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PubMed1983

Matrix vesicles in atherosclerotic calcification.

Matrix vesicles, small extracellular membranous structures, are known to be the initial loci of calcification of cartilage, bone, and dentin. Calcification is an important complication of atherosclerosis. Using histologic, ultrastructural, and cytochemical techniques, the present study has demonstrated that matrix vesicle-like structures are involved in the calcification of atherosclerotic lesions, as well as in arterial medial calcification. In aortas from autopsied humans and from rabbits and chickens on atherogenic diets, the matrix vesicles appear to be derived from intimal and medial cellular components, mainly smooth muscle cells.

Animals↗

PubMed1996

Characteristics and regulation of Pi transport in osteogenic cells for bone metabolism.

Inorganic phosphate (Pi) is an essential element in the development of osteogenic cells. The translocation of Pi from the systemic to the skeletal extracellular compartment appears to be an important function of osteoblastic cells. The plasma membrane of osteogenic cells is endowed with a sodium-dependent Pi transport system that is regulated by osteotropic factors such as parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), insulin-like growth factor-1 (IGF-1), platelet-derived growth factor (PDGF) and fluoride. A similar Pi transport system has been recently identified in matrix vesicles derived from the plasma membrane of osteogenic cells, such as epiphyseal chondrocytes or osteoblastic cells. Matrix vesicles are extracellular structures which are considered to play an important role in endochondral and membranous calcification. Pi transport appears to be the driving force responsible for the accumulation of mineral inside the matrix vesicles and thereby can be considered as a pivotal determinant in the induction of the calcification process. Furthermore, modulation of the activity of the Pi transport at the level of the plasma membrane of osteogenic cells by osteotropic factors is transferred to the matrix vesicles derived from these cells. This notion implies that hormonal and other environmental factors, such as Pi itself and calcium, which have a direct impact on the Pi transport activity of osteogenic cells can also influence the capacity of the matrix vesicles to initiate the mineralization of the bone matrix. The cellular mechanisms involved in the regulation of Pi transport by osteotropic factors have been recently investigated. For the PTH/PTHrP regulatory effect, cAMP appears to be the main mediator and the response does not require the de novo synthesis of proteins. For the effects of IGF-1, PDGF and fluoride, tyrosine phosphorylation processes are involved and responses are dependent upon the de novo synthesis of proteins. The molecules responsible for activation of these signaling pathways are currently under investigation. Such an investigation may improve our understanding of the mechanisms underlying the differentiation processes of osteogenesis such as the calcification of the extracellular matrix.

Animals↗

PubMed1983

[Relation between the number of vesicles in the vascular smooth muscle cells and extracellular calcium concentration].

The relationship between the number of vesicles in smooth muscle cells in mesenterial arteries and calcium concentration in incubation medium was studied in experiments on normotensive control rats (NKWR) and spontaneously hypertensive rats (SHR). Extracellular elevation of calcium resulted in an increase in the number of vesicles, which was more demonstrable in SHR than in NKWR. After preliminary adrenalectomy the same high concentration of calcium in incubation medium did not induce any changes in the number of vesicles in both animal groups. The decrease of calcium concentration did not produce any changes in the number of vesicles in SHR or NKWR before and after adrenalectomy. The increase of the number of vesicles in response to high extracellular concentration of calcium is assumed to be a means of raising the active surface of the smooth cells and compensates for membrane deficiency seen under pathology.

Animals↗

PubMed1996

Effect of 17 beta-estradiol on chondrocyte membrane fluidity and phospholipid metabolism is membrane-specific, sex-specific, and cell maturation-dependent.

