1,453 research outputs found

    Selectively inhibition of oxalate-stimulated Ca2+ transport by cyclopiazonic acid and thapsigargin in smooth muscle microsomes

    Get PDF
    45Ca2+ uptake and efflux studies were performed on membranes prepared from dog mesenteric artery and rat vas deferens. Oxalate-stimulated, ATP-dependent Ca2+ uptake in microsomal vesicles, a property characteristic of sarcoplasmic reticulum, was completely inhibited in a concentration-dependent manner by cyclopiazonic acid (0.1-30 (iM) and thapsigargin (10 nM - 10 |xM). Using discontinuous sucrose gradient centrifugation, rat vas deferens microsomes were separated into two fractions, one enriched in plasma membrane (F2), the other enriched in sarcoplasmic reticulum (F3). The F3 fraction had a major increase in Ca2+ uptake in the presence of oxalate, which was completely inhibited by either cyclopiazonic acid or thapsigargin. In the F2 fraction Ca2+ uptake in the presence of oxalate was lower than in F3 and was not completely inhibited by thapsigargin and cyclopiazonic acid. Instead, the F2 fraction had a thapsigargin-insensitive and cyclopiazonic acid insensitive, saponin-sensitive component of uptake, which probably represents Ca2+ uptake by plasma membrane. In the absence of oxalate, the inhibition of Ca2+ uptake by saponin and cyclopiazonic acid or thapsigargin was additive in the F2 and F3 fractions, suggesting that cyclopiazonic acid and thapsigargin selectively inhibited sarcoplasmic reticulum derived Ca2+ uptake and did not affect plasma membrane derived Ca2+ uptake. Measurement of the initial rate of Ca2+ uptake in the presence and absence of oxalate by rat vas deferens microsomes demonstrated selective inhibition of oxalate-stimulated Ca2+ uptake by cyclopiazonic acid and thapsigargin. Ca2+ efflux from rat vas deferens microsomes actively loaded with 45Ca2+ either in the presence or the absence of oxalate was not increased by cyclopiazonic acid or thapsigargin, showing that the inhibition of Ca2+ accumulation was not due to an increase in Ca2+ efflux. In both rat vas deferens and dog mesenteric artery, the maximal inhibitory effects of cyclopiazonic acid developed rapidly, whereas for maximal inhibition thapsigargin required pretreatment of microsomes prior to measurement of Ca2+ uptake. In rat vas deferens microsomes the inhibitory effects of cyclopiazonic acid could be quickly and completely reversed, whereas the effects of thapsigargin were not easily reversed. Collectively, these results suggest selectivity of cyclopiazonic acid and thapsigargin for the sarcoplasmic reticulum Ca2+ pump. Their selective inhibitory properties and differences in onset and offset of inhibition make cyclopiazonic acid and thapsigargin useful pharmacological tools in the study of the physiological and pathophysiological roles of the sarcoplasmic reticulum Ca2+ pump in regulating smooth muscle Ca2+.published_or_final_versio

    Regulation of vascular tone: cross-talk between sarcoplasmic reticulum and plasmalemma

    Get PDF
    Selected topics on the roles of sarcoplasmic reticulum (SR) in the control of vascular smooth muscle (VSM) tone are briefly reviewed with particular reference to the regulation of cytosolic concentration of free calcium ions, [Ca2+]i. Although morphological evidence and subcellular membrane studies indicate a relatively meager quantity of SR in VSM and of endoplasmic reticulum (ER) in endothelial cells (ECs) compared with skeletal muscle and cardiac muscle, contractility studies suggest that vascular tone is, to a large extent, regulated by the intracellular Ca2+ stores in smooth muscle and endothelial cells. Cytosolic Ca2+ levels control myosin light chain phosphorylation and contraction in VSM and activation of NO synthase and phospholipase A2 in ECs to regulate nitric oxide (NO) and prostaglandin I2 formation. Understanding of the importance of SR or ER in modulating the [Ca2+]i in VSM and ECs has been further advanced as a result of the new development and refinement of biophysical techniques in the measurement of cellular Ca2+ concentrations and ion currents, such as fluorescent Ca2+ indicators and patch-clamp techniques. Experimental evidence has accumulated in support of the existence of cross-talk between SR-ER and the plasma membrane (PM). Novel pharmacological tool drugs selective for the SR-ER Ca2+ pump, such as thapsigargin and cyclopiazonic acid, as well as for SR-ER Ca2+ channels, such as ryanodine (for the Ca(2+)-induced Ca2+ release channel) and inositol polyphosphates and heparin (for the inositol-1,4,5-trisphosphate activated Ca2+ channel), together with the use of blockers for selective PM Ca2+ channels have enabled better formulation and elucidation of the mechanisms of cross-talk between SR-ER and PM.(ABSTRACT TRUNCATED AT 250 WORDS)published_or_final_versio

