J. Caspar Rüegg

Smooth muscle myosin phosphatase inhibition and force enhancement by black sponge toxin

Smooth muscle contraction depends on the state of myosin phosphorylation and hence on the balance of myosin light chain kinase and phosphatase activity. Effects of okadaic acid isolated from black sponge on both enzyme activities and contractility were studied in chemically skinned fibers from guinea pig taenia coli. The toxin strongly inhibits myosin phosphatase and enhances tension development.

Counting target molecules by exponential polymerase chain reaction: copy number of mitochondrial DNA in rat tissues.

In this report, we show that the actual number of target molecules of the polymerase chain reaction can be determined by measuring the concentration of product accumulating in consecutive cycles. The equation describing product accumulation, log Nn = log eff x n + log N0, can be analyzed by linear regression and the molar concentration of target at cycle zero, N0, is obtained. Using this new approach, the actual content of mitochondrial DNA was determined in rat tissues and ranged from 116 x 10(9) molecules/g in fast-twitch skeletal muscle to 743 x 10(9) molecules/g in liver. Using morphometric data from the literature, mitochondria were found to contain 1 to 3 DNA molecules. There was no relation between the oxidative capacity of a tissue and its content of mitochondrial DNA, indicating that transcriptional and posttranscriptional mechanisms rather than gene dosage, as postulated by others, determine to what extent the mitochondrial genome is expressed.

Troponin replacement in permeabilized cardiac muscle Reversible extraction of troponin I by incubation with vanadate

Calcium‐dependent regulation of tension and ATPase activity in permeabilized porcine ventricular muscle was lost after incubation with 10 mM vanadate. After transfer from vanadate to a vanadate‐free, low‐Ca2+ solution (pCa > 8), the permeabilized muscle produced 84.8% ± 20.1% (± S.D., n=98) of the isometric force elicited by high Ca22+ (pCa ∼4.5 prior to incubation with vanadate. Transfer back to a high Ca2+ solution elicited no additional force (83.2% ± 18.7% of control force). SDS‐PAGE and immunoblot analysis of fibers and solutions demonstrated substantial extraction (>90%) of Troponin I (TnI). Calcium dependence was restored after incubation with solutions containing either whole cardiac troponin or a combination of TnI and troponin C subunits. This reversible extraction of troponin directly demonstrates the role of TnI in the regulation of striated muscle contractility and permits specific substitution of the native TnI with exogenously supplied protein.

Myosin composition and functional properties of smooth muscle from the uterus of pregnant and non-pregnant rats.

The myosin heavy chain stoichiometry and the force-velocity relation have been determined in the myometrium of the non-pregnant and pregnant rat. The relative proportions of the slower migrating heavy chain (MHC1) greatly exceeded that of the faster migrating heavy chain (MHC2) as shown by electrophoresis on SDS 4%-polyacrylamide gels. The ratios of MHC1/MHC2 were 2.2/1 in the non-pregnant rats, 2.6/1 in the pregnant rat, and contrasted with 0.8/1 in the rat portal vein. This stoichiometry was unchanged by extracting the myosin from the smooth muscle as native myosin in a salt extract, as dissociated myosin using sodium dodecyl sulphate (SDS) or by isolating the native myosin first by a non-dissociating (pyrophosphate) electrophoresis step and subsequently analysing the protein bands on the SDS 4%-polyacrylamide gel. Although the unequal proportions of the heavy chains suggested the possibility that the native myosin molecule may be arranged as homodimeric heavy chains, no evidence for or against the existence of native myosin isoforms could be obtained by electrophoresing native myosin extracts on pyrophosphate-polyacrylamide gels. The force-velocity relations of the intact electrically stimulated myometrium from the non-pregnant and pregnant rats gave isometric force of 45 and 135 mN/mm2 andVmax of 0.71 and 0.52 lengths/s (37°C) when measured at 95% of optimal length, whereas in chemically skinned uterine strips at 22°CVmax was 0.09 and 0.13 lengths/s, respectively. The length-force relationship was of similar shape in the non-gravid and gravid skinned tissues. The energetic tension cost (ATP-turnover/active stress) in skinned fibres was also similar. The mechanical and metabolic characteristics of the gravid and non-gravid uterus found in the present study do not suggest an obvious difference in the intrinsic properties of the myosin, although significant functional alterations in the tissue appear during pregnancy. This corresponds to the lack of a difference in the pattern of the heavy chains.

