Force Maintenance Research Articles

Altered isometric force control during object release in patients with Huntington's Disease

Background: Objective markers for the severity of motor dysfunction are warranted for clinical trials in patients with Huntington's Disease (HD). Specific impairments in the control of isometric grip forces were found in grasping, holding, and transport tasks in HD. In previous studies we identified „grip force variability“ (GFV) as a measure correlated (1) to the clinical severity of the disease as assessed in the UHDRS, (2) to the amount of neurodegeneration in MRI volumetry, and (3) to the CAG repeat length when corrected for the age of patients. Thus GFV may be a surrogate marker to objectively assess the severity of HD. While the initiation and maintenance of grip forces in grasping and lifting has intensively been investigated in HD, it is unknown if specific impairments occur during object release in HD.

Abstract 20: Improvement in Upper-Limb Function in Children With Hemiplegic Cerebral Palsy Following a Six-Week Motor Training Intervention

Objective: To determine if motor training will lead to improved upper-limb coordination in children with hemiplegic cerebral palsy (CP). Design: Within-subject control intervention study. Setting: Home, school, university motor control laboratory. Participants: 16 subjects with hemiplegic CP. Intervention: 2 groups of 8 subjects received 6 weeks of task-based, upper-limb motor training in a home or school setting. Subjects received either a more traditional intensity of training consisting of 30 minutes, 3 times a week, or a more intensive intensity comprised of 30 minutes of training twice a day, 5 days a week. Main Outcome Measures: Upper-limb coordination associated with reaching and hand function were assessed using 3-dimensional motion analysis and grasp force techniques prior to intervention onset, at week 3, and week 6. Reach trajectories were analyzed as a measure of multijoint coordination. Static grasp force variability was used as a measure of hand function. Results: Within-subject analysis revealed improvements in multijoint coordination ranging from 20% to 40% in 6 of the 8 children receiving the more intensive intervention. Across all subjects, a significant (P<.05) improvement from baseline values was observed at week 3 with only marginal or no further improvements at week 6. In the less intensive group, similar improvements were observed in most subjects but only at week 6. Improvement in hand function was variable in both groups, although a nonsignificant (P=.07) trend toward improved force maintenance was observed in the more intensive group. Conclusions: These findings demonstrate that a home-based, task-specific motor training program involving as little as 1.5h/wk for 6 weeks can improve upper-limb coordination in CP. These results also show that such training programs can be effectively administered outside a clinic setting, thereby increasing accessibility to rehabilitative programs.

The phage‐shock‐protein response

The phage-shock-protein (Psp) system responds to extracytoplasmic stress that may reduce the energy status of the cell. It is conserved in many different bacteria and has been linked to several important phenotypes. Escherichia coli psp mutants have defects in maintenance of the proton-motive force, protein export by the sec and tat pathways, survival in stationary phase at alkaline pH, and biofilm formation. Yersinia enterocolitica psp mutants cannot grow when the secretin component of a type III secretion system is mislocalized, and have a severe virulence defect in animals. A Salmonella enterica psp mutation exacerbates some phenotypes of an rpoE null mutant and the psp genes of S. enterica and Shigella flexneri are highly induced during macrophage infection. PspA, the most abundant of the Psp proteins, is required for most of the phenotypes associated with the Psp system. Therefore, PspA is probably an effector that may play a role in maintaining cytoplasmic membrane integrity and/or the proton-motive force. However, PspA is not required for the ability to tolerate secretin mislocalization, which suggests an important physiological role for other Psp proteins. This article summarizes our current understanding of the Psp system: inducing signals, the underlying signal transduction mechanisms, the physiological roles it may play, and a genomic analysis of its conservation.

No effect of twitchin phosphorylation on the rate of myosin head detachment in molluscan catch muscle: are myosin heads involved in the catch state?

