Myosin light chain phosphorylation and evidence for latchbridge formation in norepinephrine stimulated canine veins

The role of 20000 dalton myosin light chain phosphorylation in mediating venous smooth muscle contraction was studied in isolated preparations of canine jugular and femoral vein. One min 10(-5) M norepinephrine-induced contraction was accompanied by significant increases in phosphorylation (jugular - 21 to 46%; femoral - 19 to 54%) which were reversed within 10 min after agonist washout. During 40 min stimulation, phosphorylation and isometric force redevelopment rates declined to near basal levels while force was maintained. These findings implicate light chain phosphorylation as a prerequisite for initial tension development by crossbridge cycling in venous smooth muscle. However, long term tension can be maintained through a process similar to the latchbridge state in tracheal and arterial smooth muscle.     

Effects of ATP reduction on the pattern of force development and myosin light chain phosphorylation in intact arterial smooth muscle

The effect of reduction of ATP content on phosphorylation of the 20 kDa light chain of myosin (MLC) and force development in intact carotid arterial smooth muscle was investigated. With reduction of ATP to 23% of control by treatment with 2-deoxyglucose there was reduction in basal, in peak and 30 min MLC phosphorylation during contraction (P less than 0.001). The rate of force development was reduced, but maximal force was the same as control. By treatment with 50 microM iodoacetate, the resting ATP content was unchanged but fell to 22% after 30 min contraction. Basal MLC phosphorylation was the same as control, but peak (P less than 0.001) and 30 min phosphorylation were lower (P less than 0.005), even though the rate and magnitude of force development were greater. The results indicate that neither rate nor magnitude of force development correlate with MLC phosphorylation. Basal and initial MLC phosphorylation may play a cooperative role in contractile function.     

Growth stimulation by serum in Entamoeba histolytica is associated with protein tyrosine dephosphorylation

Very little protein tyrosine phosphorylation was observed in growing (exponential-phase) Entamoeba histolytica cells by immunoblotting and quantitative immunofluorescence. After 1 h of serum deprivation, two proteins (42 and 38 kDa in SDS-PAGE) were tyrosine phosphorylated and two more proteins (96 and 63 kDa) also showed tyrosine phosphorylation when examined after 4 h of serum deprivation. Intense enhancements of anti-phosphotyrosine immunofluorescence levels were observed during this period of serum withdrawal. Membrane-associated tyrosine kinase activity reached a peak (3.5-fold increase) 1 h after serum deprivation and decreased thereafter reaching a basal level by 2 h of serum deprivation. Interestingly, tyrosine kinase activities remained unaffected by serum stimulation (2-60 min) of serum-deprived cells. Also, during this period of serum stimulation tyrosine phosphorylated proteins of serum-deprived cells were dephosphorylated. Tyrosine phosphatase activities were suppressed during serum deprivation and on serum addition to serum-deprived cells tyrosine phosphatase activities increased significantly. Our data attest that protein tyrosine phosphorylation was associated with growth inhibition of E. histolytica and serum stimulation of E. histolytica produced tyrosine phosphatase activation and protein tyrosine dephosphorylation.     

Basal myosin light chain phosphorylation is a determinant of Ca2+ sensitivity of force and activation dependence of the kinetics of myocardial force development

It is generally recognized that ventricular myosin regulatory light chains (RLC) are approximately 40% phosphorylated under basal conditions, and there is little change in RLC phosphorylation with agonist stimulation of myocardium or altered stimulation frequency. To establish the functional consequences of basal RLC phosphorylation in the heart, we measured mechanical properties of rat skinned trabeculae in which approximately 7% or approximately 58% of total RLC was phosphorylated. The protocol for achieving approximately 7% phosphorylation of RLC involved isolating trabeculae in the presence of 2,3-butanedione monoxime (BDM) to dephosphorylate RLC from its baseline level. Subsequent phosphorylation to approximately 58% of total was achieved by incubating BDM-treated trabeculae in solution containing smooth muscle myosin light chain kinase, calmodulin, and Ca2+ (i.e., MLCK treatment). After MLCK treatment, Ca2+ sensitivity of force increased by 0.06 pCa units and maximum force increased by 5%. The rate constant of force development (ktr) increased as a function of Ca2+ concentration in the range between pCa 5.8 and pCa 4.5. When expressed versus pCa, the activation dependence of ktr appeared to be unaffected by MLCK treatment; however, when activation was expressed in terms of isometric force-generating capability (as a fraction of maximum), MLCK treatment slowed ktr at submaximal activations. These results suggest that basal phosphorylation of RLC plays a role in setting the kinetics of force development and Ca2+ sensitivity of force in cardiac muscle. Our results also argue that changes in RLC phosphorylation in the range examined here influence actin-myosin interaction kinetics differently in heart muscle than was previously reported for skeletal muscle.     