In this study we examined the hypothesis that 17 beta-estradiol exerts both rapid and direct, nongenomic effects of cells in the endochondral pathway. To do this, we used a cell culture model in which chondrocytes at two distinct stages of cell maturation are isolated from the costochondral cartilage of male and female rats, and examined the short-term effect of 17 alpha- and 17 beta-estradiol on [14C]arachidonic acid turnover in the cell layer and phospholipase A2 specific activity in plasma membranes and extracellular matrix vesicles isolated from similarly prepared cultures. In addition, the effect of 17 alpha- and 17 beta-estradiol on plasma membrane and matrix vesicle membrane fluidity was assessed. The effect of hormone on arachidonic acid turnover was rapid, time- and concentration-dependent, stereo-specific, and cell maturation-specific. Only resting zone cells from female rats were affected, and only 17 beta-estradiol elicited a response. Similarly, only female rat resting zone chondrocytes exhibited a change in phospholipase A2 activity after a 24 h exposure to hormone, causing an increase in enzyme activity in the matrix vesicles, but not plasma membranes. When isolated membranes were incubated directly with hormone, membrane fluidity was decreased in both plasma membranes and matrix vesicles isolated from female rat resting zone chondrocyte cultures. This nongenomic effect was dose-dependent and stereo-specific and differentially expressed in the two membrane fractions with respect to time course and magnitude of response. These results support the hypothesis that 17 beta-estradiol has a rapid action on chondrocyte membrane lipid metabolism and suggest that specific membrane components, characteristic of a particular sex and state of cell maturation, are involved in the nongenomic effects of this sex hormone on isolated matrix vesicles and plasma membranes.

Animals↗

PubMed1998

Carbonic anhydrase II plays a major role in osteoclast differentiation and bone resorption by effecting the steady state intracellular pH and Ca2+.

Carbonic anhydrase II (CA II) expression in characteristic for the early stage of osteoclast differentiation. To study how CA II, which is crucial in proton generation in mature osteoclasts, influences the osteoclast differentiation process we performed rat bone marrow cultures. In this model, acetazolamide, a specific CA inhibitor, decreased the 1,25 (OH)2D3-induced formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells, in a dose-dependent manner. We then performed intracellular pH (pHi) and Ca2+ (Cai2+) measurements for cultured osteoclasts and noticed that addition of acetazolamide caused a rapid, transient increase of both parameters. The increase in pHi was dependent neither on the culture substrate nor on the extracellular pH (pHe) but the increase could be diminished by DIDS or by bicarbonate removal. Membrane-impermeable CA inhibitors (benzolamide and pd5000) did not have this effect. Addition of CA II antisense oligonucleotides into the cultures reduced the pHi increase significantly. CA II inhibition was also found to neutralize the intracellular vesicles at extracellular pH (pHe) of 7.4, but at less extent at pHe 7.0. In mouse calvaria cultures, bone resorption was inhibited dose dependently by acetazolamide at pHe 7.4 while inhibition was smaller at pHe 7.0. We conclude that CA II is essential not only in bone resorption but also in osteoclast differentiation. In both processes, however, the crucial role of CA II is at least partially due to the effect on the osteoclast pHi regulation.

Acetazolamide↗

PubMed1987

Maturation process of Japanese encephalitis virus in cultured mosquito cells in vitro and mouse brain cells in vivo.

The maturation process of Japanese encephalitis (JE) virus in C6/36 cells in vitro and in mouse brain cells in vivo was studied by electron microscopy. In the C6/36 cell infection, 500 to 2250 virions per cell were released into the medium during the period of study; yet, no virus budding process was observed at the host cell membranes. JE virions at various maturation stages appeared within the cisternae of rough endoplasmic reticulum (RER) of infected cells at 24 hours p.i.; and, although C6/36 cells did not show a well-developed Golgi apparatus, the virions appeared to be carried to the cell surface within host-cell secretory vesicles for extracellular release as early as 24 hours p.i. The occurrence of a secretory-type intracellular transport of maturing JE virus particles was well recognizable in brain cells of infected mice, in which JE virus particles were found almost exclusively in the cisternae of RER, in the Golgi apparatus, and in various vesicles, including coated vesicles, in the vicinity of the Golgi apparatus. Our previous study of dengue-2 virus morphogenesis and our present study of JE virus morphogenesis differed substantially at various stages of maturation. Possible mechanisms which explain these differences were discussed.