Relaxation of chemically skinned guinea pig taenia coli smooth muscle from rigor by photolytic release of adenosine-5′-triphosphate

SummaryThe mechanical events following release of ATP from P3-1-(2-nitro)phenylethyladenosine-5′-triphosphate (caged-ATP) in skinned guinea pig taenia coli smooth muscle in rigor were investigated. A rigor force of about 25–35% of the maximal active force was obtained by removing ATP at the plateau of a maximal active contraction. In the rigor solution free-Mg2+ was 2mm, ionic strength 90mm and pH 7.0. When caged-ATP (12.5mm) was diffused into the preparation there was no change in the rigor force. Photolytic production of about 2mm ATP was achieved with a xenon flash lamp. Following illumination, force decreased with an approximate initial rate constant of 0.7 s−1. The rate of relaxation was increased in the presence of inorganic phosphate (at 3mm: 1.3 s−1; 10mm: 2.2 s−1). At higher Mg2+ concentrations the rate of relaxation was slower (5mm: 0.2 s−1) and at lower concentrations the rate was faster (0.5mm: 1.2 s−1). An increased rate of relaxation was observed when ionic strength was increased to 150mm (2.2 s−1). Phosphate increased the rate of relaxation at the different levels of Mg2+ (0.5–10mm) and ionic strength (90, 150mm). In preparations shortened (by 1–3%) to give reduced rigor force, a small transient increase in tension was recorded after ATP release. In comparison to the rates of ATP-induced dissociation of actomyosin in solution, reported in the literature, the rate of relaxation from rigor is slower. This may reflect a slow rigor cross-bridge dissociation or mechanical interactions not associated with cross-bridges in the muscle fibre. However, the results may also be interpreted on the basis of a model proposed for striated muscle by Goldmanet al. (1984) where the relaxation from rigor in the absence of Ca2+ involves a phase of reattaching cross-bridges whose lifetime in a tension-producing state is influenced by phosphate.

Okadaic acid, a phosphatase inhibitor, produces a CA2+ and calmodulin-independent contraction of smooth muscle

The effects of okadaic acid, a phosphoprotein phosphatase inhibitor, on the contractile response and on myosin light chain phosphorylation were studied in intact lamb tracheal smooth muscle. The effects of okadaic acid were compared to the response to the same fibers stimulated with 1 μM methacholine, a concentration that induces 90% of maximal force. Okadaic acid (50 μM) produced a slow but maximal contraction that was accompanied by an increase in phosphorylation of the 20 kDa light chain of myosin. The myosin light chain phosphorylation pattern induced by okadaic acid, however, differed from that induced by methacholine. Ca2+ depletion, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a protein kinase C inhibitor, blocked or attenuated methacholine-induced contractions but had no significant effect on force development or myosin light chain phosphorylation induced by okadaic acid. These results suggest that phosphorylation of the 20 kDa light chain of myosin is essential for smooth muscle contraction; they also suggest that okadaic acid either uncovers or activates an apparently Ca2+ and calmodulin-independent protein kinase activity that phosphorylates the 20 kDa light chain of myosin at multiple sites.

The positive inotropic calcium sensitizer EMD 53998 antagonizes phosphate action on cross-bridges in cardiac skinned fibers

The diazinone derivative EMD 53998 sensitizes skinned myocardial fibers to Ca2+ and enhances maximal calcium-activated force (pCa = 4.5) by approximately 100%; the EC50 is 10 microM in the absence and about 30 microM in the presence of added inorganic phosphate (10 mM). Although concentrations of added phosphate as low as 0.5 mM inhibit force, at high concentrations of EMD 53998 (> or = 50 microM), phosphate only inhibits at concentrations exceeding 20 mM. These data suggest that the effects of EMD 53998 and phosphate are mutually antagonistic. Importantly, both EMD 53998 and phosphate had similar effects on force generation in troponin I-depleted (Ca(2+)-independent) skinned fibers, thus demonstrating that these compounds are likely to affect cross-bridges directly and not via the Ca(2+)-regulatory system.

