Contractility of isolated single submucosal gland from trachea

We isolated single submucosal glands from canine and feline trachea. Examination by light and electron microscope showed that these isolated glands consist mainly of glandular tissue, and no smooth muscle. Cell components in the glandular tissue were ultrastructurally normal, and myoepithelial cells surrounded acini and secretory tubules. In response to methacholine, the mucus was squeezed from the tip of the collecting ducts in coincidence with the contraction of the glands. The contractile properties of isolated single glands were examined with a force transducer. Cholinergic agents (methacholine and acetylcholine) as well as 40-150 mM K+ showed a dose-response relationship and induced tension up to 12 mg. The length-tension relationship was also observed. The removal of Ca2+ from the medium eliminated contractile response. Caffeine induced approximately 30% of the response to methacholine, and phenylephrine, a tension less than 30% of that with methacholine. These findings suggest that squeezing of mucus due to the contraction of myoepithelial cells has an important effect on secretory response of airway submucosal glands.     

Wave-like spontaneous contractions of isolated cardiomyocytes

Spontaneous wave-like contractions in single isolated rat cardiomyocytes are investigated by means of microcinematography and frame-by-frame analysis. Some basic wave patterns are described, as well as the effects of both solution components and preceding electrical stimulation. The typical wave velocity is found to be about 160 micron/s. Possible interrelation between the wave frequency and propagation velocity is discussed.     

Nonisotonic spontaneous contraction waves in isolated single cardiomyocytes

One-dimensional contraction bands propagating spontaneously in a cardiomyocyte are studied theoretically for three experimental situations: isotonic (zero load) regime, isometric regime, and a strong adhesive contact between the cell and a supporting surface. Possible role of such a contact in the development of the cell damage is discussed.     

Methodical approaches to obtaining of isolated cardiomyocytes

Isolated cardiac cells represent a unique model system for the study of many aspects of myocardial function. In this paper, several procedures for isolation ofmyocytes from adult animals and human hearts are described and discussed. They are based on successive treatments with Ca(2+)-free medium, disaggregating enzymes and/or mechanical agiatation.     

Myeloperoxidase impairs the contractile function in isolated human cardiomyocytes

We set out to characterize the mechanical effects of myeloperoxidase (MPO) in isolated left-ventricular human cardiomyocytes. Oxidative myofilament protein modifications (sulfhydryl (SH)-group oxidation and carbonylation) induced by the peroxidase and chlorinating activities of MPO were additionally identified. The specificity of the MPO-evoked functional alterations was tested with an MPO inhibitor (MPO-I) and the antioxidant amino acid Met. The combined application of MPO and its substrate, hydrogen peroxide (H₂O₂), largely reduced the active force (F_active), increased the passive force (F_passive), and decreased the Ca²⁺ sensitivity of force production (pCa₅₀) in permeabilized cardiomyocytes. H₂O₂ alone had significantly smaller effects on F_active and F_passive and did not alter pCa₅₀. The MPO-I blocked both the peroxidase and the chlorinating activities, whereas Met selectively inhibited the chlorinating activity of MPO. All of the MPO-induced functional effects could be prevented by the MPO-I and Met. Both H₂O₂ alone and MPO + H₂O₂ reduced the SH content of actin and increased the carbonylation of actin and myosin-binding protein C to the same extent. Neither the SH oxidation nor the carbonylation of the giant sarcomeric protein titin was affected by these treatments. MPO activation induces a cardiomyocyte dysfunction by affecting Ca²⁺-regulated active and Ca²⁺-independent passive force production and myofilament Ca²⁺ sensitivity, independent of protein SH oxidation and carbonylation. The MPO-induced deleterious functional alterations can be prevented by the MPO-I and Met. Inhibition of MPO may be a promising therapeutic target to limit myocardial contractile dysfunction during inflammation.     

Stretch-activated currents in cardiomyocytes isolated from rabbit pulmonary veins

Evidence is growing of a relationship between atrial dilation and atrial fibrillation (AF), the most prevalent type of arrhythmia. Pulmonary veins, which are important ectopic foci for provoking AF, are of increasing interest in relation to the early development of AF. Here, using single cardiomyocytes isolated from rabbit pulmonary veins, we characterised the stretch-activated currents induced by swelling and axial mechanical stretching. Swelling induced both a stretch-activated nonselective cationic current (NSC) and a Cl(-) current. The swelling-induced Cl(-) current (I Cl,swell) was inhibited by DIDS, whereas the swelling-induced NSC (I NSC,swell) was inhibited by Gd3+. The cationic selectivity of the I NSC,swell was K+ >Cs+ >Na+ >Li+, whilst the PK/PNa, PCs/PNa, and PLi/PNa permeability ratios were 2.84, 1.86, and 0.85, respectively. Activation of the I NSC,swell was faster than that of the I Cl,swell. Given a high K+ concentration in the bath solution, the I NSC,swell showed limited amplitude (<-70 mV). Mechanical stretching induced an immediate Gd3+- and streptomycin-sensitive NSC (I NSC,stretch) that was permeable to Na+, K+, Cs+ and NMDG. Persistent stretching activated a DIDS-sensitive current (I Cl,stretch). The I NSC,stretch, but not the I NSC,swell, was completely blocked by 400 microM streptomycin; therefore, the two currents may not be associated with the same channel. In addition, the type of current induced may depend on the type of stretching. Thus, stretch-induced anionic and cationic currents are functionally present in the cardiomyocytes of the main pulmonary veins of rabbits, and they may have pathophysiological roles in the development of AF under stretched conditions.     