Animals↗

PubMed1989

Mechanism of mineral formation in bone.

The mechanism of mineral formation in bone is seen best where active new bone formation is occurring, e.g., in newly forming subperiosteal bone of the embryo, in the growing bone of young animals, and in healing rickets where the calcification process in osteoid is reactivated. A large body of ultrastructural evidence, using conventional and anhydrous methods for tissue preparation, has shown convincingly that extracellular matrix vesicles are present at or near the mineralization front in all of the above, and that these vesicles are the initial site of apatite mineral deposition. Thus bone resembles growth plate cartilage, predentin, and turkey tendon in having calcification initiated by matrix vesicles. Once the calcification cascade is begun, matrix vesicles are no longer needed to support mineralization and are consumed by the advancing mineralization front in which performed crystals serve as nuclei for the formation of new crystals. The rate of crystal proliferation is promoted by the availability of Ca2+, PO4(3-), and the presence of collagen, and retarded by naturally occurring inhibitors of mineralization such as proteoglycans and several noncollagenous calcium-binding proteins of bone including bone-Gla protein (osteocalcin), phosphoproteins, osteonectin, and alpha-2HS-glycoproteins. New electron microscopic immunocytochemical findings in our laboratory suggest that the origin of alkaline phosphatase-positive bone matrix vesicles is polarized to the mineral-facing side of osteoblasts and may be concentrated near the intercellular junctions of human embryonic osteoblasts.

Alkaline Phosphatase↗

PubMed1994

Liposome-like particles isolated from human atherosclerotic plaques are structurally and compositionally similar to surface remnants of triglyceride-rich lipoproteins.

Recent studies have demonstrated the presence of unesterified cholesterol-rich, liposome-like vesicles in the extracellular space of atherosclerotic lesions in humans and animals. Liposome-like vesicles accumulate in the subendothelial space in rabbits within 2 weeks of initiation of cholesterol feeding, well before foam cells appear. These observations suggest that extracellular liposome-like vesicles may play a pivotal role in atherogenesis. The origin of these particles is unknown. We report a combination of in vivo and in vitro experiments that suggest a novel origin for these liposome-like vesicles. We demonstrate that the liposome-like particles isolated from postmortem human atherosclerotic plaques are rich in intact apolipoprotein (apo) A-I, C apolipoproteins, and sphingomyelin. We show that the in vivo derived particles are virtually identical, structurally and compositionally, to liposome-like lipolytic surface remnants of triglyceride (TG)-rich lipoproteins produced during in vitro lipolysis of hypertriglyceridemic serum. In vitro lipolysis of isolated very-low-density lipoprotein has shown that the lipolytic surface remnants remain attached to the core remnants in the absence of high-density lipoprotein (HDL), dissociate to form liposome-like vesicles in the presence of low levels of HDL, and are assimilated into HDL to form larger HDL particles in the presence of excess HDL. Thus, the in vitro produced, liposome-like particles represent a complex of lipolytic surface remnants of TG-rich lipoproteins and apo A-I derived from HDL. Two possible origins have been suggested for the extracellular liposome-like vesicles in atherosclerotic plaques: (1) modified, aggregated, and/or degraded LDL particles entrapped in an intimal matrix and (2) intracellular lipid products of arterial wall cells. Neither possibility directly explains the presence of A-I and C apolipoproteins and excess sphingomyelin that we observe. We propose as an alternate explanation that the in vivo liposome-like particles are lipolytic surface remnants of TG-rich lipoproteins. We further suggest that these remnants are produced in the intimal space by undefined processes and/or are transcytosed into the intima from the plasma compartment as a product of normal lipolysis gone awry. We conjecture that one role of HDL may be to assimilate the highly atherogenic liposome-like particles in a (1) "mop-up" fashion to remove them from the artery wall and/or (2) preventive fashion in the plasma compartment to prevent their transcytosis into the artery wall. The suggestion that elevated concentrations of surface remnants act as a "sink" for apo A-I can also account for the well-established but poorly understood link between hypertriglyceridemia and low HDL.

