Polygalacturonase, PGIP and oligogalacturonides in cell-cell communication.
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As the interface between the blood and the rest of the vessel wall, the endothelium is directly affected by hemodynamic shear stress (frictional) forces that locally regulate vascular tone and are implicated in the localization of atherosclerosis. There are many diverse responses of endothelial cells to hemodynamically related mechanical stresses ranging from ion channel activation to gene regulatory events. The processes of force transmission from the blood to the cell, and force transduction within the endothelium to electrophysiologic, biochemical, and transcriptional responses are poorly understood. This article reviews the principal mechanisms currently thought to be involved and outlines the signal pathways from the endothelium to underlying smooth-muscle cells.
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In the present work, the effect of tedisamil on gap junctional conductance (gj) and conduction velocity was investigated in the failing heart of cardiomyopathic hamsters (TO-2 strain). It was found that tedisamil (10(-7) M) increased gj by 53.8+/-1% (n = 23) in cell pairs isolated from 2 months old cardiomyopathic hamsters. The effect of tedisamil was suppressed by intracellular dialysis of an inhibitor of protein kinase A and also by adenosine indicating that the drug increases gj through the activation of adenylcyclase. Tedisamil also increased the conduction velocity and cardiac refractoriness of ventricular muscle from young cardiomyopathic hamsters. At an advanced stage of the disease, however, when the beta-adrenoceptor, adenylcyclase signaling system is impaired, tedisamil was unable to increase gj. The present results indicate that the antiarrhythmic action of tedisamil is in part related to an increase in junctional conductance and conduction velocity.
BACKGROUND: Since the first morphological description of the gap junctions use electron microscopy, a considerable number of techniques has been introduced to evaluate gap junction channel functionality, many of which use dye transfer techniques, such as dye injection and fluorescent dye transfer, analyzed by flow cytometry. METHODS: To analyze dye transfer, generally one population of cells is incubated with calcein-AM (0.5 microM) for 30 min at 37 degrees C, and the other population was incubated with the lipophilic dye DiIC(18) (3) (10 microM) for 1 h at 37 degrees C; after incubation, these cells were washed five times with PBS and cocultured for different times, and then the dye transfer was analyzed by flow cytometry. RESULTS: In this short overview, we focus on some advantages and disadvantages of flow cytometry as a technique to investigate gap junction-mediated intercellular communication (GJIC). In addition, we point out some technical pitfalls that we have encountered when applying this technique to study gap junctions in immune system cells. CONCLUSIONS: Analysis of fluorescent dye transfer by flow cytometry is a useful tool to investigate GJIC. However, some points must be taken into consideration before using this methodology, which are discussed herein.
Although oxytocin (OT) was identified in the human and primate corpus luteum (CL) over a decade ago, a definitive role for this peptide has not been elucidated. Several in vitro models have been utilized to examine the most obvious role for OT in luteal function, that of its effect on progesterone (P) production. Using dispersed cells in short term incubations, cultured cells and microdialysis procedures utilizing intact tissue, variable effects of OT on P production have been obtained. We therefore hypothesized that OT may have other role(s) in this tissue. The follicle cells remaining after a successful ovulation has occurred undergo rapid luteinization and the tissue is extensively remodeled to form the CL. At this time, in most species, two types of luteal cells have been identified based on their morphology, biochemistry and size. Both cell types produce P, however only one cell type responds to the long distance modulator LH. Since both cell types appear to be needed for the overall synthesis of P in adequate quantities required for the preparation of the endometrium "information trafficking" must occur between the two cell types. Our recent studies have shown the presence of gap junctions and connexin-43, E-cadherin, an adhesion molecule and ZO-1 protein associated with tight junctions in the baboon and human CL. The effect of OT on connexin-43 in the baboon CL has been examined.