Intracellular diffusion restrictions in isolated cardiomyocytes from rainbow trout

Background  

Restriction of intracellular diffusion of adenine nucleotides has been studied intensively on adult rat cardiomyocytes. However, their cause and role in vivo is still uncertain. Intracellular membrane structures have been suggested to play a role. We therefore chose to study cardiomyocytes from rainbow trout (Oncorhynchus mykiss), which are thinner and have fewer intracellular membrane structures than adult rat cardiomyocytes. Previous studies suggest that trout permeabilized cardiac fibers also have diffusion restrictions. However, results from fibers may be affected by incomplete separation of the cells. This is avoided when studying permeabilized, isolated cardiomyocytes. The aim of this study was to verify the existence of diffusion restrictions in trout cardiomyocytes by comparing ADP-kinetics of mitochondrial respiration in permeabilized fibers, permeabilized cardiomyocytes and isolated mitochondria from rainbow trout heart. Experiments were performed at 10, 15 and 20°C in the absence and presence of creatine.     

Fourier transform IR study of enzymatically isolated tomato fruit cuticular membrane

Fourier transform ir study on enzymatically isolated tomato fruit cuticle has been performed. Assignments of the observed frequencies to functional groups present in the cuticular membrane yield information on the polyester cross-links and the interactions among the different components that form this plant protective barrier. © 1992 John Wiley & Sons, Inc.     

Seedling production using enzymatically isolated thallus cells and its application in Porphyra cultivation

Hydrobiologia - On the basis of previous achievements in Porphyra seedling production using enzymatically isolated thallus cells, we have investigated the influences of water content of thallus...     

Visualization of collagenase-sensitive acetylcholinesterase in isolated cardiomyocytes and in heart tissue

Summary Previous studies have indicated that the asymmetric form of acetylcholinesterase (collagen-tailed) is localized in the basal lamina of the neuromuscular junction of skeletal muscle. The present study shows localization of the asymmetric acetylcholinesterase in the heart of the rat. Antiserum to 14+18 S acetylcholinesterase of the electric eel was raised in rabbits. The purified antibody did not react with collagen type I or laminin. Collagenase reduced the immunoreactivity of the enzyme with the purified antibody. Isolated cardiomyocytes and frozen sections of the heart were stained for acetylcholinesterase with the antibody. Diffuse immunofluorescence appeared over the surface of the cardiomyocytes. In the frozen sections, the immunofluorescence was most intense at the cell boundaries. These data suggest that collagenase-sensitive acetylcholinesterase in the heart is present in the myocytes and occurs in the vicinity of the basal lamina.Abbreviations AChE acetylcholinesterase - BSA bovine serum albumin - PBS phosphate-buffered saline - DME Dulbecco's Modified Eagle Medium     

Arginine esterase from isolated dog prostate secretory granules is fully active enzymatically

We have isolated secretory granules from dog prostate homogenates and have determined whether a major portion of arginine esterase was localized in this fraction and if it was enzymatically active. Secretory granules were purified by density gradient centrifugation on sucrose, metrizamide, or Percoll. A major proportion of whole prostate homogenate arginine esterase was found in the granule fractions. Furthermore, the specific enzymatic activity in the granules was similar to the one observed in seminal plasma. No evidence could be found for the existence of significant amount of a zymogen inactive form of arginine esterase. These results suggest that arginine esterase could be active within the secretory granules in vivo and that it could hydrolyze protein substrates contained in this organelle.     

Photosynthesis in enzymatically isolated leaf cells from the CAM plant Sedum telephium L.

A technique has been developed for the enzymatic isolation of leaf cells from the Crassulacean acid-metabolism plant Sedum telephium. The cells exhibited high activity in both ¹⁴CO₂ incorporation (30–70 mol CO₂ mg^-1 chlorophyll h^-1) and O₂ evolution in the presence of bicarbonate (60–110 mol O₂ mg^-1 chlorophyll h^-1). Half-maximum saturation of ¹⁴CO₂ incorporation occurred at a bicarbonate concentration of ca. 2 mM (20 M CO₂) at pH 8.4 and 30°C. Two types of light-dependent O₂ evolution are reported: O₂ evolution in the absence of exogenously supplied bicarbonate (endogenous O₂ evolution), and bicarbonate-stimulated O₂ evolution. Oxygen evolution in the presence of approximately ambient concentrations of CO₂ appeared to be a combination of the endogenous O₂ evolution and O₂ evolution from fixation of the exogenously supplied CO₂.Abbreviations CAM Crassulacean acid metabolism - cirlo chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PEP phosphoenolpyruvate - RuDP ribulose-1,5-diphosphate     