Studies on the phosphorylation of the inhibitory subunit of troponin during modification of contraction in perfused rat heart.

1. Rat hearts were perfused with 32Pi, and contractile force was increased by positive inotropic agents (agents that increase contractility). The inhibitory subunit of troponin (troponin I) was then isolated by affinity chromatography in 8M-urea, and its 32P content measured. Incorporation of phosphate into the subunit was calculated on the basis of the [gamma-32P]ATP specific radioactivity in the hearts. 2. When hearts were perfused with 30 nM-DL-isoprenaline (N-isopropylnoradrenaline), there was an increase in contractile force over 30s which was paralleled by an increase in troponin I phosphorylation. When hearts were perfused for 25s with increasing concentrations of isoprenaline from 1 NM to 0.6 muM, there was again a parallel increase in contractile force and troponin I phosphorylation. The maximum phosphorylation observed was 1.5 mol of phosphate/mol of troponin I, which was reached after 25s with 0.1 muM-isoprenaline. 3. Hearts were stimulated with a 15s pulse perfusion of 30nM-DL-isoprenaline. There was an increase in contractile force which was followed by a return to the control value within 50s. Troponin I phosphorylation increased to a plateau value which was reached within 30s, and remained constant for 60s after the isoprenaline pulse. Phosphorylase a and 3':5'-cyclic AMP concentration showed changes similar to that of the contractile force. There was no change in 3':5'-cyclic GMP concentration. 4. When hearts stimulated with a 15S pulse of isoprenaline were subsequently perfused with 0.6 muM-acetylcholine, the changes in contractile force, phosphorylase a and 3':5'-cyclic AMP were very similar to those seen with the 15s pulse of isoprenaline alone. Troponin I phosphorylation increased to a maximum 30s after the end of the isoprenaline pulse, but then rapidly decreased during the subsequent 30s. This decrease was preceded by a 60% increase in the concentration of 3':5'-cyclic GMP. 5. Hearts were perfused with 0.2 muM-glucagon for periods up to 60s. Contractile force showed little change for the first 30s, but then increased rapidly. This was paralleled by changes in 3':5'-cyclic AMP concentration. Troponin I phosphorylation increased slowly, but the increase in contractile force had reached a maximum before significant phosphorylation had occurred. 6. It is concluded that under certain conditions, e.g. immediately after beta-adrenergic stimulation, there is a good correlation between contractile force and troponin I phosphorylation. However, under other conditions, e.g. when contractile force is decreasing after removal of beta-adrenergic stimulation or in the presence of glucagon, contractile force and troponin I phosphorylation are not well correlated. These results suggest that mechanisms for modifying cardiac contractility, other than troponin I phosphorylation, must be present in rat heart.     

Insulin-induced dephosphorylation of hormone-sensitive lipase. Correlation with lipolysis and cAMP-dependent protein kinase activity

