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PubMed1995

Involvement of multiple receptors in the actions of extracellular ATP: the example of vascular endothelial cells.

The role of ATP and ADP as intercellular mediators is now well established. The presence of the nucleotides in extracellular fluids can result from several mechanisms: cell lysis, selective permeabilization of the plasma membrane and exocytosis of secretory vesicles, such as platelet dense bodies. Extracellular adenine nucleotides are rapidly degraded by ectonucleotidases expressed inter alia on the surface of endothelial cells. They act on cells via the family of P2 receptors which encompasses more than 5 subtypes, some of which have been cloned recently. The P2T, P2U and P2Y receptors belong to the superfamily of receptors coupled to G proteins, whereas the P2X receptor is a cation channel and the P2Z receptor a non-selective pore. ATP and ADP stimulate the endothelial production of prostacyclin (PGI2) and nitric oxide (NO), two vasodilators and inhibitors of platelet aggregation, via an increase in cytosolic Ca2+. This action of adenine nucleotides is believed to limit the extent of intravascular platelet aggregation and to help localize thrombus formation to areas of endothelial damage. The endothelial response to nucleotides is mediated by at least two distinct subtypes of P2 receptors, P2Y and P2U, both coupled to phospholipase C.

Adenosine Diphosphate↗

PubMed1998

Translocation of the precursor of alpha-amylase into Bacillus subtilis membrane vesicles.

Bacilli vigorously secrete proteins into the extracellular environment, and are therefore used in industry for the bulk production of enzymes such as proteinases and amylases. Studies on the mechanism of protein translocation in these Gram-positive bacteria have been hampered by the lack of an in vitro system. To establish such a system for Bacillus subtilis, everted membranes were isolated from a strain deficient in the alkaline and neutral protease. Translocation-competent membrane vesicles were obtained only when a broad range proteinase-inhibitor cocktail was used during membrane isolation. This method efficiently prevented proteolysis of SecY, one of the core integral membrane components of the preprotein translocase. Translocation of the urea-denatured precursor of the Bacillus licheniformis alpha-amylase, preAmyL, and B. subtilis alkaline phosphatase, prePhoB, into the B. subtilis membrane vesicles require the B. subtilis SecA protein and are driven by ATP hydrolysis and the proton-motive force. These studies establish an authentic in vitro translocation system for protein secretion in B. subtilis.

Adenosine Triphosphate↗

PubMed1975

The matrix of the optic vesicle-presumptive lens interface during induction of the lens in the chicken embryo.

The cell coats of the presumptive lens cells and the extracellular interface between the lens rudiment and optic vesicle were investigated in the chicken embryo throughout the period during which lens induction is presumed to take place. Histochemical methods showed that the cell coats contained both glycoproteins and glycosaminoglycans. Autoradiography after [3H]glucosamine injection indicated incorporation of the precursor with subsequent localization primarily at the cell surface. No obvious changes in the properties of the coat were noted with the progression of early lens morphogenesis. The extracellular matrix at the interface between ectoderm and optic vesicle also contained glycoprotein and glycosaminoglycan. There was a heavy concentration of [3H]glucosamine-containing macromolecules in the area. Electron microscopy revealed that the interface consisted of the basement membrane systems of lens and optic vesicle, fused with their external fibrillar layers. In contrast to the findings on cell coats the density of the interfacial matrix increases appreciably during the lens induction period. Evidence suggests that the cells of the two ocular epithelia are themselves the source of the matrix materials. It is proposed that the macromolecules excreted by the epithelial cells into the interface interact at different concentrations to form aggregates of various structure by a process of self-assembly. This may be reflected in the different ultrastructure of the layers of the interfacial matrix. Quantitative changes in the density of the matrix, leading to increased adhesion between lens rudiment and optic vesicle, may restrict the lateral spreading of the lens cells and so fix the basal area of the lens rudiment. This, together with continued cell replication, may produce the cell crowding, placode formation and invagination characteristic of lens morphogenesis.

Amylases↗

PubMedDate not supplied

Cryo-FE-SEM & TEM immuno-techniques reveal new details for understanding white-rot decay of lignocellulose.