Phosphorylation of twitchin is known to abolish the catch state of anterior byssus retractor muscle (ABRM) of the bivalve mollusc Mytilus edulis. To investigate the role of myosin head involvement in force maintenance during catch, the effect of twitchin phosphorylation on myosin head detachment was studied in saponin-skinned fibre bundles of ABRM. The detachment rate of myosin heads was deduced from two types of experiments: (1) force decay after stepwise stretch of maximally Ca2+-activated fibre bundles (pCa 4.5) and (2) force decay from high-force rigor, the former induced by a stepwise increase in ATP concentration elicited by photolysis of caged ATP (pCa<8). The rate of detachment was not affected by thiophosphorylation or phosphorylation of twitchin by 0.12 mM cAMP in the presence of the phosphatase inhibitor cyclosporine A (1 microM). Conversely, measurements of the rate of stretch-induced delayed force increase (stretch activation) and of the force increase following an ATP step in low-force rigor (pCa 4.5) suggest that the rate of myosin head attachment decreases after twitchin phosphorylation. We conclude that catch is not due to myosin heads remaining attached to actin filaments, but depends on myofilament interconnections that break down when twitchin is phosphorylated.

Molecular Determinants for PspA-Mediated Repression of the AAA Transcriptional Activator PspF

The Escherichia coli phage shock protein system (pspABCDE operon and pspG gene) is induced by numerous stresses related to the membrane integrity state. Transcription of the psp genes requires the RNA polymerase containing the sigma(54) subunit and the AAA transcriptional activator PspF. PspF belongs to an atypical class of sigma(54) AAA activators in that it lacks an N-terminal regulatory domain and is instead negatively regulated by another regulatory protein, PspA. PspA therefore represses its own expression. The PspA protein is distributed between the cytoplasm and the inner membrane fraction. In addition to its transcriptional inhibitory role, PspA assists maintenance of the proton motive force and protein export. Several lines of in vitro evidence indicate that PspA-PspF interactions inhibit the ATPase activity of PspF, resulting in the inhibition of PspF-dependent gene expression. In this study, we characterize sequences within PspA and PspF crucial for the negative effect of PspA upon PspF. Using a protein fragmentation approach, we show that the integrity of the three putative N-terminal alpha-helical domains of PspA is crucial for the role of PspA as a negative regulator of PspF. A bacterial two-hybrid system allowed us to provide clear evidence for an interaction in E. coli between PspA and PspF in vivo, which strongly suggests that PspA-directed inhibition of PspF occurs via an inhibitory complex. Finally, we identify a single PspF residue that is a binding determinant for PspA.

Compensatory role of PspA, a member of the phage shock protein operon, in rpoE mutant Salmonella enterica serovar Typhimurium

Sigma(E) is an alternative sigma factor that responds to and ameliorates extracytoplasmic stress. In Salmonella enterica serovar Typhimurium (S. Typhimurium), sigma(E) is required for oxidative stress resistance, stationary-phase survival and virulence in mice. Microarray analysis of stationary-phase gene expression in rpoE mutant bacteria revealed a dramatic increase in expression of pspA, a member of the phage shock protein (psp) operon. The psp operon can be induced by filamentous bacteriophages or by perturbations of protein secretion, and is believed to facilitate the maintenance of proton motive force (PMF). We hypothesized that increased pspA expression may represent a compensatory response to the loss of sigma(E) function. Increased pspA expression was confirmed in rpoE mutant Salmonella and also observed in a mutant lacking the F(1)F(0) ATPase. Alternatively, expression of pspA could be induced by exposure to CCCP, a protonophore that disrupts PMF. An rpoE pspA double mutant strain was found to have a stationary-phase survival defect more pronounced than that of isogenic strains harbouring single mutations. The double mutant strains were also more susceptible to killing by CCCP or by a bactericidal/permeability-increasing protein (BPI)-derived anti-microbial peptide. Using fluorescence ratio imaging, differences were observed in the Deltapsi of wild-type and rpoE or pspA mutant bacteria. These findings suggest that pspA expression in S. Typhimurium is induced by alterations in PMF and a functional sigma(E) regulon is essential for the maintenance of PMF.