Striational autoantibodies in myasthenia gravis patients recognize I-band titin epitopes

Myasthenia gravis (MG) patients develop autoantibodies primarily against the acetylcholine receptor in the motor endplate, but also against intracellular striated muscle proteins, notably titin, the giant elastic protein of the myofibrillar cytoskeleton. Titin antibodies have previously been shown to be directed against a single epitope on the molecule, located at the A-band/I-band junction and referred to as the main immunogenic region (MIR) of titin. By using immunofluorescence microscopy on stretched single myofibrils, we now report that approximately 40% of the sera from 18 MG/thymoma patients and 8 late-onset MG patients with thymus atrophy contain antibodies that bind to a more central I-band titin region. This region consists of homologous immunoglobulin domains and is known to be differentially spliced dependent on muscle type. All patients with I-band titin antibodies also had antibodies against the MIR. Although a statistically significant correlation between the occurrence of I-band titin antibodies and MG severity was not apparent, the results could hint at an initial immunoreactivity to titins MIR, followed by reactivity along the titin molecule in the course of the disease.

Recombinant troponin I substitution and calcium responsiveness in skinned cardiac muscle

Using treatment with vanadate solutions, we extracted native cardiac troponin I and troponin C (cTnI and cTnC) from skinned fibers of porcine right ventricles. These proteins were replaced by exogenously supplied TnI and TnC isoforms, thereby restoring Ca2+-dependent regulation. Force then depended on the negative logarithm of Ca2+ concentration (pCa) in a sigmoidal manner, the pCa for 50% force development, pCa50, being about 5.5. For reconstitution we used fast-twitch rabbit skeletal muscle TnI and TnC (sTnI and sTnC), bovine cTnI and cTnC or recombinant sTnIs that were altered by site-directed mutagenesis. Incubation with TnI inhibited isometric tension in TnI-extracted fibers in the absence of Ca+, but restoration of Ca2+ dependence required incubation with both TnI and TnC. Relaxation at low Ca2+ levels and the steepness of the force/pCa relation depended on the concentration of exogenously supplied TnI in the reconstitution solution (range 20–150 μM), while Ca2+ sensitivity, i.e. the pCa50, was dependent on the isoform, and also on the concentration of TnC in the reconstitution solution. At pH 6.7, skinned fibers reconstituted with optimal concentrations of sTnC and sTnI (120 μM and 150 μM, respectively) were more sensitive to Ca2+ than those reconstituted with cTnC and cTnI (difference in pCa50 approx. 0.2 units). Rabbit sTnI was cloned and expressed inEscherichia coli using a high yield expression plasmid. We introduced point mutations into the TnI inhibitory region comprising the sequence of the minimal common TnC/actin binding site (-G104-K-F-K-R-P-P-L-R-R-V-R115-). The four mutants produced by substitution of T for P110, G for P110, G for L111 and G for K105 were chosen, based on previous work with synthetic peptides showing that single amino acid substitution in this region diminished the capacity of these peptides to inhibit acto-Si, ATPase or contraction of skinned fibers. Therefore, all amino acid residues of the inhibitory region are thought to contribute to biological activity of TnI. However, each of the recombinant TnIs could substitute for endogenous TnI. In combination with exogenous TnC, Ca2+ dependence could be restored whengly110sTnI,thr110sTnI orgly111sTnI was used for reconstitution. The mutantgly105sTnI, on the other hand, reduced the ability of skinned fibers to relax at low Ca2+ concentrations and it caused an increase in Ca2+ sensitivity.