Animals↗

PubMed1980

Fate of horseradish peroxidase in the secretion zone of the rat incisor enamel organ.

Adult CDF albino rats were killed from 10 min to 6 hr after a single intravenous dose of HRP. Experimental and control tissues were reacted for peroxidase activity and processed for light and electron microscopy. At 10 min, all extracellular spaces of the secretion zone showed reaction product. A reaction was also seen around Tomes' processes and in a layer of enamel spaces in the region of thin enamel. At later times intervals, reactions around Tomes' processes were also seen in regions of thicker enamel. Tracer was located preferentially at the growth fronts of rod and interrod enamel, and also diffused for some distance into enamel. From 2 to 6 hr, the enamel over the transition zone became heavily labeled. The tracer penetrated for more than 90 micrometer into the enamel and was localized mainly in the interrod enamel. Droplets of dense stippled material in the extracellular spaces between Tomes' processes did not mix with tracer, but sites which contain a light stippled material in the controls (extracellular spaces, vesicles within ameloblasts) showed a reaction. It is concluded that (1) the basal terminal bars of secretory ameloblasts do not impede the flow of large molecules, (2) the apical terminal bars are permeable in early secretion, become increasingly tight as secretion progresses, and are again permeable in the transition zone, (3) ameloblasts can shuttle large extracellular molecules towards the enamel growth fronts, (4) large molecules can diffuse into enamel; rod and interrod enamel differ with regard to the diffusion of large molecules, (5) ameloblasts phagocytose significant amounts of light stippled material. The possibility is considered that extracellular enamel precursor molecules move preferentially towards the enamel growth fronts, perhaps by a mechanism involving membrane flow, and diffuse through enamel in similar fashion as HRP.

Ameloblasts↗

PubMed1984

Isolation of a plasma membrane-enriched fraction from collagenase-suspended rachitic rat growth plate chondrocytes.

An attempt was made to concentrate plasma membranes of homogenized chondrocytes isolated by collagenase digestion of rachitic rat epiphyseal growth plate cartilage. This study reports the characterization of enzymes in the plasma membrane of isolated chondrocytes and their comparison with extracellular matrix vesicle components. The plasma membrane-enriched fractions that were obtained showed a sevenfold increase in 5'-nucleotidase and a 15-fold increase in alkaline phosphatase, both of which are regarded as plasma membrane markers. SDS-polyacrylamide gel electrophoretic profiles of proteins extracted from membrane fractions contained several major protein bands also seen in isolated matrix vesicles. These studies indicate the usefulness of concentrating plasma membrane components from isolated chondrocytes, after the chondrocytes have been enzymatically freed from investing matrix and other stromal components by collagenase.

Alkaline Phosphatase↗

PubMed1993

Tumor spheroid-induced vesicle formation on endothelial cells is associated with procoagulant properties.

Fibrin deposits in tumor beds are an intriguing phenomenon. It has been suggested that fibrin plays a role as a provisional matrix in which the tumor grows and induces development of a vascular network. On the other hand fibrin possibly protects the tumor nodule from host defense mechanisms. We therefore investigate whether tumor cells can induce a procoagulant response in endothelial cells leading to fibrin formation. For our studies we employed a modification of the matrix model of Montesano in which sprouting of endothelial cell aggregates can be followed. This system allows us to study in vitro the involvement of coagulation in tumor growth and angiogenesis. Cocultures of endothelial cell aggregates and avascular tumor spheroids in collagen type I gels results in the appearance of extracellular vesicle-like structures on the endothelial sprouts. The vesicles formed on endothelial cell sprouts upon coculturing with tumor cells exhibit an increased amidolytic activity, suggestive of factor X/Xa activity, not dependent on tissue factor exposure. Experiments using HgCl2 and Iodoacetamide point to the importance of SH groups in the factor X/Xa activity on endothelial cell sprouts.

Animals↗

PubMed1997

[Molecular immunology of voltage-gated calcium channel and Lambert-Eaton myasthenic syndrome].