High-resolution Cryo-Field Emission Scanning Electron Microscopy (HR-Cryo-FE-SEM) and immuno-cytochemistry were used to reveal novel details on the morphological events and spatial distribution of oxidoreductive enzymes during the degradation of birch wood by the white-rot fungi Phlebia radiata and mutant strain P radiata Cel 26. Cryo-observations of fractured fibres showed degradation across the cell wall by P. radiata (wild) to progress by delamination and removal of concentric orientated aggregates from the secondary S2 cell wall. Decay by P radiata Cel 26 progressed by removal of materials (lignin and hemicelluloses) between the aggregates (primarily cellulose) that remained even after advanced decay. With both decay patterns, extracellular slime materials were present uniting lumina hyphae with the attacked fibre wall. The extracellular slime material had two morphological forms: viz a fibrillar (often tripartite) and a 'gel-form', the former found in discrete bands progressing across the lumen onto the fibre wall. Using TEM immunocytochemistry, laccase, manganese peroxidase (MnP) and diarylpropane enzymes were localized in the periplasmic space of luminal hyphae, in association with the cell membrane, periplasmic vesicles and fungal cell wall. Extracellularly, the three enzymes were found associated with the slime and tripartite membranes and with the birch cell walls at all stages of attack through to middle lamella corner decay. Enzyme distribution was correlated with morphological changes in cell wall structure. The association of extracellular slime with these enzymes and sites of decay strongly suggests a major role for this matrix in fibre cell wall decomposition.

Betula↗

PubMed1985

Freeze-fracture replica study of capillary endothelium after embolization in the dog.

The underlying mechanisms that lead to brain edema following ischemic insult have been subject to much debate. In this study, experimental cerebral infarction was produced in 25 dogs by injecting 1 or 2 silicone rubber cylinders through the cervical internal carotid artery. The animals were sacrificed 24 hours after embolization. Freeze-fracture studies were conducted on the plasma membrane of the capillary endothelium from 15 control and 25 ischemic dogs. No definite findings of tight junction opening were made in the ischemic preparations. Pinocytotic vesicles were seen as concave areas on the protoplasmic face (PF) of the plasma membrane and as protrusions on the extracellular face (EF). The average pinocytotic vesicle count per square micron was increased in ischemic animals. On the luminal side, it reached 22.0 +/- 1.2/sq mu in the 50 PF samples and 29.5 +/- 1.3/sq mu in the 50 EF samples in the experimental preparations, as compared to 7.2 +/- 0.5 sq mu in the 50 PF samples and 9.0 +/- 0.6 sq mu in the 50 EF samples in normal cortex. The average area of the vesicles was also enlarged in experimental animals: 4990.7 +/- 798 sq nm in the 50 PF samples and 4762.8 +/- 878 sq nm in the 50 EF samples, as compared to 3567.7 +/- 570 sq nm in the 50 PF samples and 3404.5 +/- 573 sq nm in the 50 EF samples in normal cortex (p greater than 0.01). These results indicate that transcellular transportation by pinocytotic vesicles plays an important role in the increase of capillary permeability observed in an ischemic model.

Animals↗

PubMed1989

Lipoprotein lipase in myocytes and capillary endothelium of heart: immunocytochemical study.

Lipoprotein lipase was immunolocalized by electron microscopy in hearts of young mice; 78% of lipoprotein lipase was in myocytes, 3-6% in extracellular space, and 18% in capillary endothelium. Lipoprotein lipase in myocytes was located primarily in sarcoplasmic reticulum, Golgi sacs, and transport vesicles and also in secretory vesicles at the cell periphery. Lipoprotein lipase in extracellular space was present near the orifice of secretory vesicles of myocytes and in narrow zones spanning the space between myocytes and capillary endothelium. The lowest concentration of lipase associated with endothelial cells was at the basal plasma membrane, whereas the highest concentration was at the surface of luminal projections. Lipoprotein lipase was associated with chylomicrons at the capillary surface but not with chylomicron remnants. Fasting mice for 48 h increased, in heart, lipoprotein lipase activity by 120% and immunolocalized lipase by 270%. The greatest increase (5-fold) occurred at the surface of intraluminal endothelial projections. The findings indicate that lipoprotein lipase in heart is synthesized by myocytes, transferred across extracellular space along cell surfaces and across endothelial cells via vesicles or intracellular channels, and concentrated at the surface of luminal projections of endothelium where the enzyme hydrolyzes triacylglycerol of chylomicrons and very low-density lipoproteins.