Identification of a New Member of the Phage Shock Protein Response in Escherichia coli, the Phage Shock Protein G (PspG)

The phage shock protein operon (pspABCDE) of Escherichia coli is strongly up-regulated in response to overexpression of the filamentous phage secretin protein IV (pIV) and by many other stress conditions including defects in protein export. PspA has an established role in maintenance of the proton-motive force of the cell under stress conditions. Here we present evidence for a new member of the phage shock response in E. coli. Using transcriptional profiling, we show that the synthesis of pIV in E. coli leads to a highly restricted response limited to the up-regulation of the psp operon genes and yjbO. The psp operon and yjbO are also up-regulated in response to pIV in Salmonella enterica serovar Typhimurium. yjbO is a highly conserved gene found exclusively in bacteria that contain a psp operon but is physically unlinked to the psp operon. yjbO encodes a putative inner membrane protein that is co-controlled with the psp operon genes and is predicted to be an effector of the psp response in E. coli. We present evidence that yjbO expression is driven by sigma(54)-RNA polymerase, activated by PspF and integration host factor, and negatively regulated by PspA. PspF specifically regulates only members of the PspF regulon: pspABCDE and yjbO. We found that increased expression of YjbO results in decreased motility of bacteria. Because yjbO is co-conserved and co-regulated with the psp operon and is a member of the phage shock protein F regulon, we propose that yjbO be renamed pspG.

Effects of Vocationally Oriented Medical Rehabilitation for Aircraft Maintenance Personnel—A Preliminary Study of Long-Term Effects with 5-year Follow-Up

Changes in the physical capacity, musculoskeletal symptoms, and perceived work ability of Finnish Air Force maintenance personnel were studied after vocationally oriented medical rehabilitation (VOMR). Twenty persons with chronic musculoskeletal symptoms in their back or neck took part in VOMR courses. The measurements were carried out at the beginning of the rehabilitation course and after two follow-up periods (0.5 and 5 years). The subjects worked most of the time in a bent position and often with their backs twisted and their arms above their shoulders. The severity of low-back pain and the number of days of sick leave decreased significantly (p < 0.05-0.01) during the 5-years follow-up. Also the exercise breaks at work increased (p < 0.01). After half a year of medical rehabilitation the measured range of the cervical spine and the dynamic and endurance strength of the upper and lower extremities was greater (p < 0.05-0.001) than at the beginning of the rehabilitation. There were no statistically significant differences in the use of physical therapy, experienced work strain, physical exercise or maximal oxygen consumption during the follow-up. This is a preliminary follow-up study lasting 5 years showed some significant changes in physical capacity, musculoskeletal symptoms and work ability. However, controlled studies are needed to evaluate these preliminary findings of this kind of rehabilitation model.

Twitchin, a thick-filament protein from molluscan catch muscle, interacts with F-actin in a phosphorylation-dependent way

Twitchin belongs to the titin-like giant proteins family, it is co-localized with thick filaments in molluscan catch muscles and regulates the catch state depending on its level of phosphorylation. The mechanism by which twitchin controls the catch state remains to be established. We report for the first time the ability of twitchin to interact with F-actin. The interaction is observed at low and physiological ionic strengths, irrespective of the presence or absence of Ca 2+. It was demonstrated by viscosity and turbidity measurements, low- and high-speed co-sedimentation, and with the light-scattering particle size analysis revealing the specific twitchin–actin particles. The twitchin–actin interaction is regulated by twitchin phosphorylation: in vitro phosphorylated twitchin does not interact with F-actin. We speculate that the catch muscle twitchin might provide a mechanical link between thin and thick filaments, which contributes to catch force maintenance.

Force maintenance with submaximal fatiguing contractions.

