Electron probe X-ray microanalysis of post-tetanic Ca2+ and Mg2+ movements across the sarcoplasmic reticulum in situ

Ca2+ and Mg2+ movements across the sarcoplasmic reticulum (SR) of frog skeletal muscle fibers were measured in situ by electron probe microanalysis of muscles rapidly frozen following a tetanus. At 400 ms following a 1.2-s tetanus at room temperature, the force had relaxed to base-line, and 0.3 mmol of Ca2+/liter of cytoplasmic H2O had been pumped by the SR, indicating that the in situ pumping of the SR Ca-ATPase is sufficiently high to account for the removal of Ca2+ from the Ca2+-specific sites of troponin (0.18 mmol of Ca2+-specific sites/liter of cytoplasmic H2O) and for the rate of relaxation from a tetanus at room temperature. The half-time of the return of the total 1.0 mmol of Ca2+/liter of cytoplasmic H2O released during a tetanus was 1.1 s, comparable to the slow Koff rate of Ca2+ from (carp) parvalbumin (1.0 s-1) and consistent with the hypothesis that the return of this Ca2+ to the terminal cisternae is rate-limited by the Ca2+ off-rate from parvalbumin. The return of the Mg2+ taken up by the terminal cisternae during a tetanus to resting levels was significantly slower than the time course of the Ca2+ movements, suggesting that the Mg2+ permeability of the SR in situ is low and may be transiently increased during tetanic stimulation.     

The crystal structure of RhoA in complex with the DH/PH fragment of PDZRhoGEF, an activator of the Ca(2+) sensitization pathway in smooth muscle

Calcium sensitization in smooth muscle is mediated by the RhoA GTPase, activated by hitherto unspecified nucleotide exchange factors (GEFs) acting downstream of Galphaq/Galpha(12/13) trimeric G proteins. Here, we show that at least one potential GEF, the PDZRhoGEF, is present in smooth muscle, and its isolated DH/PH fragment induces calcium sensitization in the absence of agonist-mediated signaling. In vitro, the fragment shows high selectivity for the RhoA GTPase. Full-length fragment is required for the nucleotide exchange, as the isolated DH domain enhances it only marginally. We crystallized the DH/PH fragment of PDZRhoGEF in complex with nonprenylated human RhoA and determined the structure at 2.5 A resolution. The refined molecular model reveals that the mutual disposition of the DH and PH domains is significantly different from other previously described complexes involving DH/PH tandems, and that the PH domain interacts with RhoA in a unique mode. The DH domain makes several specific interactions with RhoA residues not conserved among other Rho family members, suggesting the molecular basis for the observed specificity.     

Cortical potential imaging using L-curve and GCV method to choose the regularisation parameter

Background

The electroencephalography (EEG) is an attractive and a simple technique to measure the brain activity. It is attractive due its excellent temporal resolution and simple due to its non-invasiveness and sensor design. However, the spatial resolution of EEG is reduced due to the low conducting skull. In this paper, we compute the potential distribution over the closed surface covering the brain (cortex) from the EEG scalp potential. We compare two methods – L-curve and generalised cross validation (GCV) used to obtain the regularisation parameter and also investigate the feasibility in applying such techniques to N170 component of the visually evoked potential (VEP) data.

Methods

Using the image data set of the visible human man (VHM), a finite difference method (FDM) model of the head was constructed. The EEG dataset (256-channel) used was the N170 component of the VEP. A forward transfer matrix relating the cortical potential to the scalp potential was obtained. Using Tikhonov regularisation, the potential distribution over the cortex was obtained.

Results

The cortical potential distribution for three subjects was solved using both L-curve and GCV method. A total of 18 cortical potential distributions were obtained (3 subjects with three stimuli each – fearful face, neutral face, control objects).

Conclusions

The GCV method is a more robust method compared to L-curve to find the optimal regularisation parameter. Cortical potential imaging is a reliable method to obtain the potential distribution over cortex for VEP data.