1. Diffusion-mediated changes in ion channel function within blood vessels have not been demonstrated directly in a patch-clamp study. Here, we examined the hypothesis that endothelium-derived diffusible bioactive substances would modify endothelin-1 (ET-1)-evoked membrane currents in smooth muscle cells situated within intact arterioles. 2. In pieces of arterioles dissected from the rat cerebral pial membrane, patch electrodes were placed on single smooth muscle cells identified under the microscope. Under perforated patch-clamp conditions, ET-1 evoked an oscillatory inward current at negative potentials in such cells in the presence of the gap junction disrupter 18alpha-glycyrrhetinic acid. ET-1 also elicited an oscillation superimposed on a membrane depolarization in current-clamp mode. 3. The oscillatory current exhibited an outwardly rectifying current-voltage relationship, a sensitivity to niflumic acid, a requirement for inositol 1,4,5-trisphosphate (IP(3))- and caffeine-sensitive Ca(2+) stores and for external Ca(2+) and a rank order of anion permeabilities characteristic of Ca(2+)-activated Cl(-) currents (I(Ca(Cl))). 4. This oscillatory response was inhibited by bradykinin (an effect distinct from the electrical propagation of hyperpolarization) and this effect was attenuated by the NO-synthase inhibitor N(omega)-nitro-L-arginine and by the NO scavenger oxyhaemoglobin but not by the cyclo-oxygenease inhibitor indomethacin. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) and nitroprusside closely mimicked the effect of bradykinin. 5. The present patch-clamp study has revealed diffusion-mediated cell-to-cell interaction in an intact blood vessel: bradykinin appears to cause NO to move from endothelium to smooth muscle, there to inhibit an ET-1-evoked oscillatory I(Ca(Cl)) via the NO-cGMP pathway.
Oocyte maturation is a key issue of current animal biotechnology. This study was designed to examine the morphodynamics of the cumulus-oocyte association during oocyte maturation. Porcine cumulus-oocyte complexes were recovered from slaughterhouse ovaries; matured in vitro for 0, 24, 36, and 44 h; and evaluated by scanning electron microscopy either combined or not combined with the osmium-dimethyl sulfoxide-osmium maceration (ODO) method. The cytoskeleton distribution was also observed by fluorescence staining. Prior to maturation culture (0 h), the spherical cumulus cells were tightly clustered around the oocyte, with narrow intercellular spaces. They showed active secretion at 36 h and were fully expanded at 44 h of culture. The ODO methods revealed that the cumulus cells projected numerous long and thin transzonal projections at 0 h, but these were largely disconnected at 44 h. The outer surface of the zona pellucida showed a meshwork surface regardless of time of incubation, whereas the inner surface changed from a fine fibrous surface to a spongy surface that was coated with mucin. The vitelline surface changed from a sparse distribution of short microvilli (MV) to a dense distribution of well-developed MV. Fluorescence staining showed that the cumulus cell projections consisted mainly of microfilaments, which were abundant at the germinal vesicle and metaphase-I (M-I) stages (0-24 h) but which were decreased in number at the M-II stage (36-44 h). We conclude that the cumulus-oocyte transzonal projections became disconnected between the M-I and M-II stages as a result of cumulus expansion. The cumulus-cumulus communications, however, remained intact at these stages, although the biological functions of these communications were not clear.
In a process called quorum sensing, groups of bacteria communicate with one another to coordinate their behavior and function like a multicellular organism. A diverse array of secreted chemical signal molecules and signal detection apparatuses facilitate highly productive intra- and interspecies relationships.
wingless, a segment polarity gene required in every segment for the normal development of the Drosophila embryo, encodes a cysteine-rich protein with a signal peptide. A polyclonal antiserum localizes the wingless protein in approximately the same region of the embryo as the wingless mRNA. The pattern of antigen localization changes rapidly during development. In the extended germband stage, stripes of wingless staining are present in the trunk region just anterior to the parasegment boundary; wingless-expressing cells abut engrailed-expressing cells across that boundary. wingless antigen is seen both inside and outside the cell by electron microscopy: inside the cell, in small membrane-bound vesicles and in multivesicular bodies; outside the cell, close to or on the plasma membrane and associated with material in the intercellular space. The multivesicular bodies containing the wingless protein are occasionally found in engrailed-positive cells, suggesting that the wingless protein behaves as a paracrine signal.