Lambert-Eaton myasthenic syndrome(LEMS), an autoimmune disease that is often associated with small cell lung carcinoma(SCLC), impairs the quantal release of acetylcholine by directing antibodies against voltage-gated calcium channels (VGCC) in the motor nerve terminal. We focused attention on the P/Q type VGCC, to which there are antibodies in LEMS patients in higher frequency than antibodies to other types of VGCC. To search for antigenic sites in the molecular structure of alpha 1A subuuit of P/Q type VGCC in LEMS, we synthesized 4 peptides corresponding to the extracellular region (S5-S6 linker) of each of 4 domains that form alpha 1A subunit of VGCC. Also, LEMS patients' sera were studied by immunoprecipitation assay using these antigens. Peptides corresponding to the extracellular region (S5-S6 linker) of domains II and IV were specifically reactive with LEMS antibodies; their titiers respectively correlated with those of anti-P/Q type calcium channel (omega-conotoxin MVIIC-sensitive human cerebellum extret). Lewis rats were immunized with the domain II S5-S6 linker peptides conjugated with KLH. The immunized rats showed LEMS features characterized by reduced acetylcholine quantum content of endplate potentials and antibodies reactive with P/Q type VGCC. Our observations suggest 2 potential epitopes of LEMS antibodies. Synaptotagmin is a Ca2+ and phospholipid binding protein integrated in synaptic vesicle membranes. It plays a crucial role in neurotransmitter release, probably as a Ca2+ sensor for exocytosis of synaptic vesicles. The extracellular region of synaptotagmin was found antigenic for the induction of a rat model of LEMS. A proportion of human LEMS antibodies reacted with the recombinant synaptotagmin in immunoblot.

Acetylcholine↗

PubMedDate not supplied

Tissue transglutaminase expression and activity in placenta.

Tissue transglutaminase (tTG) expression, distribution and activity were examined in human placenta and derived cells. Immunochemical techniques and RT-PCR were used to demonstrate tTG protein and mRNA in stromal cells and trophoblast in first trimester and at term, with higher levels later in pregnancy. Decidual cells also produce tTG. The data were confirmed using primary cultures of trophoblast, fibroblasts and decidual stromal cells. Substrate incorporation studies indicated tTG activity in association with fibroblast extracellular matrix and the syncytial microvillous membrane (MVM), where several target polypeptides could be observed. tTG is a major autoantigen in Coeliac disease (CoD) which is associated with poor pregnancy outcome. tTG at the placental MVM is a plausible target of maternal autoantibody action.

Biotinylation↗

PubMed1993

KCl contractions in the rat isolated seminal vesicle.

Potassium chloride (K+) produced dose-dependent contractions of the rat isolated seminal vesicle with no sign of tachyphylaxis. The contractile response was biphasic in nature and composed of an early rapid phasic contraction and a slowly developing, but more sustained, tonic response separated by a transient relaxation. Neither cocaine nor depletion of tissue catecholamines by reserpine influenced responses to K+. Moreover, guanethidine, phentolamine, phenoxybenzamine, atropine and physostigmine all failed to modify responses of the tissue to K+. Thus, the possibility of K+ acting via release of endogenous noradrenaline or acetylcholine is excluded. Calcium-free conditions with or without EGTA reduced both the components of K(+)-induced contraction. The rate of reduction of both responses was faster in the presence of EGTA with part of the tonic response being resistant even to calcium deprivation in the presence of EGTA. On the other hand, verapamil reduced both responses in a similar manner, whereas nifedipine produced dose-dependent rightward shifts of the concentration-response curves of both the phasic and tonic responses to K+. However, in the presence of nifedipine, the maximum response of only the phasic contraction was significantly lowered. It is concluded that both phases of KCl contraction in the rat seminal vesicle use extracellular Ca2+, of which some is tightly bound with high affinity, probably to plasma membranes. In addition, two subtypes of voltage-sensitive Ca2+ channels may exist, one of which is preferentially sensitive to nifedipine and both are sensitive to verapamil.