Animals↗

PubMed1996

Vesicular transport of histamine in stimulated human basophils.

Human basophils participating in experimentally produced contact allergy display progressive secretion of electron-dense secretory granule contents and retention of cytoplasmic granule containers in the absence of entire granule extrusion, a process termed piecemeal degranulation (PMD) and postulated to be effected by vesicular transport (Dvorak HF, Dvorak AM: Clin Hematol 4:651, 1975). Proof of this hypothesis was sought using models of human basophil-stimulated secretion, partially purified human peripheral blood basophils, and a morphometric analysis of the fraction of total cellular cytoplasmic vesicles loaded with histamine, a major proinflammatory mediator present in basophil secretory granules. The subcellular localization of histamine was accomplished using a new ultrastructural enzyme-affinity-gold method based on the affinity of diamine oxidase for its substrate, histamine (Dvorak et al: J Histochem Cytochem 41:787, 1993). Two models were selected for a kinetic analysis of stimulated vesicle transport of histamine based on known biochemical and ultrastructural characteristics (MacGlashan et al: J Immunol 136:2231, 1986; Warner et al: J Leukoc Biol 45:558, 1989; Dvorak et al: Am J Pathol 141:1309, 1992; Dvorak et al: Lab Invest 64:234, 1991). These models were selected to include the rapid release reaction stimulated by the bacterial peptide, FMLP, and the slow release reaction stimulated by the phorbol diester tumor promoter, TPA. The results of this study showed that the fraction of histamine-loaded cytoplasmic vesicles (%VG/TV/micron2) in TPA-stimulated basophils significantly exceeded the fraction in unstimulated cells, a process that persisted for 45 minutes after TPA stimulation and was associated with extensive PMD and no morphologic evidence of recovery. Similarly, the fraction of histamine-loaded cytoplasmic vesicles after FMLP stimulation significantly exceeded the fraction in unstimulated cells, a process that persisted for 10 minutes after FMLP stimulation and was associated with the morphologic continuum of PMD-->anaphylactic degranulation (characterized by extrusion of granules)-->recovery, a process largely complete in the 10-minute samples. These studies establish for the first time that an important proinflammatory mediator, histamine, traffic from secretory granules to the extracellular milieu in small cytoplasmic vesicles in stimulated human basophils. The association of this process with the ultrastructural release reaction defined as PMD produced primarily by TPA and in part by FMLP establishes vesicular transport as the mechanism for effecting this type of regulated secretion. Vesicular transport of histamine was also significant in the more complex stimulated secretory and recovery model produced by exposure of human basophils to the bacterial peptide FMLP.

Basophils↗

PubMed2006

G protein betagamma-subunits activated by serotonin mediate presynaptic inhibition by regulating vesicle fusion properties.

Neurotransmitters are thought to be released as quanta, where synaptic vesicles deliver packets of neurotransmitter to the synaptic cleft by fusion with the plasma membrane. However, synaptic vesicles may undergo incomplete fusion. We provide evidence that G protein-coupled receptors inhibit release by causing such incomplete fusion. 5-hydroxytryptamine (5-HT) receptor signaling potently inhibits excitatory postsynaptic currents (EPSCs) between lamprey reticulospinal axons and their postsynaptic targets by a direct action on the vesicle fusion machinery. We show that 5-HT receptor-mediated presynaptic inhibition, at this synapse, involves a reduction in EPSC quantal size. Quantal size was measured directly by comparing unitary quantal amplitudes of paired EPSCs before and during 5-HT application and indirectly by determining the effect of 5-HT on the relationship between mean-evoked EPSC amplitude and variance. Results from FM dye-labeling experiments indicate that 5-HT prevents full fusion of vesicles. 5-HT reduces FM1-43 staining of vesicles with a similar efficacy to its effect on the EPSC. However, destaining of FM1-43-labeled vesicles is abolished by lower concentrations of 5-HT that leave a substantial EPSC. The use of a water-soluble membrane impermeant quenching agent in the extracellular space reduced FM1-43 fluorescence during stimulation in 5-HT. Thus vesicles contact the extracellular space during inhibition of synaptic transmission by 5-HT. We conclude that 5-HT, via free Gbetagamma, prevents the collapse of synaptic vesicles into the presynaptic membrane.