Smooth muscle: PKC‐induced Ca2+ sensitisation by myosin phosphatase inhibition

When arterial smooth muscles are stimulated with certain agonists such as phenylephrine contractile force increases while the intracellular free Ca2+ concentration may increase very little or even not at all (cf. Bradley & Morgan, 1987). In this case, force may be enhanced by raising the responsiveness of the contractile machinery or - for that matter - the sensitivity of the myofilaments to intracellular free Ca2+ (some 100 nM under resting conditions). The nature of mechanisms underlying Ca2+ sensitisation in pharmacomechanical coupling has been shown to involve various quite different intracellular signalling pathways and signalling molecules, such as small G-proteins (e.g. Rho A) and enzymes such as Rho-associated kinase, MAP kinase and various isoforms of protein kinase C (PKC) (cf. Somlyo & Somlyo, 1994; for review see Arner & Pfitzer, 1999). Based on the observation that phorbol esters, which are well known activators of PKC, cause a sustained contraction of arterial smooth muscle (Rasmussen et al. 1984) and also increase the sensitivity of the contractile apparatus to added Ca2+ in permeabilised smooth muscle preparations (Chatterjee & Tejeda, 1986), a role for PKC-induced Ca2+ sensitisation in tonic smooth muscle contraction was proposed long ago. During smooth muscle activation, PKC has been shown to be translocated from the cytosol to the cell membrane (Khalil et al. 1992), where it may be activated by diacylglycerol (DAG). It then initiates a signal sequence leading to the phosphorylation of specific intracellular receptors called receptors for activated C-kinase (RACKs). Thus the question arises as to the nature of RACKs involved in Ca2+ sensitisation of the myofilamets by PKC. The primary mechanism of smooth muscle contractile activation is the phosphorylation of myosin light chain (MLC) though, admittedly, the thin filament proteins caldesmon and calponin may also play a role (Arner & Pfitzer, 1999). It is now well established that the physiological increase in the phosphorylation of MLC is caused not only via Ca2+/calmodulin activation of myosin light chain kinase but also by a decrease in the dephosphorylating enzyme activity (e.g. Kitazawa et al. 1991). In a paper appearing in this issue of The The Journal of Physiology, Kitazawa et al. (1999) now show that in permeabilised (skinned) preparations of arterial smooth muscle, PKC enhances contractile force at a given submaximal concentration of free Ca2+ by increasing the level of MLC phosphorylation via inhibition of myosin phosphatase. This inhibition is mediated by phosphorylation of a novel RACK called CPI-17. This smooth muscle specific myosin phosphatase inhibitor exerts its phosphatase inhibitory and hence Ca2+ sensitising action in permeabilised preparations only when phosphorylated by PKC. These discoveries were made by using a differential skinning method: while intact arterial smooth muscle or preparations permeabilised by Staphylococcus aureusα-toxin or with the saponin ester β-escin became sensitised to Ca2+ by phorbol esters and other activators of PKC, fibres demembranated with the detergent Triton X-100 did not. The reason was that Triton-skinned fibres had lost the smooth muscle specific phosphatase-1 inhibitor protein CPI-17 and most of PKC. However, reconstitution of these fibres with PKC and CPI-17 increased MLC phosphorylation and the response to Ca2+ suggesting that, indeed, this kinase and the phosphatase inhibitor were the key players, causing increased myosin phosphorylation and contraction at submaximal Ca2+ concentration. These important new findings challenge current ideas (cf. Singer, 1996) suggesting that the physiological activation of PKC in vascular smooth muscle and its sensitisation to Ca2+ are mediated by mechanisms involving mitogen activated protein kinase-dependent caldesmon phosphorylation rather than phosphorylation of MLC by PKC. Now, the question arises as to what extent the proposed mechanisms may be operating in smooth muscle in vivo. It seems quite puzzling how in intact cells PKC can phosphorylate CPI-17 which, apparently, is located in the cytosol, while PKC activation requires its translocation to the cell membrane. Perhaps, CPI-17 shuttles between membrane-bound active PKC and thick filament-bound myosin phosphatase. Alternatively, membrane-bound (activated) PKC may be in rapid equilibrium with cytosolic PKC. In the cytosolic compartment the quantity of activated PKC, though low, may then be still sufficient to cause the Ca2+ sensitising downstream effect mediated by the phosphorylation of the smooth muscle specific phosphatase-1 inhibitor CPI-17 targeted to the thick filaments.