Skinning smooth muscle

LOnnbro, P. Hellstrand, P. (1991). Heat production in chemically skinned smooth muscle of guinea-pig Taenia coli. J. Physiol. 440, 385-401. 

V [al(V)2a2(V)], [al(V)3l 390-nm-long fibrils with globular N-terminal extension often with type I Cornea, teeth, bone, placenta, skin, smooth muscle... 

The use of saponin, a plant glycoside, for permeabilization was introduced by Endo and coworkers (1977). Saponin removes the surface membrane without impairment of the functions of SR. The plasma membrane, unlike that of triton skinned smooth muscle (Spedding, 1983), appears fairly intact in electron microscopic pictures of cross sections of permeabilized cells. However, when patches of isolated membranes were viewed face on in homogenates of permeabilized cells, numerous 70- to 80-A holes were visible (Kargacin and Lay, 1987). 

III. PROBING THE PHOSPHORYLATION THEORY IN TRITON SKINNED SMOOTH MUSCLE ... 

In intact smooth muscle, phosphorylation often declines while force is maintained, the so-called "latch" state (Dillon etal., 1981). Regulation of the latch state is still poorly understood. It has been very difficult to induce a latch state in skinned smooth muscle. The closest may be triton skinned chicken gizzard, which contracts independent of MLC phosphorylation (Wagner and Ruegg, 1986). In skinned chicken gizzard, but also in other types of smooth muscle (Bialojan et al., 1987 Siegman et al., 1989 Kenney et al., 1990 Schmidt et al, 1995), a steep and nonlinear relation between force and MLC phosphorylation was observed, which was postulated by the "latch model" of Hai and Mur-... 

In triton skinned and in a-toxin-permeabilized smooth muscle preparations, the Ca + sensitivity of force production is also decreased by cGMP (Pfitzer et al., 1984,1986 Nishimura efflZ., 1992). This may be due to an up-regulation of MLCP (Pfitzer etal., 1986). Clear evidence that modulation of the activity of MLCP would affect Ca + sensitivity of tension development was in fact obtained in triton skinned smooth muscle when it was shown that inhibition of MLCP by the black sponge toxin, okadaic acid, increased Ca + sensitivity (Takai et al., 1987 Bialojan et al., 1988). On the other hand, incubation of triton skinned chicken gizzard fibers with a purified phosphatase decreased Ca + sensitivity (Bialojan et al., 1987). 

Pharmacomechanical coupling was primarily studied by measuring [Ca +J and myosin regulatory light chain phosphorylation in intact and skinned smooth muscle. Several groups found that stimuli increased... 

The 1,4,5-IP3 hypothesis has been extensively tested in smooth muscle. In skinned smooth muscle, physiological concentrations of 1,4,5-IP3 have been shown to release sufficient Ca + rapidly enough to account for transient contractions (Somlyo et al, 1985 Suematsu et al., 1984 Walker et al, 1987 Baron et al., 1989 Abdel-Latif, 1989). This response can be blocked by intracellular heparin (Kobayashi et al., 1988b), a competitive blocker of cerebellar 1,4,5-IP3 receptors (Worley et al., 1987). Agonist stimulation increased the apparent concentration of 1,4,5-IP3 when measured as either [ HJIPg in cells (Griendling etal., 1986) or tissues (Howe etal., 1986 Long and Stone, 1987 Takuwa etal., 1986 Miller-Hance et al., 1988 Salmon and Bolton,... 

Direct inhibitors of MLCK act via one of two modes. A series of small peptide substrate (MLC) and/or calmodulin inhibitors have been described by two groups (Moreland and Hunt, 1987 Foster and Gaeta, 1988). Since these are peptides, they are not active as inhibitors in intact cell or tissue studies, but can be used as tools in skinned smooth muscle preparations, as well as with purified contractile proteins. 

Applegate D, Pardee JD (1992) Actin-facilitated assembly of smooth muscle myosin induces formation of actomyosin fibrils. J Cell Biol 117 1223-1230 Arner A, Hellstrand P (1985) Effects of calcium and substrate on force-velocity relation and energy turnover in skinned smooth muscle of the guinea-pig. J Physiol 360 347-365... 