Mouse and monkey adrenal glands were used to study the relationships between gap junction protein expression, intercellular communication and adrenal zonation. Dye communication patterns were determined by incubating freshly excised and hemisected adrenal glands in Lucifer yellow, a gap junction permeable fluorescent dye. Immunohistochemical techniques were used to localize adrenal gap junction proteins. The combination of these two techniques permitted the correlation of gap junction proteins with dye transfer and hormone responses in specialized regions of the adrenal cortex. Lucifer yellow dye communication was most pronounced in the inner glucocorticoid/androgen-producing regions (zona fasciculata/zona reticularis), but was virtually absent in the outer mainly mineralocorticoid-producing region (zona glomerulosa). This pattern of dye communication was coincident with immunohistochemical localization of the gap junction protein, alpha(1)Cx43. The variations in communication and alpha(1)Cx43 expression within the adrenal cortex are thought to be relevant to normal physiological regulation of the adrenal gland.
PURPOSE: Lens fiber gap junctions comprise approximately equal molar amounts of connexin46 (Cx46) and connexin50 (Cx50), both of which contribute significantly to coupling in the lens cortex and nucleus. The current study was conducted to test the hypothesis that regulation of lens coupling by activation of protein kinase Cgamma (PKCgamma) affects the number of channels composed of Cx46, Cx50, or both connexins. METHODS: Whole rat lenses were treated with phorbol-12-myristate-13-acetate (TPA) to activate PKCgamma or the inactive analogue 4alpha-phorbol,12,13-didecaneote (PDD) as a control. The superficial cortical fibers were studied morphologically by quantitative freeze-fracture immunolabeling (FRIL); functionally by Lucifer yellow dye transfer assay; and chemically by measuring PKCgamma activity, connexin phosphorylation and coimmunoprecipitation. RESULTS: Treatment with TPA activated PKCgamma and uncoupled the lens cortex by approximately 60%. PDD had no effect. Activation of PKCgamma decreased the density of Cx50 channels assembled in gap junctions, increased the density of Cx50 hemichannels in the plasma membrane and induced circular voids measuring 22 to 300 nm in diameter within the remaining plaques. Coimmunoprecipitation studies indicated that the soluble PKCgamma was translocated into membrane fractions that contained Cx46, Cx50, and the lipid raft marker caveolin (Cav)-1. In the membrane environment, PKCgamma phosphorylated Cx50 at serines and threonines and Cx46 only at threonines. CONCLUSIONS: The studies provide experimental support for the hypothesis that gap junctions comprising mixtures of Cx46 and Cx50 channels provide malleable communicating pathways between the lens nucleus and the metabolically active fibers in the surface. The findings also suggest that Cx50 channel disassembly occurs in distinct lipid microdomains.
A complex histochemical, morphometric and immunohistochemical study of communication systems, i.e. histophysiological clusters of vegetative nervous terminals, the microcirculatory bed and their immediate cellular microenvironments has been conducted. It was found that the structural and functional characteristics of communication systems play a significant role in the development of morphogenesis of stromal component of breast cancer and, therefore, may be used as a criterion of individual prognosis.