Whereas many definitions of fatigue include externally measurable decrements in force or performance, fatigue can be present with no change in the external output of the muscle. The maintenance of submaximal forces can be considered a compromise between neuromuscular force enhancement and competing inhibitory influences. An example of a muscle facilitatory process includes postactivation potentiation that results in an increased sensitivity to Ca++. The neuromuscular system copes with metabolic disruption and subsequent loss of force by recruiting additional motor units and increasing the firing frequency. If the contraction persists, firing frequency may decrease so as to optimize the stimulus rate with the prolonged duration of the muscle fibre action potential (muscle wisdom). The insertion of additional neural impulses into the train of stimuli can result in force potentiation (catch-like properties). Furthermore, there is evidence of neural potentiation and a dissociation of muscle activity with submaximal fatigue. Conversely, inhibition may be derived supraspinally or at the spinal level. While there may be some evidence of intrinsic motoneuronal fatigue, inhibitory afferent influences from chemical, tensile, pressure, and other factors play an important role in the competing influences on force output.

Measurements Of Complexity In Motor Unit Discharge Behavior Of Young And Older Adults

0838 Complexity measures such as approximate entropy (ApEn) and detrended fluctuation analysis (DFA) may provide more information about the variability in motor unit inter-spike intervals (ISIs) than magnitude measures such as the standard deviation. PURPOSE: The purpose of the present study was 1) to compare the complexity of motor unit discharge behavior in young and older adults and 2) to test the utility of two complexity measures using motor unit data from two different force matching conditions. METHODS: Old (n = 12, 72.6 years) and young (n = 12, 18.8 years) subjects performed each of the following isometric force-matching tasks involving the first dorsal interosseous muscle. A linear, 20% maximal voluntary contraction (MVC) task was used to investigate the fluctuations that occur during the maintenance of constant force. A compound sinusoidal task (20 +/− 4.14 %MVC) was used to investigate motor unit discharge variability amidst substantial modulation of motor unit discharge rates. RESULTS: ApEn was similar in both conditions (F = 1.70, p = 0.207) and there was greater complexity in older adults (F = 13.97, p = 0.001). DFA suggested age-related differences in complexity, but only in the sinusoidal condition (age × condition: F = 4.91, p = 0.039). In the linear condition, DFA indicated random behavior that was similar in young and older adults (F = 0.68, p = 0.420). In the sinusoidal condition, young adults exhibited less randomness in their motor unit discharge than older adults (F = 4.67, p = 0.042). CONCLUSION: Both measures provided evidence of greater complexity in motor unit discharge of older adults while only DFA was sensitive to differences between the two conditions. That ApEn did not discriminate between is evidence that the ApEn provides a measure of complexity that is independent of periodicity within in the data. The greater DFA in young adults compared to old in the sinusoidal condition likely reflects their greater ability to match the periodic components in the task. NIH Fellowship Grant 1 F31 NS4 3069–01.

Alteration of contractile and regulatory proteins following partial bladder outlet obstruction

This paper reviews the contractility and the expression of contractile and regulatory proteins in the detrusor smooth muscle (DSM) following partial bladder outlet obstruction (PBOO) in rabbits. PBOO was surgically induced by partial ligation of the urethra in adult male New Zealand White rabbits. The force generated by DSM strips from normal and obstructed bladders which showed bladder dysfunction, despite detrusor hypertrophy (decompensated bladder, DB) was measured. The expression of contractile and regulatory proteins was analyzed by reverse transcriptase-polymerase chain reaction and Western blotting. The DSM from obstructed DB revealed an overexpression of SM-A myosin heavy chain isoform (associated with decreased maximum velocity of shortening). DSM from sham-operated rabbits showed phasic contractions, whereas the detrusor from DB was tonic, exhibiting slow development of force, a longer duration of force maintenance, and slow relaxation. Rho-kinase inhibitor Y-27632 enhanced the relaxation of precontracted (with 125 mM KCl) DSM strips from DB. The enhancement of relaxation of DB by Y-27632 was associated with dephosphorylation of myosin light chain. The detrusor from normal bladders expresses predominantly the smooth muscle caldesmon (h-CaD), a thin filament-associated protein. However, the DSM from DB shows an overexpression of l-CaD, the non-muscle isoform of CaD. The l-CaD colocalizes with myosin in the cytoplasmic filaments in myocytes. These results show that the alteration of contractility of the detrusor following PBOO is associated with changes in the expression of proteins that form the contractile apparatus and regulate the actomyosin ATPase activity and contraction.