The concept that the Ca +-sensitivity of contraction could be modulated was developed when it was shown that cAMP relaxed skinned smooth muscle strips at constant submaximal [Ca +] and shifted the calcium force relation to the right towards higher values (cf. Riiegg 1992). It has also been shown that excitatory agonists that activate heterotrimeric G-proteins can increase LC20 phosphorylation and force at constant [Ca ] (Somlyo and Somlyo 1994). In this context, a decrease in Ca -sensitivity refers to a shift in the relation between [Ca +] and phosphorylation or force towards higher [Ca +] an increase in Ca +-sensitivity is a shift towards lower [Ca " ]. The physiological relevance of the Ca +-sensitivity modulation became apparent when simultaneous measurements of intracellular [Ca +] and force in intact smooth muscle showed that there was no unique rela-... 

In Triton skinned smooth muscle, cAMP or the catalytic subunit of cAMP dependent protein kinase inhibits tension development or induces relaxation at submaximal [Ca +] thereby decreasing the Ca -sensitivity of contraction (Kerrick and Hoar 1981, Riiegg et al. 1981, Riiegg and Paul 1982, Sparrow et al. 1984). Inhibition of force was associated with a decrease in LC20 phosphorylation (Riiegg and Pfitzer 1985). The inhibitory... 

An important problem with any latch theory is that the latch-bridge in many ways remains descriptive and has not been coupled to specific biochemical actomyosin states or mode of actomyosin turnover. Skinned fibres could provide a way to explore this problem but at present there is a striking lack of a latch state in skinned smooth muscle preparations with a few exceptions. The hyperbolic phosphorylation force relationship has been demonstrated in e.g. skinned smooth muscle strips from chicken... 

Arheden H, Arner A (1992) Effects of magnesium pyrophosphate on mechanical properties of skinned smooth muscle from the guinea pig taenia coli. Biophys J 61 1480-1494... 

Arner A, Hellstrand P, Rflegg JC (1987b) Influence of ATP, ADP and AMPPNP on the energetics of contraction in skinned smooth muscle. In Siegman MJ, Somiyo AP, Stephens. NL (eds) Regulation and Contraction of Smooth Muscle. Alan Liss, New York pp 43-57... 

Barsotti RJ, Ikebe M, Hartshome DJ (1987) Effects of Ca, Mg ", and myosin phosphorylation on skinned smooth muscle fibers. Am J Physiol 252 C543-C554 Bengur AR, Robinson EA, Appella E, Sellers JR (1987) Sequence of the sites phospho-rylated by protein kinase C in the smooth muscle myosin li t chain. J Biol Chem 262 7613-7617... 

Cassidy PS, Kerrick WG, Hoar PE, Malencik DA (1981) Exogenous calmodulin increases Ca " sensitivity of isometric tension activation and myosin phosphorylation in skinned smooth muscle. Pflugers Arch 392 115-120 Cavaille F, Janmot C, Ropert S, d Albis A (1986) Isoforms of myosin and actin in human, monkey and rat myometrium.Comparison of pregnant and non-preghant uterus proteins. Eur J Biochem 160 507-513... 

Fujiwara T, Itoh T, Kubota Y, Kuriyama H (1989) Effects of guanosine nucleotides on skinned smooth muscle tissue of the rabbit mesenteric artery. J Physiol Lond 408 535-547... 

Itagaki M, Komori S, Unno T, Syuto B, OhashiH (1995) Possible involvement of a small G-protein sensitive to exoenzyme C3 of Clostridium botulinum in the regulation of myofilament Ca -sensitivity in p-escin skinned smooth muscle of guinea pig ileum. Jap J Pharmacol 67 1-7... 

Itoh T, Suzuki S, Suzuki A, Nakamura F, Naka M, Tanaka T (1994) Effects of exogenously applied calponin on Ca(2+)-regulated force in skinned smooth muscle of the rabbit mesenteric artery. Pfliigers Arch 427 301-308... 

Kemp BE, Pearson RB, Guerriero V Jr, Bagchi 1C, Means AR (1987) The calmodulin binding domain of chicken smooth muscle myosin light chain kinase contains a pseudosubstrate sequence. J Biol Chem 262 2542-2548 Kenney RE, Hoar PE, Kerrick WG (1990) The relationship between ATPase activity, isometric force, and myosin l ht-chain phosphorylation and thiophosphorylation in skinned smooth muscle fiber bundles from chicken gizzard. J Biol Chem 265 8642-8649... 

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