

The purpose of this study was to determine the quantitative relationship between the number of myosin molecules that increase their ATPase activity and the degree of myosin light chain phosphorylation in smooth muscle. Single turnover experiments on the nucleotide bound to myosin were performed in the permeabilized rabbit portal vein. In the resting muscle, the rate of exchange of bound nucleoside diphosphate was biphasic and complete in approximately 30 min. When approximately 80% of the myosin light chain was thiophosphorylated, the nucleoside diphosphate exchange occurred at a much faster rate and was almost complete in 2 min. Thiophosphorylation of 10% of the myosin light chains caused an increase in the rate of ADP exchange from much more than 10% of the myosin subfragment-1. Less than 20% thiophosphorylation of the total myosin light chains resulted in the maximum increase in ADP exchanged in 2 min. It appears that a small degree of myosin light chain phosphorylation cooperatively turns on the maximum number of myosin molecules. Interestingly, even though less than 20% thiophosphorylation of the myosin light chain caused the maximum exchange of ADP within 2 min, higher degrees of thiophosphorylation were associated with further increases in the ATPase rates. We conclude that a small degree of myosin light chain thiophosphorylation cooperatively activates the maximum number of myosin molecules, and a higher degree of thiophosphorylation makes the myosin cycle faster. A kinetic model is proposed in which the rate constant for attachment of unphosphorylated cross bridges varies as a function of myosin light chain phosphorylation.

The anterior byssus retractor muscle of Mytilus edulis was used to characterize the myosin cross-bridge during catch, a state of tonic force maintenance with a very low rate of energy utilization. Addition of MgATP to permeabilized muscles in high force rigor at pCa > 8 results in a rapid loss of some force followed by a very slow rate of relaxation that is characteristic of catch. The fast component is slowed 3-4-fold in the presence of 1 mM MgADP, but the distribution between the fast and slow (catch) components is not dependent on [MgADP]. Phosphorylation of twitchin results in loss of the catch component. Fewer than 4% of the myosin heads have ADP bound in rigor, and the time course (0.2-10 s) of ADP formation following release of ATP from caged ATP is similar whether or not twitchin is phosphorylated. This suggests that MgATP binding to the cross-bridge and subsequent splitting are independent of twitchin phosphorylation, but detachment occurs only if twitchin is phosphorylated. A similar dependence of detachment on twitchin phosphorylation is seen with AMP-PNP and ATPgammaS. Single turnover experiments on bound ADP suggest an increase in the rate of release of ADP from the cross-bridge when catch is released by phosphorylation of twitchin. Low [Ca(2+)] and unphosphorylated twitchin appear to cause catch by 1) markedly slowing ADP release from attached cross-bridges and 2) preventing detachment following ATP binding to the rigor cross-bridge.

Catch is characterized by maintenance of force with very low energy utilization in some invertebrate muscles. Catch is regulated by phosphorylation of the mini-titin, twitchin, and a catch component of force exists at all [Ca2+] except those resulting in maximum force. The mechanism responsible for catch force was characterized by determining how the effects of agents that inhibit the low to high force transition of the myosin cross-bridge (inorganic phosphate, butanedione monoxime, trifluoperazine, and blebbistatin) are modified by twitchin phosphorylation and [Ca2+]. In permeabilized anterior byssus retractor muscles from Mytilus edulis, catch force was identified as being sensitive to twitchin phosphorylation, whereas noncatch force was insensitive. In all cases, inhibition of the low to high force transition caused an increase in catch force. The same relationship exists between catch force and noncatch force whether force is varied by changes in [Ca2+] and/or agents that inhibit cross-bridge force production. This suggests that myosin in the high force state detaches catch force maintaining structures, whereas myosin in the low force state promotes their formation. It is unlikely that the catch structure is the myosin cross-bridge; rather, it appears that myosin interacts with the structure, most likely twitchin, and regulates its attachment and detachment.

The endothelium-dependent contractile responses of subepicardial coronary resistance arteries (286 +/- 18 microns ID, n = 22) from rabbits fed either a 0.5 or 2.0% cholesterol-enriched diet or a control diet for 10-12 wk were determined under isometric conditions at the optimum length for active force production (Lo). After the development of tone with 29 mM K+-Krebs, arteries from control rabbits treated with acetylcholine (0.1-10 microM) showed a concentration-dependent relaxation, with a maximum decrease in tone of 63%. In contrast, coronary arteries from animals fed 0.5 and 2.0% cholesterol contracted to acetylcholine (approximately 210% increase in tone). A similar phenomenon was seen with arteries precontracted with 10 nM 9,11-methanoepoxy-prostaglandin H2 (U 46,619), a thromboxane A2 mimetic. The contractile responses to acetylcholine occurred in arteries in which the endothelium was structurally intact and which were devoid of plaque. Arteries from cholesterol-fed animals were poorly responsive to ADP (0.01-10.0 microM), whereas arteries from normal animals relaxed. All arteries relaxed to an equal degree when exposed to acidified nitrite, which produces nitric oxide (NO). The data suggest that as a result of hypercholesterolemia, there may be a dysfunction in the synthesis or release of endothelium-derived relaxing factor (EDRF) by the endothelial cells of coronary resistance arteries, rather than an abnormality of the smooth muscle cells per se.

High-energy phosphate utilization (A~P) associated with force development, force maintenance, and relaxation has been determined during single isometric tetani in the rabbit taenia coli. ATP resynthesis from glycolysis and respiration was stopped without deleterious effects on the muscle. At 18~ and a muscle length of 95% 10, the resting rate of energy utilization is 1.8 + 0.2 nmol/g,s -1, or 0.85 _+ 0.2 mmol ~P/mol of total creatine (Ct).s -1, where Ct = 2.7 ~mol/g wet wt. During the initial 25 s of stimulation when force is developed, the average rate of'A~P was -8.2 + 0.8 mmol/mol Ct.s -1, some four times greater than during the subsequent 35 s of force maintenance, when the rate was -2.0 ztz 0.6 mmol ~P/tool Ct.s -1. The energy cost of force redevelopment (0 to 95% P0) after a quick release from the peak of a tetanus is very low compared with the initial force development. Therefore, the high rate of energy utilization during force development is not due only to internal work done against the series elasticity nor to any high rate of cross-bridge cycling inherently associated with force development. The high economy of force maintenance compared with other muscle types is undoubtedly due to a slower cross-bridge cycle time. The energy utilization during 45 s of relaxation was not statistically significant, and fPdt/A~P was higher during relaxation than during force maintenance in the stimulated muscle.