Twitchin from Molluscan Catch Muscle: PRIMARY STRUCTURE AND RELATIONSHIP BETWEEN SITE-SPECIFIC PHOSPHORYLATION AND MECHANICAL FUNCTION

The phosphorylation state of the myosin thick filament-associated mini-titin, twitchin, regulates catch force maintenance in molluscan smooth muscle. The full-length cDNA for twitchin from the anterior byssus retractor muscle of the mussel Mytilus was obtained using PCR and 5'rapid amplification of cDNA ends, and its derived amino acid sequence showed a large molecule ( approximately 530 kDa) with a motif arrangement as follows: (Ig)11(IgFn2)2Ig(Fn)3Ig(Fn)2Ig(Fn)3(Ig)2(Fn)2(Ig)2 FnKinase(Ig)4. Other regions of note include a 79-residue sequence between Ig domains 6 and 7 (from the N terminus) in which more than 60% of the residues are Pro, Glu, Val, or Lys and between the 7th and 8th Ig domains, a DFRXXL motif similar to that thought to be necessary for high affinity binding of myosin light chain kinase to F-actin. Two major phosphorylation sites, i.e. D1 and D2, were located in linker regions between Ig domains 7 and 8 and Ig domains 21 and 22, respectively. Correlation of the phosphorylation state of twitchin, using antibodies specific to D1 and D2, with mechanical properties suggested that phosphorylation of both D1 and D2 is required for relaxation from the catch state.

Energy cost of contraction in rat urinary bladder smooth muscle during anoxia.

1. The aim of the present study was to investigate the effects of hypoxia on energy metabolism and contraction of rat urinary bladder smooth muscle, thereby gaining insight into the capacity of this smooth muscle to maintain contractile function when rendered hypoxic. 2. Isometric force, oxygen consumption, lactate production, heat production and unloaded shortening velocity were measured in isolated muscle strips under both aerobic and anaerobic conditions. Muscle strips were bathed in physiological saline solution with the anaerobic condition being created by replacing the oxygen bubbling the solution with nitrogen. 3. During contraction under anaerobic conditions, the rate of lactate production was increased 2.5-fold above that observed under aerobic conditions. This, however, only provided for a rate of ATP production of approximately 30% of that measured under aerobic conditions. Despite this, force maintenance was only slightly depressed, indicating that the metabolic cost of contraction was reduced in hypoxia. In support of this, the rate of heat production during contractions in anoxia was only approximately half of that under aerobic conditions, whereas, again, force was only slightly lower. Unloaded shortening velocity was significantly lower in anoxia, suggesting a slower cross-bridge turnover rate. 4. The results indicate that the economy of force maintenance is increased in bladder smooth muscle under hypoxic conditions and that this is due, at least in part, to a reduced rate of cross-bridge cycling. This may help to preserve bladder contractile function during periods of ischaemia that may be associated with bladder filling and emptying.

Obstruction-induced changes in urinary bladder smooth muscle contractility: a role for Rho kinase.