Animals↗

PubMed1975

On degeneration of peptidergic neurosecretory fibres in the albino rat.

Three types of degenerating peptidergic neurosecretory fibres have been found in the posterior pituitary of chronically dehydrated albino rats. "Dark" neurosecretory fibres and their swellings contain neurosecretory granules, neurotubules, shrunken mitochondria and diffusely distributed fine dense material. Some swellings are filled with synaptic vesicles and/or conglomerations of dense membranes. The transitional forms exist between these fibres and extracellular accumulations of electron dense material. Synaptic vesicles, single neurosecretory granules, lipid-like droplets and lamellar bodies occur in the latter. Some neurosecretory fibres and swellings have numerous polymorphous inclusions arising due to degradation of secretory inclusions and organelles, mitochondria and neurotubules in particular. "Dark" neurosecretory elements and those with numerous polymorphous inclusions are enveloped by pituicyte cytoplasm. Sometimes the plasma membranes both of the pituicytes and neurosecretory fibres are destroyed or transformed into a multi-membrane complex. It is assumed that pituicytes may phagocytize degenerating neurosecretory elements. N urosecretory fibres with a locally dissolved neuroplasm and/or large lucent vacuoles seem to be due to axonal degeneration by the "light" type. These neurosecretory elements, the largest of them in particular, may transform into large cavities bordered by a membrane and containing flake-like material and single-membrane vacuoles. Degeneration of neurosecretory elements seems to occur mainly due to hyperfunction of the hypothalamo-hypophysial neurosecretory system.

Animals↗

PubMed1994

Hormone storage vesicle proteins. Transcriptional basis of the widespread neuroendocrine expression of chromogranin A, and evidence of its diverse biological actions, intracellular and extracellular.

Chromogranin A (CgA) is an acidic soluble protein found in the core of secretory vesicles throughout the neuroendocrine system, from which it is coreleased by exocytosis with a variety of amine and peptide hormones and neurotransmitters. Much has now been learned about the structure of CgA, and there is emerging evidence that it plays several biological roles, both within secretory granules and after release from neuroendocrine cells. Factors governing its gene's widespread yet restricted (neuroendocrine) pattern of expression are only now being explored. In an attempt to understand how cells throughout the neuroendocrine system (but not exocrine or other nonendocrine cells) turn on and control the expression of CgA, we have isolated and begun to characterize functional 5' promoter elements from the rodent CgA genes. Within the sympathoadrenal system, interest focuses on a recently proposed (though as yet incompletely investigated) function of CgA: its ability to suppress catecholamine release from adrenal chromaffin cells when such cells are stimulated by their usual physiologic secretagogue. We anticipate that such studies will contribute to an understanding of this abundant, yet previously mysterious protein's role in neuroendocrine function.

Adrenal Medulla↗

PubMed1998

Ultrastructural aspects of cartilage formation, mineralization, and degeneration during primary antler growth in fallow deer (Dama dama).