     

Insights into the molecular activation mechanism of the RhoA-specific guanine nucleotide exchange factor, PDZRhoGEF

PDZRhoGEF (PRG) belongs to a small family of RhoA-specific nucleotide exchange factors that mediates signaling through select G-protein-coupled receptors via Gα(12/13) and activates RhoA by catalyzing the exchange of GDP to GTP. PRG is a multidomain protein composed of PDZ, regulators of G-protein signaling-like (RGSL), Dbl-homology (DH), and pleckstrin-homology (PH) domains. It is autoinhibited in cytosol and is believed to undergo a conformational rearrangement and translocation to the membrane for full activation, although the molecular details of the regulation mechanism are not clear. It has been shown recently that the main autoregulatory elements of PDZRhoGEF, the autoinhibitory "activation box" and the "GEF switch," which is required for full activation, are located directly upstream of the catalytic DH domain and its RhoA binding surface, emphasizing the functional role of the RGSL-DH linker. Here, using a combination of biophysical and biochemical methods, we show that the mechanism of PRG regulation is yet more complex and may involve an additional autoinhibitory element in the form of a molten globule region within the linker between RGSL and DH domains. We propose a novel, two-tier model of autoinhibition where the activation box and the molten globule region act synergistically to impair the ability of RhoA to bind to the catalytic DH-PH tandem. The molten globule region and the activation box become less ordered in the PRG-RhoA complex and dissociate from the RhoA-binding site, which may constitute a critical step leading to PRG activation.     

The cAMP-responsive Rap1 guanine nucleotide exchange factor, Epac, induces smooth muscle relaxation by down-regulation of RhoA activity

Agonist activation of the small GTPase, RhoA, and its effector Rho kinase leads to down-regulation of smooth muscle (SM) myosin light chain phosphatase activity, an increase in myosin light chain (RLC(20)) phosphorylation and force. Cyclic nucleotides can reverse this process. We report a new mechanism of cAMP-mediated relaxation through Epac, a GTP exchange factor for the small GTPase Rap1 resulting in an increase in Rap1 activity and suppression of RhoA activity. An Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP ("007"), significantly reduced agonist-induced contractile force, RLC(20), and myosin light chain phosphatase phosphorylation in both intact and permeabilized vascular, gut, and airway SMs independently of PKA and PKG. The vasodilator PGI(2) analog, cicaprost, increased Rap1 activity and decreased RhoA activity in intact SMs. Forskolin, phosphodiesterase inhibitor isobutylmethylxanthine, and isoproterenol also significantly increased Rap1-GTP in rat aortic SM cells. The PKA inhibitor H89 was without effect on the 007-induced increase in Rap1-GTP. Lysophosphatidic acid-induced RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts, consistent with Epac signaling through Rap1B to down-regulate RhoA activity. Isoproterenol-induced increase in Rap1 activity was inhibited by silencing Epac1 in rat aortic SM cells. Evidence is presented that cooperative cAMP activation of PKA and Epac contribute to relaxation of SM. Our findings demonstrate a cAMP-mediated signaling mechanism whereby activation of Epac results in a PKA-independent, Rap1-dependent Ca(2+) desensitization of force in SM through down-regulation of RhoA activity. Cyclic AMP inhibition of RhoA is mediated through activation of both Epac and PKA.     

Expression of CPI-17 in smooth muscle during embryonic development and in neointimal lesion formation

Ca²⁺ sensitivity of smooth muscle (SM) contraction is determined by CPI-17, an inhibitor protein for myosin light chain phosphatase (MLCP). CPI-17 is highly expressed in mature SM cells, but the expression level varies under pathological conditions. Here, we determined the expression of CPI-17 in embryonic SM tissues and arterial neointimal lesions using immunohistochemistry. As seen in adult animals, the predominant expression of CPI-17 was detected at SM tissues on mouse embryonic sections, whereas MLCP was ubiquitously expressed. Compared with SM α-actin, CPI-17 expression doubled in arterial SM from embryonic day E10 to E14. Like SM α-actin and other SM marker proteins, CPI-17 was expressed in embryonic heart, and the expression was down-regulated at E17. In adult rat, CPI-17 expression level was reduced to 30% in the neointima of injured rat aorta, compared with the SM layers, whereas the expression of MLCP was unchanged in both regions. Unlike other SM proteins, CPI-17 was detected at non-SM organs in the mouse embryo, such as embryonic neurons and epithelium. Thus, CPI-17 expression is reversibly controlled in response to the phenotype transition of SM cells that restricts the signal to differentiated SM cells and particular cell types. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Photolytic release of MgADP reduces rigor force in smooth muscle