Detrusor smooth muscle (DSM) undergoes hypertrophy after partial bladder outlet obstruction (PBOO) in male rabbits, as it does in men with PBOO induced by benign prostatic hyperplasia. Despite detrusor hypertrophy, some bladders are severely dysfunctional (decompensated). In this study, the rabbit model for PBOO was used to determine the biochemical regulation of the contractile apparatus and force maintenance by the detrusor from decompensated bladders (DB). Bladders from sham-operated rabbits served as a control. On stimulation with 125 mM KCl, the DSM from sham-operated (SB) rabbits showed phasic contractions, whereas the detrusor from DB was tonic, exhibiting slow development of force, a longer duration of force maintenance, and slow relaxation. The Rho kinase (ROK) inhibitor Y-27632 enhanced the relaxation of precontracted DSM strips from DB. The enhancement of relaxation of the KCl-induced contraction of DB by Y-27632 was associated with dephosphorylation of myosin light chain (MLC20). The DSM extract from DB showed low phosphatase activity compared with that from SB. The DB also showed more Ca2+-independent MLC20 phosphorylation, which was partially inhibited by Y-27632. RT-PCR and Western blotting revealed similar expression levels of MLC kinase and ROK-alpha in SB and DB, but ROK-beta was overexpressed in DB. These results suggest that the ROK-mediated pathway is partly responsible for the high degree of force maintenance and slow relaxation in the detrusor from DB.

Transduction of biologically active motifs of the small heat shock-related protein HSP20 leads to relaxation of vascular smooth muscle.

Activation of cyclic nucleotide-dependent signaling pathways leads to phosphorylation of the small heat shock-related protein, HSP20, on serine 16, and relaxation of vascular smooth muscle. In this study, we used an enhanced protein transduction domain (PTD) sequence to deliver HSP20 phosphopeptide analogs into porcine coronary artery. The transduction of phosphoHSP20 analogs led to dose-dependent relaxation of coronary artery smooth muscle. Peptides containing the protein transduction domain coupled to a random orientation of the same amino acids did not. Direct fluorescence microscopy of arterial rings incubated with fluorescein isothiocyanate (FITC)-PTD or FITC-PTD-HSP20 peptides showed a diffuse peptide uptake. Mass spectrometric immunoassays (MSIAs) of smooth muscle homogenates were used to determine whether the phosphopeptide analogs affected the phosphorylation of endogenous HSP20. Treatment with the phosphodiesterase inhibitor papaverine led to a mass shift of 80 Da. However, there was no mass shift of HSP20 in muscles treated with phosphoHSP20 analogs. This suggests that the PTD-phosphoHSP20 peptide alone is sufficient to inhibit force maintenance and likely has a direct effect on the target of phosphorylated HSP20. These results suggest that transduction of phosphopeptide analogs of HSP20 directly alters physiological responses of intact muscles. The data also support a direct role for phosphorylated HSP20 in mediating vasorelaxation.

DISRUPTION OF TONIC DISCHARGE IS A SOURCE OF MOTOR UNIT DISCHARGE VARIABILITY IN OLDER ADULTS

During sustained muscular contractions, motor units discharge about a mean rate with variability (c.f. Galganski et al., 1993; Laidlaw et al. 2000; Patten & Kamen, 2000; Vaillancourt et al., 2002). The nature of the variability may be linked to age-related changes in motor unit morphology and impaired control of muscular force. Typically, only stable segments of motor unit discharge time series are used in variability computations and this selection may result in the loss of information. PURPOSE The purpose was to compare motor unit discharge variability among young and older adults with specific interest in the momentary disruption of tonic discharge. METHODS Young (n = 12, mean 18.8 years) and older (n = 12, mean 72.6 years) adults performed each of two different isometric force-matching tasks involving the first dorsal interosseous muscle (FDI). Motor unit discharge behavior was recorded using a quadrifilar needle electrode. A linear, 20% maximal voluntary contraction (MVC) force-matching task was used to investigate the fluctuations that occur during the maintenance of constant force. A complex sinusoidal force-matching task (20% +/− 3.65%MVC) was used to investigate motor unit discharge during force control requiring substantial modulation of motor unit discharge rates. RESULTS Older adults were less proficient in both force-matching conditions (F= 8.58, p= 0.008) and there was a trend towards greater motor unit discharge variability in this group (F= 2.36, p= 0.118). The most notable difference between the two groups was a greater positive skewness of motor unit inter-spike interval (ISI) distributions in the older adults (F= 11.63, p= 0.003). In both conditions, these skewness results were driven by atypically long ISIs. CONCLUSION Momentary disruptions in tonic motor unit discharge occur more often in older adults. These disruptions could occur due to either the inherent discharge variability associated with activity around the recruitment threshold or some other control mechanism. One of these mechanisms may account for part of the impaired force control.