The effect of insulin on the state of phosphorylation of hormone-sensitive lipase, cellular cAMP-dependent protein kinase activity and lipolysis was investigated in isolated adipocytes. Increased phosphorylation of hormone-sensitive lipase in response to isoproterenol stimulation was closely paralleled by increased lipolysis. Maximal phosphorylation and lipolysis was obtained when the cAMP-dependent protein kinase activity ratio was greater than or equal to 0.1, and this corresponded to a 50% increase in the state of phosphorylation of hormone-sensitive lipase. Insulin (1 nM) reduced cAMP-dependent protein kinase activity and also reduced lipolysis with both cAMP-dependent and cAMP-independent antilipolytic effects up to an activity ratio of approximately 0.4, above which the antilipolytic effect was lost. Insulin caused a decrease in the state of phosphorylation of hormone-sensitive lipase at all levels of cAMP-dependent protein kinase activity. Under basal conditions, with cAMP-dependent protein kinase activity at a minimum, this reflected a dephosphorylation of the basal phosphorylation site of hormone-sensitive lipase in a manner not mediated by cAMP. When the cAMP-dependent protein kinase was stimulated to phosphorylate the regulatory phosphorylation site of hormone-sensitive lipase, the insulin-induced dephosphorylation occurred both at the basal and regulatory sites. At low levels of cAMP-dependent protein kinase activity ratios (0.05-0.1), dephosphorylation of the regulatory site correlated with reduced cAMP-dependent protein kinase activity, but not at higher activity ratios (greater than 0.1). Stimulation of cells with isoproterenol produced a transient (1-5 min) peak of cAMP-dependent protein kinase activity and of phosphorylation of hormone-sensitive lipase. The state of phosphorylation also showed a transient peak when the protein kinase was maximally and constantly activated. In the presence of raised levels of cellular cAMP, insulin (1 nM) caused a rapid (t1/2 approximately 1 min) dephosphorylation of hormone-sensitive lipase. In unstimulated cells the reduction in phosphorylation caused by insulin was distinctly slower (t1/2 approximately 5 min). These findings are interpreted to suggest that insulin affects the state of phosphorylation of hormone-sensitive lipase and lipolysis through a cAMP-dependent pathway, involving reduction of cAMP, and through a cAMP-independent pathway, involving activation of a protein phosphatase activity that dephosphorylates both the regulatory and basal phosphorylation sites of hormone-sensitive lipase.     

Imaging epidermal growth factor receptor phosphorylation in human colorectal cancer cells and human tissues

In tumor cells, high phosphorylation levels of receptor tyrosine kinases may occur in the absence of exogenous ligands due to autocrine signaling or enhanced tyrosine kinase activity. Here we show that the phosphorylation state of the endogenous epidermal growth factor receptor (EGFR) can be quantitatively imaged in tumor cells and tissues by detecting fluorescence resonance energy transfer between fluorophores conjugated to antibodies against the receptor and phosphotyrosine, respectively. Five different human colorectal cell lines were analyzed for activity and expression of EGFR. All cell lines exhibited basal EGFR phosphorylation under serum starvation conditions. Phosphorylation levels increased after stimulation with EGF or pervanadate, dependent on the level of basal EGFR phosphorylation in the respective cell lines. This basal activity correlated inversely with receptor expression. Using the acceptor photobleaching fluorescence resonance energy transfer imaging approach, a significantly higher phosphorylation state of EGFR was also found in resected human colorectal tumor samples as compared with adjacent healthy tissue. Imaging of EGFR phosphorylation may thus serve as a valuable tool to investigate the role of receptor tyrosine kinase activity in malignant cell growth.     

Isoproterenol attenuates myosin phosphorylation and contraction of tracheal muscle

The effects of isoproterenol on isometric force, unloaded shortening velocity, and myosin phosphorylation were examined in thin muscle bundles (0.1-0.2 mm diam) dissected from lamb tracheal smooth muscle. Methacholine (10(-6) M) induced rapid increases in isometric force and in phosphorylation of the 20,000-Da myosin light chain. Myosin phosphorylation remained elevated during steady-state maintenance of isometric force. The shortening velocity peaked at 15 s after stimulation with methacholine and then declined to approximately 45% of the maximal value by 3 min. Isoproterenol pretreatment inhibited methacholine-stimulated myosin light chain phosphorylation, shortening velocity, and force during the early stages of force generation. However, the inhibitory effect of isoproterenol on force and myosin phosphorylation is proportionally greater than that on shortening velocity. Isoproterenol pretreatment also caused a rightward non-parallel shift in the methacholine dose-response curves for both isometric tension and myosin light chain phosphorylation. These data demonstrate that isoproterenol attenuates the contractile properties of airway smooth muscles by affecting the rate and extent of myosin light chain phosphorylation, perhaps through a mechanism that involves the synergistic interaction of myosin light chain kinase phosphorylation and Ca2+ metabolism.     

Phosphorylation of microtubule-associated protein-2 in GH3 cells. Regulation by cAMP and by calcium.