Due to their rapid growth, regular replacement and easy accessibility, deer antlers are considered a useful model for the study of cartilage and bone differentiation and mineralization in mammals. The present study describes, for the first time, the cellular and extracellular matrix changes associated with cartilage formation, mineralization and degeneration in primary antlers on the ultrastructural level. Growing primary antlers of 3 to 4 cm length were obtained from six fallow bucks, aged about 10 months. It was shown that the chondroblasts were derived from progenitor cells of the antler perichondrium and differentiated into mature chondrocytes that subsequently underwent hypertrophic changes. Concomitant with cell hypertrophy, formation of a lacunar and a perilacunar extracellular matrix was observed, the latter containing numerous collagenous fibers. Mineralization of the extracellular matrix occurred via matrix vesicles and the formation of apatite crystals at distinct sites of the collagenous fibers. The hypertrophic chondrocytes of the mineralized cartilage then degenerated, a process that was also occasionally observed in more distally located cells surrounded by still unmineralized matrix. No morphological indications of a transdifferentiation of hypertrophic chondrocytes into bone forming cells, i.e., co-occurrence of a degenerating chondrocyte and a viable osteogenic cell in intact lacunae, were found. The cellular and extracellular matrix changes seen in primary antlers resemble those described for secondary antlers. Our results further indicate that the hypertrophic chondrocytes of primary antlers eventually undergo apoptosis, thereby providing further evidence that metaplastic conversion of cartilage into bone does not play a role in antler growth.

Animals↗

PubMed1994

Immunoelectron microscopy of epitopes on Na,K-ATPase catalytic subunit. Implications for the transmembrane organization of the C-terminal domain.

The transmembrane folding of the alpha subunit of Na,K-ATPase was studied by using immunoelectron microscopy to determine whether monoclonal antibodies with defined epitopes bind to the extracellular or cytoplasmic surface. In double labeling experiments, an antibody and a reference marker were bound to purified membrane-associated Na,K-ATPase and were visualized by employing colloidal gold particles of two different sizes. Wheat germ agglutinin and a previously characterized monoclonal antibody were used as control markers for the exoplasmic and cytoplasmic surfaces, respectively. Three antibodies, VG4, VG2, and IIC9, unambiguously bound to the extracellular surface. Previously IIC9 had been assigned to the cytoplasmic surface because, in immunofluorescence studies, it stained intact cells only when they were detergent-permeabilized. To investigate the basis for this contradiction, a third assay for sidedness was used: competition binding in solution to right-side-out renal medullary vesicles. IIC9 was found to bind to the extracellular surface of sealed vesicles, but only in certain experimental conditions. It was concluded that IIC9 has an epitope that is not always accessible and that in this instance, studies of binding to intact and detergent-treated cells had given misleading results. An extracellular disposition for all three antibodies is not compatible with existing folding models, and new models are presented.

Amino Acid Sequence↗

PubMed1992

Bidirectional mechanism of plasma membrane transport of reduced glutathione in intact rat hepatocytes and membrane vesicles.

We determined the trans effects of extracellular reduced glutathione (GSH) on the rate of efflux of endogenous labeled GSH from freshly isolated rat hepatocytes. The presence of GSH (10 mM) in the medium significantly stimulated the fractional rate of efflux of [35S]GSH from 5.2 to 12.6%/15 min (p < 0.01). This effect was concentration-dependent, had sigmoid type of kinetics (D50 of 0.32 mM), and was reversible upon removal of external GSH. trans-Stimulation (counter-transport) was also observed with 5 mM oxidized glutathione (GSSG) and ophthalmic acid (fractional [35S] GSH efflux: 13.4% +/- 4.1 and 8.8% +/- 2.3 in 15 min, respectively, compared with control: 4.7 +/- 2.5/15 min). Bromosulphthalein-glutathione (BSP-GSH, 5 mM) in Krebs buffer inhibited the fractional [35S]GSH efflux (1.1%/15 min), whereas in Cl(-)-free buffer, GSH efflux was stimulated (14.2%/15 min) compared with control. trans-Stimulation was independent of chloride. BSP-GSH cis-inhibited and trans-stimulated the initial rate of GSH transport in basolateral-enriched membrane vesicles (bLPM) but not in canalicular-enriched membrane vesicles (cLPM). gamma-Glutamyl compounds also cis-inhibited and trans-stimulated GSH transport in bLPM vesicles. GSH-depleted hepatocytes incubated with 10 mM [35S]GSH accumulated more GSH than repleted cells, but the initial rate of uptake of radioactivity was faster in repleted cells. In contrast, repleted hepatocytes incubated with tracer or 50 microM [35S]GSH did not take up GSH. Thus, the sinusoidal membrane GSH transporter exhibits low affinity kinetics with sigmoid features for both GSH uptake and trans-stimulation of efflux, explaining the lack of uptake of GSH at low physiologic extracellular concentrations. Therefore, our findings support and explain the widely held view that GSH transport is unidirectional under physiologic conditions. However, the efflux of GSH may also occur in exchange for the uptake of organic anions and gamma-glutamyl compounds.

