Ute Gröschel-Stewart

Localisation of P2X5 and P2X7 receptors by immunohistochemistry in rat stratified squamous epithelia.

Abstract In order to investigate whether purinoceptors are involved in the physiological renewal and regeneration of epithelia, we used immunohistochemical techniques on fresh frozen sections of various stratified squamous epithelial tissues (cornea, tongue, soft palate, oesophagus, vagina and footpad) of the rat and specific polyclonal antibodies to unique peptide sequences of P2X1–7 receptors. Only two of the antibodies, anti-P2X5 and anti-P2X7, reacted with epithelial structures. P2X5 immunoreactivity was mainly associated with the membranes of the proliferating and differentiating cell layers (spinous and granular layer) in both keratinised and non-keratinised epithelia and growing hair follicles. In contrast, P2X7 immunoreactivity was clearly associated with the keratinisation process, the staining being most intense in the upper keratinised and the exfoliated layers. These findings suggest, for the first time, that P2X5 and P2X7 receptors play an important role in the physiological turnover of continuously regenerating cells, and further, raise the possibility that they represent novel targets for the development of pharmacological tools of potential benefit for diseases of epithelial dysfunction.

Distinction between smooth muscle, fibroblasts and endothelial cells in culture by the use of fluoresceinated antibodies against smooth muscle actin

SummaryFITC-labelled antibodies against native actin from chicken gizzard smooth muscle (Gröschel-Stewart et al., 1976) have been used to stain cultures of guinea-pig vas deferens and taenia coli, rabbit thoracic aorta, rat ventricle and chick skeletal muscle. The I-band of myofibrils of cardiac muscle cells and skeletal muscle myotubes stains intensely. In isolated smooth muscle cells, the staining is located exclusively on long, straight, non-interrupted fibrils which almost fill the cell. Smooth muscle cells which have undergone morphological “dedifferentiation” to resemble fibroblasts with both phase-contrast microscopy and electronmicroscopy still stain intensely with the actin antibody. In those muscle cultures which contain some fibroblasts or endothelial cells, the non-muscle cells are not stained with the actin antibody even when the reactions are carried out at 37° C for 1 h or after glycerination. Prefusion skeletal muscle myoblasts also do not stain with this antibody.It is concluded that the actin antibody described in this report is directed against a particular sequence of amino acids in muscle actin which is not homologous with non-muscle actin. The usefulness of this antibody in determining the origin of cells in certain pathological conditions such as atherosclerosis is discussed.

Comparison of the reaction of cultured smooth and cardiac muscle cells and fibroblasts to specific antibodies to myosin

SummaryImmunofluorescent staining with anti-smooth or anti-striated muscle myosin was carried out for 30 minutes at room temperature (18–20° C) on cultures of smooth muscle cells and fibroblasts from guinea-pig vas deferens, taenia coli and ureter, rabbit aorta and chicken gizzard and of cardiac muscle cells and fibroblasts from rat ventricle. With antismooth muscle myosin, smooth muscle cells showed an intense fluorescent staining in fine fibrils with an “interrupted” appearance running parallel to the longitudinal axis of the cell throughout the cytoplasm, and also in coarser, “non-interrupted” fibrils (termed here “attachment fibrils”) concentrated at the surface of the cell adjacent to the glass coverslip. Fibroblasts in the same cultures showed similar, but much weaker, reactions. When anti-striated myosin was added to the smooth muscle cultures, staining of neither cell type was observed. In contrast, cardiac muscle cells in cultures of rat ventricle did not react with anti-smooth muscle myosin, but gave bright fluorescent A-band staining with anti-striated myosin. Fibroblasts in the ventricle cultures were unreactive with anti-striated muscle myosin but gave the characteristic weak reaction with anti-smooth muscle myosin. Thus immunofluorescent staining with anti-smooth muscle myosin is useful for distinguishing between isolated smooth muscle cells and fibroblasts in tissue culture.

Immunochemistry of Cytoplasmic Contractile Proteins

Publisher Summary The chapter discusses the immunological aspects of cytoplasmic contractile protein. It describes the effect of the antibodies on contractile proteins in vitro and in vivo , as well as the localization of contractile elements in cells and tissues by immunohistochemical methods. The immunofluorescent technique is used to describe antibodies to smooth muscle in patients with liver disease and with malignant disease. Some of these antibodies react with microfilament bundles of cultured cells and with hepatocyte membranes. Transmembrane linkages, either directly or via a mediator, bring up additional aspects about remote control interactions of external stimuli and the internal contractile system, such as in capping and phagocytosia. Ca 2+ ions are important mediators of all these functions and impressive treatise on a unified theory of the control of actin and myosin in nonmuscle movements has gained additional weight by the recent findings that a Ca 2+ -dependent protein acts as the modulator of myosin kinases, which regulate the interaction of actin and myosin in nonmuscle cells.

P2X receptors in the rat duodenal villus

Abstract Immunohistochemical techniques were performed on freshly frozen sections of the duodenum of the rat using specific polyclonal antibodies to unique peptide sequences of P2X1–7 receptors. Of the antibodies to the seven known P2X receptor subtypes that mediate extracellular signalling by nucleotides, three reacted with discrete structures in the duodenal villus of the rat. Anti-P2X1 reacted with the capillary plexus in the intestinal villus, which did not extend to the crypt region, suggesting that nucleotides may be involved in the uptake and transport of metabolites. Anti-P2X5 immunostained the membranes of the narrow ”stem” of villus goblet cells, where the nucleus and cell organelles reside, possibly influencing synthesis and release of mucins. P2X7 receptor immunoreactivity was only seen in the membranes of enterocytes and goblet cells at the tip of the villus, where cells are exfoliated into the lumen, consistent with earlier findings that P2X7 is involved in apoptotic events. Thus, in complex structures such as the intestinal villus, purinoceptors appear to participate in several and diverse signalling functions.