The sodium pump keeps us going.

This invited lecture reviews recent evidence that, in skeletal muscle, excitability and contractility depend on the transmembrane distribution of Na(+) and K(+) and the membrane potential, which in turn are determined by the operation of the Na(+)-K(+) pump. Action potentials are elicited by passive fluxes of Na(+) and K(+). Because of their size and sudden onset, these transport events constitute the major challenge for the Na(+)-K(+) pumps. When the Na(+)-K(+) pumps cannot readily restore the Na(+)-K(+) gradients, working muscle cells often undergo net loss of K(+) and gain of Na(+). This leads to loss of excitability and force, in particular, in muscles where excitation-induced passive Na(+)-K(+) fluxes are large. Thus, excitability depends on the leak/pump ratio for Na(+) and K(+). When this ratio is increased by inhibition or downregulation of the Na(+)-K(+) pumps, the force decline seen during continued stimulation is accelerated. This effect is highly significant already within the first seconds of electrical stimulation. Fortunately, electrical stimulation also increases Na(+)-K(+) pumping rate within seconds. Thus, maximum increase (20-fold above the resting level) may be reached in 10 seconds, with utilization of all available Na(+)-K(+) pumps. In muscles, where excitability was inhibited by exposure to high [K(+)](o) (10-12.5 mM), activation of the Na(+)-K(+) pumps by hormones or electrical stimulation restored excitability and contractile force. In working muscles, the Na(+)-K(+) pumps, because of rapid activation of their large transport capacity, play a dynamic regulatory role in the second-to-second ongoing restoration and maintenance of excitability and force. The Na(+)-K(+) pumps become a limiting factor for contractile endurance, in particular, if their capacity is reduced by inactivity or disease.

The role of RhoA and Rho-associated kinase in vascular smooth muscle contraction.

A variety of contractile agonists trigger activation of the small GTPase RhoA. An important target of activated RhoA in smooth muscle is Rho-associated kinase (ROK), one of the downstream targets that is the myosin binding subunit (MYPT1) of myosin light chain phosphatase (MLCP). Phosphorylation of MYPT1 at T695 by activated ROK results in a decrease in phosphatase activity of MLCP and an increase in myosin light chain (LC(20)) phosphorylation catalyzed by Ca(2)(+)/calmodulin-dependent myosin light chain kinase and/or a distinct Ca(2)(+)-independent kinase. LC(20) phosphorylation in turn triggers cross-bridge cycling and force development. ROK also phosphorylates the cytosolic protein CPI-17 (at T38), which thereby becomes a potent inhibitor of MLCP. The RhoA/ROK pathway has been implicated in the tonic phase of force maintenance in response to various agonists, with no evident role in the phasic response, suggesting this pathway as a potential target for antihypertensive therapy. Indeed, ROK inhibitors restore normal blood pressure in several rat hypertensive models.

Effects of hydrogen peroxide on contraction of skinned aorta from guinea pigs.

We examined the effects of hydrogen peroxide (H2O2) on the contractile force of cell membrane permeabilized descending thoracic aorta from guinea pigs. H2O2 enhanced generated force in the Ca2+-induced contraction at any given concentration of Ca2+, and maintained force level in the 30 mM Mg2+-induced Ca2+-independent force maintenance. H2O2 seems to directly enhance the cross-bridge interaction of aortic smooth muscles.