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...

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The time course of [Ca~+]i, tension, and myosin light chain phosphorylation were determined during prolonged depolarization with high K + in intact tonic (rabbit pulmonary artery) and phasic (longitudinal layer of guinea pig ileum) smooth muscles. [CaZ+]i was monitored with the 340 nm/380 nm signal ratio of the fluorescent indicator fura-2. The fluorescence ratio had a similar time course in both muscle types during depolarization with 109 mM [K+]o; after a transient peak, there was a decline to 70% of its peak value in tonic smooth muscle, and to 60% in phasic smooth muscle. Tension, however, continued to increase in the pulmonary artery, while in the ileum it declined in parallel with the [CaZ+] i. On changing [K+]o from 109 to 20 raM, tension and [CaZ+]i either remained unchanged or declined in parallel in the pulmonary artery. Phosphorylation of the 20-kD myosin light chain, measured during stimulation of muscle strips with 109 mM [K+]o in another set of experiments, increased from 3% to a peak of 50% in the intact pulmonary artery, and then declined to a steady state value of 23%. In the intact ileum, a very rapid, early transient phosphorylation (up to 50%) at 2-3 s was seen. This transient declined by 30 s to a value that was close to the resting level (7%), while tension remained at 55% of its peak force. A quick release during maintained stimulation induced no detectable change in the [CaZ+]i in either type of smooth muscle. We discuss the possibility that the slowly rising tonic tension in pulmonary artery could be due to cooperativity between phosphorylated and nonphosphorylated crossbridges.

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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.

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