Fate of isolated adult cardiomyocytes undergoing starvation-induced autophagic degeneration

We induced autophagy in isolated adult rat ventricular cardiomyocytes by incubating them in glucose-free medium supplemented with mannitol for up to 4 days. The up-regulation of LC3, and vacuoles containing partially degraded subcellular organelles were readily apparent in glucose-starved cells. Most dead cells in both groups showed features of necrosis, although the survival rate was significantly lower among glucose-starved cells than among the controls. In contrast, the rate of apoptosis was about the same in both groups. Two inhibitors of autophagy, 3-methyladenine (3-MA) and leupeptin, significantly reduced the viability of both control and glucose-starved cells in a dose-dependent manner and caused specific morphological alterations: 3-MA reduced the number of autophagic vacuoles, whereas leupeptin greatly increased their number and size. Conversely, rapamycin, an enhancer of autophagy, improved the survival of glucose-starved cells. The reduction in cellular ATP caused by glucose depletion were exacerbated by the inhibitors but mitigated by rapamycin, suggesting that inhibition of autophagy may accelerate energy depletion, leading to necrosis. Our findings suggest that in cardiomyocytes autophagy is a compensatory, prosurvival response to stress and that autophagic death is an unsuccessful outcome brought on by necrosis.

Addendum to: Maruyama R, Goto K, Takemura G, Ono K, Nagao K, Horie T, Tsujimoto A, Kanamori H, Miyata S, Ushikoshi H, Nagashima K, Minatoguchi S, Fujiwara T, Fujiwara H. Morphological and biochemical characterization of basal and starvation-induced autophagy in isolated adult rat cardiomyocytes. Am J Phsyiol Heart Circ Physiol 2008; 295:H1599-607; PMID: 18708438; DOI:10.1152/ajpheart.91449.2007.     

Observations on enzymatically isolated,living and fixed embryo sacs in several angiosperm species

A technique has been developed for isolating embryo sacs (ESs) by enzymatic maceration. Ovules were macerated in a mixture of pectinase, cellulase and, in some cases, snailase and pectolyase Y-23. The ovular tissues were removed and the ESs were isolated in toto. Embryo sacs were isolated from both fixed and fresh ovules of Antirrhinum majus L., Helianthus annuus L. and Nicotiana tabacum L. Fluorochromasia by fluorescein diacetate showed that the ESs isolated from fresh ovules were viable. The method has promise for various histochemical and cell-physiological studies and quite possibly also for in-vitro culture of ESs.Abbreviations ES embryo sac - FDA fluorescein diacetate - FPA formalin-propionic acid 50% alcohol (5:5:10, by vol.) - H33258 Hoechst 33258     

Anisotropic propagation of Ca2+ waves in isolated cardiomyocytes.

Digital imaging microscopy of fluor-3 fluorescence was used to study the propagation of intracellular Ca2+ waves in isolated adult rat cardiomyocytes from 17 to 37 degrees C. Ca2+ waves spread in both transverse and longitudinal direction of a myocyte. Transverse propagation was pronounced in waves starting from a focus at the edge of a myocyte and in waves following an irregular, curved path (spiral waves). For the former type of waves, propagation velocities were determined. Both transverse and longitudinal wave components propagated at constant velocity ranging from 30 to 125 micron/s. Myocytes were anisotropic with respect to wave propagation: waves propagated faster in the longitudinal than in the transverse direction. The ratio between longitudinal and transverse velocity increased from 1.30 at 17 degrees C to 1.55 at 37 degrees C. Apparent activation energies for transverse and longitudinal wave propagation were estimated to be -20 kJ/mol, suggesting that these processes are limited by diffusion of Ca2+. Direction-dependent propagation velocities are interpreted to result from the highly ordered structure of the myocytes, especially from the anisotropic arrangement of diffusion obstacles such as myofilaments and mitochondria.     

Myofibrillogenesis in the first cardiomyocytes formed from isolated quail precardiac mesoderm

De novo assembly of myofibrils was investigated in explants of precardiac mesoderm from quail embryos to address a controversy about different models of myofibrillogenesis. The sequential expression of sarcomeric components was visualized in double- and triple-stained explants before, during, and just after the first cardiomyocytes began to beat. In explants from stage 6 embryos, cultured for 10 h, ectoderm, endoderm, and the precardiac mesoderm displayed arrays of stress fibers with alternating bands of the nonmuscle isoforms of alpha-actinin and myosin IIB. With increasing time in culture, mesoderm cells contained fibrils composed of actin, nonmuscle myosin IIB, and sarcomeric alpha-actinin. Several hours later, before beating occurred, both nonmuscle and muscle myosin II localized in some of the fibrils in the cells. Concentrations of muscle myosin began as thin bundles, dispersed in the cytoplasm, often overlapping one another, and progressed to small, aligned A-band-sized aggregates. The amount of nonmuscle myosin decreased dramatically when Z-bands formed, the muscle myosin became organized into A-bands, and the cells began beating. The sequential changes in protein composition of the fibrils in the developing muscle cells supports the model of myofibrillogenesis in which assembly begins with premyofibrils and progresses through nascent myofibrils to mature myofibrils.