The rat pituitary cell line GH3 contains a high molecular weight microtubule-associated protein with properties characteristic of microtubule-associated protein-2 (MAP-2). The 280-kDa protein is selectively immunoprecipitated by antibodies to authentic bovine brain MAP-2 and is phosphorylated at appropriate sites by cAMP-dependent protein kinase (cAMP kinase) and multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Although MAP-2 is a minor cellular constituent, it can be immunoprecipitated from [32P]Pi-labeled GH3 cells and shown to contain a high level of basal phosphorylation. Vasoactive intestinal peptide, forskolin, 3-isobutyl-1-methylxanthene, or cholera toxin, treatments which increase cellular cAMP levels, or dibutyryl cAMP stimulate phosphorylation of specific sites on MAP-2 without significantly increasing its high state of basal phosphorylation. Phosphopeptide mapping reveals that the sites phosphorylated by cAMP kinase in vitro are the same sites whose phosphorylation in situ increases following stimulation of GH3 with agents that activate cAMP kinase. Increasing intracellular Ca2+ levels in GH3 cells also stimulates phosphorylation of MAP-2 but at sites distinct from those phosphorylated following treatment with cAMP inducing agonists. Phosphopeptide mapping indicates that the sites phosphorylated by CaM kinase in vitro are the same sites whose phosphorylation in situ increases following Ca2(+)-mediated stimulation. We conclude that activation of cAMP- and Ca2(+)-based signaling pathways leads to phosphorylation of MAP-2 in GH3 cells and that cAMP kinase and CaM kinase mediate phosphorylation by these pathways, respectively.     

Effect of oestradiol and tamoxifen on the testosterone response in male rats to a single injection of hCG

A single s.c. injection of hCG (100 i.u.) produced a biphasic serum testosterone response in adult male rats, peaks being noted at 2 h (24 ng/ml) and 3 days (16 ng/ml). The levels fell to control during the intervening interval (8 ng/ml), although there were elevated levels of serum hCG. Maintenance of high oestradiol levels by a s.c. injection of 50 micrograms oestradiol benzoate given on Day 2 after the initial hCG injection failed to prolong the refractory period and the secondary peak of testosterone (16 ng/ml) occurred on Day 3. Administration of the antioestrogen, tamoxifen (2 mg or 3 micrograms), 24 h before or simultaneously with hCG did not prevent testicular refractoriness in vivo because serum testosterone levels still declined after 2 h to reach a nadir at 2 days. The basal in-vitro testosterone production by decapsulated testes from animals injected with hCG was enhanced at 2 h. Stimulation by hCG increased the amount of testosterone produced (X 1.5 that in controls). By 12 h basal production decreased and there was no further increment in testosterone in the presence of hCG. This refractoriness to further hCG stimulation prevailed until Day 3, but the total production of testosterone fell so that at 24 h and 2 days testes were producing basal amounts of testosterone. Testes recovered from refractoriness at 4 and 5 days, when basal and stimulated testosterone production were greater than in controls. Injection of 50 micrograms oestradiol benzoate at 2 days did not prolong the in-vitro refractory period and 2 mg or 3 micrograms tamoxifen had no effect on the in-vitro steroidogenic activity, since testes were still refractory to further hCG stimulation from 12 h to 3 days. The results of the present study do not support the hypothesis that oestradiol is involved in the hCG-induced refractoriness of the Leydig cell. The nadir between the peaks of serum testosterone in vivo corresponds to the period during which the testis is refractory to in-vitro stimulation by hCG.     

Cytoplasmic free calcium, myosin light chain phosphorylation, and force in phasic and tonic smooth muscle

The time course of [Ca2+]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. [Ca2+]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 [Ca2+]i. On changing [K+]o from 109 to 20 mM, tension and [Ca2+]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 [Ca2+]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.     

Phosphorylation and internalization of gp130 occur after IL-6 activation of Jak2 kinase in hepatocytes.