Photolytic release of MgADP (25-300 microM) from caged ADP in permeabilized tonic (rabbit femoral artery-Rfa) and phasic (rabbit bladder-Rbl) smooth muscle in high-tension rigor state, in the absence of Ca(2+), caused an exponential decline (approximately 1.5% in Rfa and approximately 6% in Rbl) of rigor force, with the rate proportional to the liberated [MgADP]. The apparent second-order rate constant of MgADP binding was estimated as approximately 1.0 x 10(6) M(-1) s(-1) for both smooth muscles. In control experiments, designed to test the specificity of MgADP, photolysis of caged ADP in the absence of Mg(2+) did not decrease rigor force in either smooth muscle, but rigor force decreased after photolytic release of Mg(2+) in the presence of ADP. The effects of photolysis of caged ADP were similar in smooth muscles containing thiophosphorylated or non-phosphorylated regulatory myosin light chains. Stretching or releasing (within range of 0.1-1.2% of initial Ca(2+)-activated force) did not affect the rate or relative amplitude of the force decrease. The effect of additions of MgADP to rigor cross-bridges could result from rotation of the lever arm of smooth muscle myosin, but this need not imply that ADP-release is a significant force-producing step of the physiological cross-bridge cycle.     

LPP,a LIM protein highly expressed in smooth muscle

An 80-kDa protein, prominently expressed in smooth muscle, was microsequenced and identified as LPP, the product of the lipoma-preferred partner gene (Petit MMR, Mols R, Schoenmakers EFPM, Mandahl N, and Van de Ven WJM. Genomics 36: 118–129, 1996). Using a specific anti-LPP antibody, we showed, in Western blots and with immunofluorescence microscopy, the selective expression of LPP in vascular and visceral smooth muscles (0.5–1 ng/µg total protein). In other mature (noncultured) tissues, including heart and skeletal muscle, the protein is present only in trace amounts and is closely correlated with the levels of the smooth muscle marker -actin. In freshly isolated guinea pig bladder smooth muscle cells, immunofluorescence images showed LPP as linear arrays of punctate, longitudinally oriented staining superimposed with vinculin staining on the plasma membrane surface. A corresponding pattern of periodic labeling at the membrane in transverse sections of bladder smooth muscle suggested an association of LPP with peripheral dense bodies. In cultured rat aortic smooth muscle cells, LPP colocalized with vinculin at focal adhesions but not with p120 catenin or -actinin. Overexpression of the protein increased EGF-stimulated migration of vascular smooth muscle cells in Transwell assays, suggesting the participation of LPP in cell motility. The Rho-kinase inhibitor Y-27632 dissociated focal adhesions and LPP staining at the cell periphery and enhanced the nuclear accumulation of LPP induced by leptomycin B, indicating that LPP has a potential for relocating to the nucleus through a shuttling mechanism that is sensitive to inhibition of Rho-kinase. LIM protein; dense plaque; Rho-kinase; nuclear transport; cell migration     

Calcium content of peripheral and central mitochondria in the guinea pig myocardium: electron probe analysis

We quantitated subcellular elemental concentrations in stimulated and resting guinea pig myocardium to determine whether species-specific properties of guinea pigs or the subcellular localization of mitochondria accounted for reports of higher mitochondrial Ca in guinea pigs than in other species. Small papillary muscles or trabeculae isolated from guinea pig ventricles were stimulated to raise cytosolic [Ca(2+)](i) by two methods: (1). tetanizing by rapid pacing preparations in which Ca(2+) uptake by the sarcoplasmic reticulum was inhibited with cyclopiazonic acid or (2). freeze trapping paced muscles near-peak systole. Electron probe X-ray microanalysis showed no significant difference between the (low, approximately 0.4 mmol/kg dry weight) mitochondrial Ca content of stimulated guinea pig hearts, compared to mitochondria of other species, such as rat and hamsters, and the Ca contents of peripheral and central mitochondria were also not significantly different.