Alanine↗

PubMed1977

Extracellular matrix synthesis in blastula and gastrula stages of normal and hybrid frog embryos. II. Autoradiographic observations on the sites of synthesis and mode of transport of galactose- and glucosamine-labelled materials.

Pulse-chase labelling experiments and light- and electron-microscopic autoradiography were used to examine the sites of synthesis, mode of transport, and sites of deposition of galactose- and glucosamine-labelled materials in different developmental stages of normal developing Rana pipiens embryos and interspecific hybrid embryos formed by fertilizing the eggs of R. pipiens with the sperm of R. catesbeiana. In both normal and hybrid embryos, after 15-min pulse, grains are closely associated with juxtanuclear and cytoplasmic collections of membrane-bound vesicles which resemble the Golgi apparatus. In normal embryos following a 15-30 min pulse and a 60-min chase, grains are largely cleared from the cytoplasmic vesicles and deposited in the extracellular spaces or along cell surfaces where the extracellular spaces are relatively large. In contrast, arrested hybrid embryos given a 15-30-min pulse and a 60-min chase show a marked accumulation of grains over cytoplasmic structures such as the Golgi apparatus and vesicular elements in the cell cortex. Finally, early gastrula stage normal embryos are most active in the synthesis of galactose-labelled materials in cells above the dorsal lip of the blastopore, where cell migration is initiated.

Animals↗

PubMed1997

Protrusion of cell surface coupled with single exocytotic events of secretion of the slime in Physarum plasmodia.

Exocytosis has been proposed to participate in the formation of pseudopods. Using video-enhanced microscopy, we directly visualized exocytosis of single vesicles in living Physarum plasmodia migrating on a substrate. Vesicles containing slime, the plasmodial extracellular matrix, of approximately 3.5 microm in diameter, shrank at the cell periphery at the average rate of approximately 1 microm/second, and became invisible. Immediately after exocytotic events, the neighboring cell surface extended to form a protrusion. The rate of extension was approximately 1 microm/second. The protrusion showed lamella-like morphology, and contained actin microfilaments. Electron microscopy suggested that the organization of microfilaments in such protrusions may be a random meshwork rather than straight bundles. These morphologies suggest that protruded regions are pseudopods. Importantly, only the slime-containing vesicle preferentially invaded the hyaline layer that consists of dense actin microfilaments while the other vesicular organelles remained in the granuloplasm. Quantitative analysis demonstrated a linear relationship in terms of their surface area, between individual protrusions and single slime-containing vesicles. It is, therefore, likely that most of the plasma membrane of the protrusion was supplied by fusion of the slime-containing vesicle during exocytosis.

Actins↗

PubMed2003

Steroid hormone action in musculoskeletal cells involves membrane receptor and nuclear receptor mechanisms.

Steroid hormones regulate target cells through traditional nuclear mechanisms as well as by membrane mechanisms. 1alpha,25(OH)2D3 and 24R,25(OH)2D3 bind membrane receptors (mVDR) and mediate their effects on the physiological responses of musculoskeletal cells via protein kinase C (PKC). In cultures of costochondral growth plate chondrocytes, 1alpha,25(OH)2D3 binds the 1,25-mVDR in growth zone cells, activating phospholipase C (PLC), leading to diacylglycerol (DAG) production and PKC translocation to the plasma membrane. It also activates PLA2, increasing arachidonic acid release and prostaglandin synthesis. 24R,25(OH)2D3 binds its membrane receptor in resting zone chondrocytes, activating phospholipase D (PLD), and increasing DAG and PKC activity, but translocation does not occur. PLA2 activity is decreased, reducing arachidonic acid and prostaglandin production. 17Beta-estradiol (E2) activates PKC in both cartilage cells, but DAG is not involved. 1alpha,25(OH)2D3 and 24R,25(OH)2D3 also increase PKC in osteoblasts in a cell-specific manner. Antibodies to the 1,25-mVDR block PKC activation. Membrane-mediated events influence gene expression via signaling cascades, including the ERK1/2 MAP kinases. The ability of steroid hormones to initiate events nongenomically is important for regulation of matrix vesicle (MV) function in the extracellular matrix. MVs have mVDRs, but ligand binding inhibits PKC-zeta (PKCzeta) via a mechanism that differs from PKCalpha activation in the plasma membranes. Treatment of MVs from growth zone chondrocyte cultures with 1alpha,25(OH)2D3 releases stromelysin-1 (MMP-3) and increases TGF-beta activation. MMP-3 is also involved in proteoglycan degradation, facilitating calcification. 24R,25(OH)2D3 inhibits PKCzeta in MV from resting zone cell cultures and inhibits MMP-3 release. Chondrocytes and osteoblasts produce 1,25(OH)2D3, 24,25(OH)2D3, and E2; thus, locally produced steroids may function as autocrine regulators of matrix events, including matrix vesicle enzyme activity and matrix protein remodelling during longitudinal growth, calcification, and growth factor activation.