Recent evidence has shown that members of the Jak kinase family are activated after IL-6 binds to its receptor complex, leading to a tyrosine phosphorylation of gp130, the IL-6 signal-transducing subunit. The different members of the IL-6 cytokine subfamily induce distinct patterns of Jak-Tyk phosphorylation in different cell types. Using monospecific antibodies to gp130, Jak2 kinase, and phosphotyrosine, we investigated the kinetics of IL-6 stimulation of members of this pathway in primary hepatocytes. Our findings show that Jak 2 is maximally activated within 2 min of exposure to IL-6, followed by gp130 phosphorylation that reaches its peak in another 2 min then declines to basal level by 60 min. In vitro phosphorylation experiments show that activated Jak 2 is able to phosphorylate both native gp130 and a fusion peptide containing its cytoplasmic domain, demonstrating gp130 is a direct substrate of Jak 2 kinase. Experiments designed to explore the cell surface expression of gp130 show that > or = 2 h are required to get a second round of phosphorylation after the addition of more cytokines. This finding suggests that activated gp130 is internalized from the cell surface after IL-6 stimulation. Additional experiments using protein synthesis inhibitors reveal that new protein synthesis is required to get a second cycle of gp130 phosphorylation indicating gp130 must be synthesized de novo and inserted into the membrane. These findings provide strong evidence that down regulation of the IL-6 signal in hepatocytes involves the internalization and cytosol degradation of gp130.     

Biochemical events associated with activation of smooth muscle contraction

Biochemical events associated with activation of smooth muscle contraction were studied in neurally stimulated bovine tracheal smooth muscle. A latency period of 500 ms preceded increases in isometric force and myosin light chain phosphorylation. However, stimulation resulted in the rapid hydrolysis of inositol phospholipids as demonstrated by increases in inositol phosphates by 500 ms. Inositol trisphosphate increased 2-fold with no significant change in inositol tetrakisphosphate. The apparent activation state of myosin light chain kinase was assessed indirectly through measurements of the fractional activation of a second calmodulin-dependent enzyme, cyclic nucleotide phosphodiesterase. The fractional activation of cyclic nucleotide phosphodiesterase increased after neural stimulation to a maximal extent by 500 ms and remained at this level for at least 4 s. The monophosphorylation of myosin light chain increased after 500 ms and reached a maximum value by 2 s. Diphosphorylation also occurred but to a much lesser extent. Fractional activation of cyclic nucleotide phosphodiesterase and myosin light chain phosphorylation both decreased after 10 min continuous stimulation, although the force response remained at a maximal level. These observations demonstrate that inositol trisphosphate formation and activation of cyclic nucleotide phosphodiesterase (and hence most likely myosin light chain kinase) by calmodulin precede myosin light chain phosphorylation and that these events are sufficiently rapid to mediate the contractile response of neurally stimulated tracheal smooth muscle.     

Role of vimentin in smooth muscle force development

Vimentin intermediate filaments undergo spatial reorganization in cultured smooth muscle cells in response to contractile activation; however, the role of vimentin in the physiological properties of smooth muscle has not been well elucidated. Tracheal smooth muscle strips were loaded with antisense oligonucleotides (ODNs) against vimentin and then cultured for 2 days to allow for protein degradation. Treatment with vimentin antisense, but not sense, ODNs suppressed vimentin protein expression; neither vimentin antisense nor sense ODNs affected protein levels of desmin and actin. Force development in response to ACh stimulation or KCl depolarization was lower in vimentin-deficient tissues than in vimentin sense ODN- or non-ODN-treated muscle strips. Passive tension was also depressed in vimentin-depleted muscle tissues. Vimentin downregulation did not attenuate increases in myosin light chain (MLC) phosphorylation in response to contractile stimulation or basal MLC phosphorylation. In vimentin sense ODN-treated or non-ODN-treated smooth muscle strips, the desmosomal protein plakoglobin was primarily localized in the cell periphery. The membrane-associated localization of plakoglobin was reduced in vimentin-depleted muscle tissues. These studies suggest that vimentin filaments play an important role in mediating active force development and passive tension, which are not regulated by MLC phosphorylation. Vimentin downregulation impairs the structural organization of desmosomes, which may be associated with the decrease in force development. intermediate filaments; cytoskeleton; contraction; desmin     

Angiotensin II activation of focal adhesion kinase and pp60c-Src in relation to mitogen-activated protein kinases in hepatocytes