    Health related quality of life and psychological variables among a sample of asthmatics in Ile-Ife South-Western Nigeria

    Get PDF
    Background: Assessment of health related quality of life (HRQL) has become central to assessing the selfperceived impact of physical and mental impairment on patient’s health. Studies have reported a high rate of psychological disturbances among asthmatics; however, the impact of these psychological factors on HRQL remains unexplored. Objectives: To assess the health related quality of life among a sample of asthmatics and to identify the psychological and clinical variables that affect quality of life among asthmatics. Method: A total of 81 patients attending the clinic were assessed using the Mini-Asthma Quality of Life questionnaire (Mini-AQLQ), and the Hospital Anxiety and Depression Scale (HADS). Sociodemographic and clinical variables were also obtained from the patients, the lung function was assessed using Peak Expiratory Flow Rate (PEFR). Results: Mean age of all the patients was 35.22 (SD±14.36) with a mean duration of asthma symptoms of 17.5 (SD±14.4) years. Mean peak expiratory flow was 336 l/min (SD±74.12). Anxiety was present in 44.4% of respondents, while 40% of respondents reported the presence of depressive symptoms, 48.1% of the respondents reported low scores on the asthma quality of life questionnaire. Poor quality of life was associated with the presence of psychological symptoms, female sex, and lower educational level. Conclusion: Psychosocial variables are just as important as clinical variables as determinants of health related quality of life among asthmatics.Keywords: quality of life; asthma; anxiety; depression; psychosocia

    Towards a More Robust Lower Neck Compressive Injury Tolerance - An Approach Combining Multiple Test Methodologies

    Get PDF
    Objective.The compressive tolerance of the cervical spine has traditionally been reported in terms of axial force at failure. Previous studies suggest that axial compressive force at failure is particularly sensitive to the alignment of the cervical vertebra and the end conditions of the test methodology used. The present study was designed to develop a methodology to combine the data of previous experiments into a diverse dataset utilizing multiple test methods to allow for the evaluation of the robustness of current and proposed eccentricity based injury criteria. Methods. Data was combined from two studies composed of dynamic experiments including whole cervical spine and head kinematics that utilized different test methodologies with known end conditions, spinal posture, injury outcomes and measured kinetics at the base of the neck. Loads were transformed to the center of the C7-T1 intervertebral disc and the eccentricity of the sagittal plane resultant force relative to the center of the disc was calculated. The correlation between sagittal plane resultant force and eccentricity at failure was evaluated and compared to the correlation between axial force and sagittal plane moment and axial force alone. Results. Accounting for the eccentricity of the failure loads decreased the scatter in the failure data when compared to the linear combination of axial force and sagittal plane moment and axial force alone. A correlation between axial load and sagittal plane flexion moment at failure (R2 = 0.44) was identified. The sagittal plane extension moment at failure did not have an identified correlation with the compressive failure load for the tests evaluated in this data set (R2 = 0.001). The coefficients of determination for the linear combinations of sagittal plane resultant force with anterior and posterior eccentricity are 0.56 and 0.29 respectively. These correlations are an improvement compared to the combination of axial force and sagittal plane moment. Conclusions. Results using the outlined approach indicate that the combination of lower neck sagittal plane resultant force and the anterior-posterior eccentricity at which the load is applied generally correlate with the type of cervical damage identified. These results show promise at better defining the tolerance for compressive cervical fractures in male Post Mortem Human Subjects (PMHS) than axial force alone. The current analysis requires expansion to include more tolerance data so the robustness of the approach across various applied loading vectors and cervical postures can be evaluated