Acetylcholine↗

PubMed2025

Nanoscale Epigenetic Profiling of Colorectal Cancer Cell-Derived Exosomes via Single-Vesicle Nanoscopy.

Exosomes play critical roles in cancer diagnosis and treatment as they carry molecular information that reflects the epigenetic state of their parent cells. For the first time, nanoscale epigenetic profiling of individual exosomes derived from colorectal cancer cell lines is demonstrated via photo-induced force microscopy (PiFM). Exosomes from three cell lines with distinct CpG island methylator phenotype (CIMP) status are analyzed at the single-vesicle level. The nano-IR method provides simultaneous high-resolution topographical and spectroscopic data, revealing detailed vibrational signatures that distinguish CIMP-high (HCT116 and HT29) exosomes from CIMP-negative (SW480) ones. Notably, exosomes from CIMP-high cells exhibit red-shifted amide I and nucleic acid region compared to those from CIMP-negative cells, a shift attributed to increased 5-methylcytosine (5mC) modifications, as verified by quantum chemical calculations. Furthermore, these measurements reveal heterogeneity among individual exosomes, suggesting the presence of distinct subpopulations with unique epigenetic profiles, demonstrating the importance of single-vesicle resolution to detect molecular variations that remain obscured in ensemble studies. These findings present the potential of PiFM-based single-vesicle analysis to identify epigenetic markers in exosomes, laying the groundwork for its application in refined cancer diagnostics and targeted therapeutic strategies.

Humans↗

PubMed2000

Pulsed electromagnetic field stimulation of MG63 osteoblast-like cells affects differentiation and local factor production.

Pulsed electromagnetic field stimulation has been used to promote the healing of chronic nonunions and fractures with delayed healing, but relatively little is known about its effects on osteogenic cells or the mechanisms involved. The purpose of this study was to examine the response of osteoblast-like cells to a pulsed electromagnetic field signal used clinically and to determine if the signal modulates the production of autocrine factors associated with differentiation. Confluent cultures of MG63 human osteoblast-like cells were placed between Helmholtz coils and exposed to a pulsed electromagnetic signal consisting of a burst of 20 pulses repeating at 15 Hz for 8 hours per day for 1, 2, or 4 days. Controls were cultured under identical conditions, but no signal was applied. Treated and control cultures were alternated between two comparable incubators and, therefore, between active coils; measurement of the temperature of the incubators and the culture medium indicated that application of the signal did not generate heat above the level found in the control incubator or culture medium. The pulsed electromagnetic signal caused a reduction in cell proliferation on the basis of cell number and [3H]thymidine incorporation. Cellular alkaline phosphatase-specific activity increased in the cultures exposed to the signal, with maximum effects at day 1. In contrast, enzyme activity in the cell-layer lysates, which included alkaline phosphatase-enriched extracellular matrix vesicles, continued to increase with the time of exposure to the signal. After 1 and 2 days of exposure, collagen synthesis and osteocalcin production were greater than in the control cultures. Prostaglandin E2 in the treated cultures was significantly reduced at 1 and 2 days, whereas transforming growth factor-beta1 was increased; at 4 days of treatment, however, the levels of both local factors were similar to those in the controls. The results indicate enhanced differentiation as the net effect of pulsed electromagnetic fields on osteoblasts, as evidenced by decreased proliferation and increased alkaline phosphatase-specific activity, osteocalcin synthesis, and collagen production. Pulsed electromagnetic field stimulation appears to promote the production of matrix vesicles on the basis of higher levels of alkaline phosphatase at 4 days in the cell layers than in the isolated cells, commensurate with osteogenic differentiation in response to transforming growth factor-beta1. The results indicate that osteoblasts are sensitive to pulsed electromagnetic field stimulation, which alters cell activity through changes in local factor production.

Alkaline Phosphatase↗

PubMed1996

The transient pore formed by homologous terminal complement complexes functions as a bidirectional route for the transport of autocrine and paracrine signals across human cell membranes.