We have investigated signaling pathways leading to angiotensin II (Ang II) activation of mitogen-activated protein kinase (MAPK) in hepatocytes. MAPK activation by Ang II was abolished by the Ang II type 1 (AT1) receptor antagonist losartan, but not by the Ang II type 2 (AT2) receptor antagonist PD123319. Ang II (100 nM) induced a rapid phosphorylation of Src (peak approximately 2 min) and focal adhesion kinase (FAK, peak approximately 5 min) followed by a decrease to basal levels in 30 min. An increased association between FAK and Src in response to Ang II was detected after 1 min, which declined to basal levels after 30 min. Treatment with the Src kinase inhibitor PP-1 inhibited FAK phosphorylation. Downregulation of PKC, intracellular Ca2+ chelator BAPTA or inhibitors of PKC, Src kinase, MAPK kinase (MEK), Ca2+/calmodulin dependent protein kinase, phosphatidylinositol 3-kinase all blocked Ang II-induced MAPK phosphorylation. In contrast to other cells, there was no evidence for the role of EGF receptor transactivation in the activation of MAPK by Ang II. However, PDGF receptor phosphorylation is involved in the Ang II stimulated MAPK activation. Furthermore, Src/FAK and Ca/CaM kinase activation serve as potential links between the Ang II receptor and MAPK activation. These studies offer insight into the signaling network upstream of MAPK activation by AT1 receptor in hepatocytes.     

Ca²⁺ sensitization of cardiac myofilament proteins contributes to exercise training-enhanced myocardial function in a porcine model of chronic occlusion

Exercise training has been shown to improve cardiac dysfunction in both patients and animal models of coronary artery disease; however, the underlying cellular and molecular mechanisms have not been completely understood. We hypothesized that exercise training would improve force generation in the myocardium distal to chronic coronary artery occlusion via altered intracellular Ca(2+) concentration ([Ca(2+)](i)) cycling and/or Ca(2+) sensitization of myofilaments. Ameroid occluders were surgically placed around the proximal left circumflex coronary artery of adult female Yucatan pigs. Twenty-two weeks postoperatively, the myocardium was isolated from nonoccluded (left anterior descending artery dependent) and collateral-dependent (formerly left circumflex coronary artery dependent) regions of sedentary (pen confined) and exercise-trained (treadmill run, 5 days/wk for 14 wk) pigs. Force measurements in myocardial strips showed that the percent change in force at stimulation frequencies of 3 and 4 Hz relative to 1 Hz was significantly higher in exercise-trained pigs compared with sedentary pigs. β-Adrenergic stimulation with dobutamine significantly improved force kinetics in myocardial strips of sedentary but not exercise-trained pigs at 1 Hz. Additionally, time to peak and half-decay of intracellular Ca(2+) (340-to-380-nm fluoresence ratio) responses at 1 Hz were significantly decreased in the collateral-dependent region of exercise-trained pigs with no difference in peak [Ca(2+)](i) between groups. Furthermore, the skinned myocardium from exercise-trained pigs showed an increase in Ca(2+) sensitivity compared with sedentary pigs. Immunoblot analysis revealed that the relative levels of cardiac troponin T and β(1)-adrenergic receptors were decreased in hearts from exercise-trained pigs independent of occlusion. Also, the ratio of phosphorylated to total myosin light chain-2, basal phosphorylation levels of cardiac troponin I (Ser(23) and Ser(24)), and cardiac myosin binding protein-C (Ser(282)) were unaltered by occlusion or exercise training. Thus, our data demonstrate that exercise training-enhanced force generation in the nonoccluded and collateral-dependent myocardium was associated with improved Ca(2+) transients, increased Ca(2+) sensitization of myofilament proteins, and decreased expression levels of β(1)-adrenergic receptors and cardiac troponin T.     

Potentiation in mouse lumbrical muscle without myosin light chain phosphorylation: Is resting calcium responsible?