Production of specific antibodies to contractile proteins and their use in immunofluorescence microscopy

SummaryThe preparation of highly purified myosin from surgical specimen of human uterine muscle is described. Antibodies were raised in rabbits against this immunogen. In immunodiffusion, they react with uterine and chicken gizzard muscle myosin, no reaction is observed between uterine myosin and the anti-chicken-gizzard- myosin. In immunofluorescence, antiuterine-myosin stains smooth muscle in the contractile and “modulated” state and non-muscle cells such as fibroblasts, platelets and endothelium of various species. Thus, these antibodies contrast anti-gizzard-myosin, which has previously been shown to be specific for contractile state muscle cells. We therefore conclude that the uterine myosin preparation consists of two immunogens, the one being associated with cell contractility and the other, termed cytoplasmic myosin, with motility and mitosis. The latter is indistinguishable from the myosin present in non-muscle cells and can be absorbed specifically with actomyosin from blood platelets.

Expression of two ATP-gated ion channels, P2X5 and P2X6, in developing chick skeletal muscle

Physiological and pharmacological studies have shown that ATP has potent effects on developing chick skeletal muscle. These effects have previously been shown to be developmentally regulated, and the responses were characteristic of activation of the P2X ligand‐gated ion‐channel family of ATP receptors. Here, using immunohistochemistry, we describe the expression patterns of two members of the P2X receptor family, P2X5 and P2X6, during development of skeletal muscle in the chick embryo. These receptors were first expressed at early stages of skeletal muscle development, and expression disappeared immediately before the stage at which fusion of myoblasts to form myotubes occurs. P2X5 was also demonstrated in nerves supplying developing skeletal muscle, in some dorsal root ganglion cells, and in dorsal and ventral spinal cord. No expression of the other five members of the P2X family were demonstrated in developing skeletal muscle. Dev Dyn 1999;216:442–449. ©1999 Wiley‐Liss, Inc.

Fitc-Labelled Antibody Staining of Tropomyosin-Containing Fibrils in Smooth, Cardiac and Skeletal-Muscle Cells, Prefusion Myoblasts, Fibroblasts, Endothelial Cells and 3t3 Cells in Culture

SummaryFITC-labelled antibodies to purified chicken gizzard smooth muscle tropomyosin were prepared and used to stain muscle and non-muscle cells in culture.Skeletal muscle myoblasts stained both diffusely throughout the cytoplasm and in fine filamentous structures. Once myotubes developed the staining was localized exclusively in the I-band region of the myofibrils. Similarly, cardiac muscle cells stained in the I-band alone.Primary and subcultured smooth muscle cells, irrespective of their state of differentiation, stained exclusively in long, straight fibrils. The staining of the fibrils was interrupted with stained regions 1–2 μm long and unstained spacings 0.5 μm.Interrupted fibrils were also observed in fibroblasts and endothelial cells, however their staining reaction was very weak (almost indistinguishable from that with pre-immune serum) and they were few in number.3T3 cells demonstrated moderate staining in interrupted fibrils. Sheaths of very fine fibrils staining with a similar intensity were also found throughout the cytoplasm. Interruptions in these fine fibrils were often aligned to give the whole cell a striated appearance. Sheaths of fibrils were not found in the other cell types studied

Histochemical and immunochemical studies on mammalian striated muscle fibres

SummaryIn 15 rabbit and 2 human distinct types of striated muscle, it was shown that all fibres classified as type I (“red”) by NADH-diaphorase and myofibrillar ATPase reaction exhibit intense fluorescence when treated with a FITC-labelled antibody against human pectoral actomyosin. Type II (“white”) fibres showing minimal fluorescence, an intermediate fibre type can be differentiated by all three methods. In the rabbit, all three fibre types are found, whereas, in the human muscles studied, only type I and intermediate type fibres were found.

Characterization of the ATPase activities of myosins isolated from the membrane and the cytoplasmic fractions of human platelets

Myosin was purified from the membrane fraction and the cytoplasm of human platelets, and the K+(EDTA)- and Ca2+-dependent ATPase activities were studied under various experimental conditions. The ATPase activity of the myosin from the membrane fraction was slightly lower than that of its cytoplasmic counterpart, regardless of the different assay conditions (pH, ionic strength, and temperature). Both myosins showed the same pH optima and a similar ionic strength dependence for the two ATPase activities measured. In addition, they exhibited the same substrate specificity using ATP, CTP, and GTP as substrates. The activation energy of the Ca2+-dependent ATPase activity was essentially the same for the two myosins, while the activation energy of the K+(EDTA)-dependent ATPase activity of the membrane myosin was higher than that of the cytoplasmic myosin. The ATPase activity of the membrane myosin was found to be more sensitive to freezing and thawing than the cytoplasmic myosin. The alkylation of the thiol groups by N-ethylmaleimide or N-iodoacetyl-N-(5-sulfo-1-naphtyl)ethylenediamine, and the trinitrophenylation of the lysyl residues by 2,4,6-trinitrobenzenesulfonate caused a significant decrease in the K+(EDTA)-dependent ATPase activity of the two myosins. However, the membrane myosin was much less affected than the cytoplasmic myosin. Actin induced inhibition of the K+ (EDTA) ATPase of both myosins, and much smaller quantities of actin were needed to inhibit the cytoplasmic myosin ATPase compared to quantities needed to inhibit the myosin ATPase from the membrane fraction. This indicates that the membrane myosin has a lower affinity toward actin. The observed variations in the ATPase activity of the myosins isolated from the membrane and the cytoplasm fractions of human platelets may reflect differences in their respective physiological functions.