    Axonal growth arrests after an increased accumulation of Schwann cells expressing senescence markers and stromal cells in acellular nerve allografts

    Get PDF
    Acellular nerve allografts (ANAs) and other nerve constructs do not reliably facilitate axonal regeneration across long defects (>3 cm). Causes for this deficiency are poorly understood. In this study, we determined what cells are present within ANAs before axonal growth arrest in nerve constructs and if these cells express markers of cellular stress and senescence. Using the Thy1-GFP rat and serial imaging, we identified the time and location of axonal growth arrest in long (6 cm) ANAs. Axonal growth halted within long ANAs by 4 weeks, while axons successfully regenerated across short (3 cm) ANAs. Cellular populations and markers of senescence were determined using immunohistochemistry, histology, and senescence-associated β-galactosidase staining. Both short and long ANAs were robustly repopulated with Schwann cells (SCs) and stromal cells by 2 weeks. Schwann cells (S100β(+)) represented the majority of cells repopulating both ANAs. Overall, both ANAs demonstrated similar cellular populations with the exception of increased stromal cells (fibronectin(+)/S100β(−)/CD68(−) cells) in long ANAs. Characterization of ANAs for markers of cellular senescence revealed that long ANAs accumulated much greater levels of senescence markers and a greater percentage of Schwann cells expressing the senescence marker p16 compared to short ANAs. To establish the impact of the long ANA environment on axonal regeneration, short ANAs (2 cm) that would normally support axonal regeneration were generated from long ANAs near the time of axonal growth arrest (“stressed” ANAs). These stressed ANAs contained mainly S100β(+)/p16(+) cells and markedly reduced axonal regeneration. In additional experiments, removal of the distal portion (4 cm) of long ANAs near the time of axonal growth arrest and replacement with long isografts (4 cm) rescued axonal regeneration across the defect. Neuronal culture derived from nerve following axonal growth arrest in long ANAs revealed no deficits in axonal extension. Overall, this evidence demonstrates that long ANAs are repopulated with increased p16(+) Schwann cells and stromal cells compared to short ANAs, suggesting a role for these cells in poor axonal regeneration across nerve constructs

    Finely tuned temporal and spatial delivery of GDNF promotes enhanced nerve regeneration in a long nerve defect model

    Get PDF
    The use of growth factors, such as glial cell line-derived neurotrophic factor (GDNF), for the treatment of peripheral nerve injury has been useful in promoting axon survival and regeneration. Unfortunately, finding a method that delivers the appropriate spatial and temporal release profile to promote functional recovery has proven difficult. Some release methods result in burst release profiles too short to remain effective over the regeneration period; however, prolonged exposure to GDNF can result in axonal entrapment at the site of release. Thus, GDNF was delivered in both a spatially and temporally controlled manner using a two-phase system comprised of an affinity-based release system and conditional lentiviral GDNF overexpression from Schwann cells (SCs). Briefly, SCs were transduced with a tetracycline-inducible (Tet-On) GDNF overexpressing lentivirus before transplantation. Three-centimeter acellular nerve allografts (ANAs) were modified by injection of a GDNF-releasing fibrin scaffold under the epineurium and then used to bridge a 3 cm sciatic nerve defect. To encourage growth past the ANA, GDNF-SCs were transplanted into the distal nerve and doxycycline was administered for 4, 6, or 8 weeks to determine the optimal duration of GDNF expression in the distal nerve. Live imaging and histomorphometric analysis determined that 6 weeks of doxycycline treatment resulted in enhanced regeneration compared to 4 or 8 weeks. This enhanced regeneration resulted in increased gastrocnemius and tibialis anterior muscle mass for animals receiving doxycycline for 6 weeks. The results of this study demonstrate that strategies providing spatial and temporal control of delivery can improve axonal regeneration and functional muscle reinnervation
    corecore