The increase in isometric twitch force observed in fast-twitch rodent muscles during or after activity, known universally as potentiation, is normally associated with myosin regulatory light chain (RLC) phosphorylation. Interestingly, fast muscles from mice devoid of detectable skeletal myosin light chain kinase (skMLCK) retain a reduced ability to potentiate twitch force, indicating the presence of a secondary origin for this characteristic feature of the fast muscle phenotype. The purpose of this study was to assess changes in intracellular cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) after a potentiating stimulus in mouse lumbrical muscle (37°C). Lumbricals were loaded with the Ca²⁺-sensitive fluorescent indicators fura-2 or furaptra to detect changes in resting and peak, respectively, intracellular Ca²⁺ levels caused by 2.5 s of 20-Hz stimulation. Although this protocol produced an immediate increase in twitch force of 17 ± 3% (all data are n = 10) (P < 0.01), this potentiation dissipated quickly and was absent 30 s afterward. Fura-2 fluorescence signals at rest were increased by 11.1 ± 1.3% (P < 0.01) during potentiation, indicating a significant increase in resting [Ca²⁺]_i. Interestingly, furaptra signals showed no change to either the amplitude or the duration of the intracellular Ca²⁺ transients (ICTs) that triggered potentiated twitches during this time (P < 0.50). Immunofluorescence work showed that 77% of lumbrical fibers expressed myosin heavy chain isoform IIx and/or IIb, but with low expression of skMLCK and high expression of myosin phosphatase targeting subunit 2. As a result, lumbrical muscles displayed no detectable RLC phosphorylation either at rest or after stimulation. We conclude that stimulation-induced elevations in resting [Ca²⁺]_i, in the absence of change in the ICT, are responsible for a small-magnitude, short-lived potentiation of isometric twitch force. If operative in other fast-twitch muscles, this mechanism may complement the potentiating influence of myosin RLC phosphorylation.     

Mechanical force regulation of myofibroblast differentiation in cardiac fibroblasts

The myocardium responds to chronic pressure or volume overload by activation and proliferation of cardiac fibroblasts and their differentiation into myofibroblasts. Because alpha-smooth muscle actin (SMA) expression is the classical marker for myofibroblast differentiation, we examined force-induced SMA expression and regulation by specific MAPK pathways. Rat cardiac fibroblasts were separated from myocytes and smooth muscle cells, cultured, and phenotyped by using the presence of SMA, vimentin, and ED-A fibronectin and the absence of desmin as myofibroblast markers. Static tensile forces (0.65 pN/microm2) were applied to fibroblasts via collagen-coated magnetite beads. In neonatal cardiac fibroblasts cultured for 1 day, immunostaining and Western and Northern blotting showed very low basal levels of SMA. After the application of force, there were 1.5- to 2-fold increases of SMA protein and mRNA within 4 h. Force-induced SMA expression was dependent on ERK phosphorylation and on intact actin filaments. In contrast to cells cultured for 1 day, cells grown for 3 days on rigid substrates showed prominent stress fibers and high basal levels of SMA, which were reduced by 32% within 4 h after force application. ERK was not activated by force, but p38 phosphorylation was required for force-induced inhibition of SMA expression. These results indicate that mechanical force-induced regulation of SMA content is dependent on myofibroblast differentiation and by selective activation of MAPKs.     

Effects of statin treatment and withdrawal on angiotensin II-induced phosphorylation of p38 MAPK and ERK1/2 in cultured vascular smooth muscle cells

Abrupt discontinuation of 3-hydroxy-3-methylglutaryl-coenzyme-A-reductase inhibitors (statins) is associated with increased cardiovascular risk. To investigate the molecular mechanisms determining the increased cardiovascular risk after statin withdrawal, we studied the effects of statin treatment and withdrawal on angiotensin II (AII) actions in rat aortic vascular smooth muscle cells (VSMC) in culture. In VSMC, AII stimulated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), and of p38 mitogen-activated protein kinase (p38 MAPK), with an EC50% of 0.86 and 3 nM, respectively. Maximal stimulation was observed after 5-10 min of exposure to AII. Pretreatment with 1-3 microM simvastatin for 24h inhibited AII-mediated stimulation of ERK1/2 and p38 MAPK phosphorylation; without affecting the levels on non-phosphorylated MAPK. Washout of simvastatin produced a rebound increase above control levels of AII-mediated phosphorylation of ERK1/2 and p38 MAPK. As previously reported for other agonists, the rebound increase of AII effects was observed from 1 to 3h after statin withdrawal, and was lost at later times. The basal levels of phosphorylation and the amount of non-phosphorylated kinases were unaffected by statin withdrawal. Similar effects were observed with lovastatin. Our results suggest that statins modulate AII effects in VSMC, and that transient increases in AII effects mediated via the MAPK pathway may play a role in the vascular dysfunction associated with statin withdrawal.