BACKGROUND: We have previously shown that the membrane attack complex (MAC) of complement stimulates cell proliferation and that insertion of homologous MAC into the membranes of endothelial cells results in the release of potent mitogens, including basic fibroblast growth factor (bFGF). The mechanism of secretion of bFGF and other polypeptides devoid of signal peptides, such as interleukin 1 (IL-1) is still an open problem in cell biology. We have hypothesized that the homologous MAC pore itself could constitute a transient route for the diffusion of biologically active macromolecules in and out of the target cells. MATERIALS AND METHODS: Human red blood cell ghosts and artificial lipid vesicles were loaded with labeled growth factors, cytokines and IgG, and exposed to homologous MAC. The release of the 125I-macromolecules was followed as a function of time. The incorporation of labeled polypeptides and fluorescent dextran (MW: 10,000) was measured in MAC-impacted human red blood cells and human umbilical endothelial cells (HUVEC), respectively. RESULTS: Homologous MAC insertion into HUVEC resulted in the massive uptake of 10-kD dextran and induced the release of bFGF, in the absence of any measurable lysis. Red blood cell ghosts preloaded with bFGF, IL-1 beta, and the alpha-chain of interferon-gamma (IFN-gamma) released the polypeptides upon MAC insertion, but they did not release preloaded IgG. MAC-impacted ghosts took up radioactive IFN-gamma from the extracellular medium. Vesicles loaded with IL-I released the polypeptide when exposed to MAC. CONCLUSIONS: The homologous MAC pore in its nonlytic form allows for the export of cytosolic proteins devoid of signal peptides that are not secreted through the classical endoplasmic reticulum/Golgi exocytotic pathways. Our results suggest that the release, and perhaps the uptake, of biologically active macromolecules through the homologous MAC pore is a novel biological function of the complement system in mammals.

Biological Transport↗

PubMed1996

Nongenomic regulation of protein kinase C isoforms by the vitamin D metabolites 1 alpha,25-(OH)2D3 and 24R,25-(OH)2D3.

Prior studies have shown that vitamin D regulation of protein kinase C activity (PKC) in the cell layer of chondrocyte cultures is cell maturation-dependent. In the present study, we examined the membrane distribution of PKC and whether 1 alpha,25-(OH)2D3 and 24R,25-(OH)2D3 can directly regulate enzyme activity in isolated plasma membranes and extracellular matrix vesicles. Matrix vesicle PKC was activated by bryostatin-1 and inhibited by a PKC-specific pseudosubstrate inhibitor peptide. Depletion of membrane PKC activity using isoform-specific anti-PKC antibodies suggested that PKC alpha is the major isoform in cell layer lysates as well as in plasma membranes isolated from both cell types; PKC zeta is the predominant form in matrix vesicles. This was confirmed in Western blots of immunoprecipitates as well as in studies using control peptides to block binding of the isoform specific antibody to the enzyme and using a PKC zeta-specific pseudosubstrate inhibitor peptide. The presence of PKC zeta in matrix vesicles was further verified by immunoelectron microscopy. Enzyme activity in the matrix vesicle was insensitive to exogenous lipid, whereas that in the plasma membrane required lipid for full activity. 1,25-(OH)2D3 and 24,25-(OH)2D3 inhibited matrix vesicle PKC, but stimulated plasma membrane PKC when added directly to the isolated membrane fractions. PKC activity in the matrix vesicle was calcium-independent, whereas that in the plasma membrane required calcium. Moreover, the vitamin D-sensitive PKC in matrix vesicles was not dependent on calcium, whereas the vitamin D-sensitive enzyme in plasma membranes was calcium-dependent. It is concluded that PKC isoforms are differentially distributed between matrix vesicles and plasma membranes and that enzyme activity is regulated in a membrane-specific manner. This suggests the existence of a nongenomic mechanism whereby the effects of 1,25-(OH)2D3 and 24,25-(OH)2D3 may be mediated via PKC. Further, PKC zeta may be important in nongenomic, autocrine signal transduction at sites distal from the cell.

24,25-Dihydroxyvitamin D 3↗