Rap phosphatases are a recently discovered family of protein aspartate phosphatases that dephosphorylate the Spo0F--P intermediate of the phosphorelay, thus preventing sporulation of Bacillus subtilis. They are regulators induced by physiological processes that are antithetical to sporulation. The RapA phosphatase is induced by the ComP-ComA two-component signal transduction system responsible for initiating competence. RapA phosphatase activity was found to be controlled by a small protein, PhrA, encoded on the same transcript as RapA. PhrA resembles secreted proteins and the evidence suggests that it is cleaved by signal peptidase I and a 19-residue C-terminal domain is secreted from the cell. The sporulation deficiency caused by the uncontrolled RapA activity of a phrA mutant can be complemented by synthetic peptides comprising the last six or more of the C-terminal residues of PhrA. Whether the peptide controls RapA activity directly or by regulating its synthesis remains to be determined. Complementation of the phrA mutant can also be obtained in mixed cultures with a wild-type strain, suggesting the peptide may serve as a means of communication between cells. Importation of the secreted peptide required the oligopeptide transport system. The sporulation deficiency of oligopeptide transport mutants can be suppressed by mutating the rapA and rapB genes or by introduction of a spo0F mutation Y13S that renders the protein insensitive to Rap phosphatases. The data indicate that the sporulation deficiency of oligopeptide transport mutants is due to their inability to import the peptides controlling Rap phosphatases.
Self-incompatibility, a mechanism that prevents self-fertilization in plants, is based on the ability of the pistil to discern the presence of self-pollen and on the female tissue's capacity to inhibit the growth or germination of self-related, but not of genetically unrelated, pollen. As a self-recognition system, self-incompatibility responds to specific cellular products and signals and thus offers a unique system in which to study the components of cellular communication in plants. The cytological manifestations of self-incompatibility have been well studied, and, with the cloning of cDNAs for several proteins associated with this recognition process, a detailed molecular view of self-incompatibility is emerging.
The contractility of corporal smooth muscle plays a critical role in the entire erectile process in man. Moreover, in the absence of severe vascular disease, or congenital or other structural abnormalities/malformations, relaxation of the corporal smooth muscle is both necessary and sufficient to elicit a sustained erection. As such, understanding the initiation, maintenance and modulation of corporal smooth muscle tone is an absolute prerequisite to the improved understanding, diagnosis and treatment of erectile dysfunction. Despite this fact, identification of both the precise mechanistic basis by which endogenous and exogenous vasomodulators exert their effects on individual corporal smooth muscle cells, and moreover, the process by which these signals are spread among the diverse array of parenchymal cells in the paired corpora, remains somewhat of a physiological enigma. Therefore, the goal of this report is two-fold: first, to review current knowledge of the regulation of corporal smooth muscle tone at the cellular and molecular level; and second, to outline a cogent explanation for the rapid and syncytial integration of the effects of diverse stimuli among corporal smooth muscle cells in the human penis.
The tumor-promoting ability of clonazepam (CZP), a widely used benzodiazepine anticonvulsant, was investigated in an in vivo mouse liver tumor promotion assay and an in vitro mouse hepatocyte intercellular communication assay. The development of preneoplastic hepatocellular foci of cellular alteration and hepatocellular neoplasms was studied in male B6C3F1 mice initiated, at 5 weeks of age, with a single i.p. injection of N-nitrosodiethylamine (NDEA; 90 mg/kg body weight) in tricaprylin, followed by administration of either phenobarbital (PB; 0.05%) or CZP (0.068% or 0.136%) in diet beginning 2 weeks after carcinogen injection and continuing to 60 weeks of age. Several mice from each group were killed after 9, 21, 33 or 53 weeks on test diet, and portions of liver and other organs were fixed in formalin and examined histologically. Unlike PB, CZP did not promote the development of preneoplastic hepatocellular foci or neoplasms (adenomas and carcinomas) in NDEA-initiated mice. Following limited (2 weeks) dietary exposure at 0.15%, CZP was a potent inducer of hepatic P450IIB1-mediated alkoxyresorufin O-dealkylase activities. In contrast, the degree of induction in hepatic tissue from mice fed 0.136% CZP for 53 weeks was markedly lower than that in mice fed 0.05% PB for 53 weeks. In the in vitro assay, diazepam, a strong tumor promoter in mouse liver, significantly inhibited mouse hepatocyte gap junctional intercellular communication, while CZP had no significant effect on this parameter. Thus, CZP, a drug structurally related to diazepam, is inactive as a liver tumor promoter in mice.