24,25-Dihydroxyvitamin D 3↗

PubMed2006

Vacuolar sequential exocytosis of large dense-core vesicles in adrenal medulla.

Individual exocytic events in intact adrenal medulla were visualized by two-photon extracellular polar-tracer imaging. Exocytosis of chromaffin vesicles often occurred in a sequential manner, involving first vesicles located at the cell periphery and then those present deeper within the cytoplasm. Sequential exocytosis occurred preferentially at regions of the plasma membrane facing the intercellular space. The compound vesicles swelled to more than five times their original volume and formed vacuolar exocytic lumens as a result of expansion of intravesicular gels and their confinement within the lumen by the fusion pore and the narrow intercellular space. Such luminal swelling greatly promoted sequential exocytosis. The SNARE protein SNAP25 rapidly migrated from the plasma membrane to the membrane of fused vesicles. These data indicate that vesicles present deeper within the cytoplasm can be fusion ready like those at the cell periphery, and that swelling of exocytic lumens promotes assembly of the fusion machinery. We suggest the existence of two molecular configurations for fusion-ready states in Ca2+ -dependent exocytosis.

Adrenal Medulla↗

PubMed1993

Decreased synthesis and delayed processing of sulfated glycoproteins by cells from human polycystic kidneys.

BACKGROUND: In polycystic kidney disease, there is progressive distention of tubules and remodeling of an altered extracellular matrix. Recent findings indicate a defect(s) in the synthesis, post-translational modification, cellular transport, and extracellular matrix incorporation of proteoglycans in PKD that may have a fundamental role in its pathogenesis. EXPERIMENTAL DESIGN: Pulse-chase techniques and quantitative autoradiography were used to investigate in vitro the de novo synthesis and the kinetics of intracellular transport and extracellular matrix assembly of sulfated glycoproteins by epithelial cells from normal human kidneys (NK) and from cysts of autosomal dominant polycystic kidneys (ADPKD). NK and ADPKD cell monolayers were pulsed with [35S]sulfate for 150 min and chased for four intervals between 15 and 120 min. RESULTS: In ADPKD versus NK cells there was a significant reduction in the de novo synthesis of sulfated glycoproteins and, during the chase, a marked prolongation of the disappearance time for cellular sulfated glycoproteins. Transport kinetics revealed a substantial delay in processing and release of these macromolecules by the Golgi apparatus in ADPKD cells. During the chase, kinetics for the cellular transport of sulfated glycoproteins by secretory vesicles and their assembly into extracellular matrix were comparable in ADPKD and NK cells. However, the percentage of total sulfated glycoproteins incorporated into the matrix of ADPKD versus NK cell monolayers was notably diminished. CONCLUSIONS: The findings indicate that in ADPKD versus NK cell monolayers, synthesis of sulfated glycoproteins is impaired, processing of sulfated glycoproteins by the Golgi apparatus is prolonged, and assembly of these macromolecules into the extracellular matrix is reduced. These alterations may have a fundamental role in the pathogenesis of autosomal dominant polycystic kidney disease.

Autoradiography↗