scholarly journals Phospholipase C activation by ethanol in rat hepatocytes is unaffected by chronic ethanol feeding

1990 ◽  
Vol 272 (1) ◽  
pp. 59-64 ◽  
Author(s):  
J B Hoek ◽  
T F Taraschi ◽  
K Higashi ◽  
E Rubin ◽  
A P Thomas
Keyword(s):  
Phospholipase C ◽  
Order Parameter ◽  
Plasma Membranes ◽  
Structural Changes ◽  
Rat Hepatocytes ◽  
Maximal Response ◽  
Molecular Order ◽  
Transient Activation ◽  
Ethanol Feeding

The activation of phosphoinositide-specific phospholipase C by ethanol was compared in hepatocytes isolated from ethanol-fed rats and from pair-fed control animals. Ethanol (100-300 mM) caused a dose-dependent transient increase in cytosolic free Ca2+ levels in indo-1-loaded hepatocytes from both groups of animals. The rate of Ca2+ increase was similar in hepatocytes from control and ethanol-fed rats, but the decay of the Ca2+ increase was somewhat slower in the latter preparation. The ethanol-induced Ca2+ increase caused activation of glycogen phosphorylase, with 50% response at 50 mM-ethanol and a maximal response at 150-200 mM-ethanol, not significantly different in hepatocytes from control and ethanol-fed animals. Ins(1,4,5)P3 formation in response to ethanol (300 mM) or vasopressin (2 nM or 40 nM) was also similar in the two preparations. It is concluded that long-term ethanol feeding does not lead to an adaptive response with respect to the ethanol-induced phospholipase C activation in rat hepatocytes. The ability of ethanol in vitro to decrease membrane molecular order in liver plasma membranes from ethanol-fed and control rats was measured by e.s.r. Membranes from ethanol-fed animals had a significantly lower baseline order parameter compared with control preparations (0.313 and 0.327 respectively), indicative of decreased membrane molecular order. Addition of 100 mM-ethanol significantly decreased the order parameter in control preparations by 2.1%, but had no effect on the order parameter of plasma membranes from ethanol-fed rats, indicating that the plasma membranes had developed tolerance to ethanol, similar to other membranes in the liver. Thus the membrane structural changes associated with this membrane tolerance do not modify the ethanol-induced activation of phospholipase C. The transient activation of phospholipase C by ethanol in hepatocytes may play a role in maintaining an adaptive phenotype in rat liver.

scholarly journals Reversible particle movements associated with unstacking and restacking of chloroplast membranes in vitro.

1976 ◽  
Vol 71 (1) ◽  
pp. 136-158 ◽  
Author(s):  
L A Staehelin
Keyword(s):  
Plasma Membranes ◽  
Structural Changes ◽  
Freeze Fracture ◽  
Chloroplast Membranes ◽  
Ps Ii ◽  
Membrane Particles ◽  
Intramembranous Particles ◽  
Membrane Surfaces ◽  
Ps Ii Activity

Freeze-fracture and freeze-etch techniques have been employed to study the supramolecular structure of isolated spinach chloroplast membranes and to monitor structural changes associated with in vitro unstacking and restacking of these membranes. High-resolution particle size histograms prepared from the four fracture faces of normal chloroplast membranes reveal the presence of four distinct categories of intramembranous particles that are nonrandomly distributed between grana and stroma membranes. The large surface particles show a one to one relationship with the EF-face particles. Since the distribution of these particles between grana and stroma membranes coincides with the distribution of photosystem II (PS II) activity, it is argued that they could be structural equivalents of PS II complexes. An interpretative model depicting the structural relationship between all categories of particles is presented. Experimental unstacking of chloroplast membranes in low-salt medium for at least 45 min leads to a reorganization of the lamellae and to a concomitant intermixing of the different categories of membrane particles by means of translational movements in the plane of the membrane. In vitro restacking of such experimentally unstacked chloroplast membranes can be achieved by adding 2-20 mM MgCl2 or 100-200 mM NaCl to the membrane suspension. Membranes allowed to restack for at least 1 h at room temperature demonstrate a resegregation of the EF-face particles into the newly formed stacked membrane regions to yield a pattern and a size distribution nearly indistinguishable from the normally stacked controls. Restacking occurs in two steps: a rapid adhesion of adjoining stromal membrane surfaces with little particle movement, and a slower diffusion of additional large intramembranous particles into the stacked regions where they become trapped. Chlorophyll a:chlorophyll b ratios of membrane fraction obtained from normal, unstacked, and restacked membranes show that the particle movements are paralleled by movements of pigment molecules. The directed and reversible movements of membrane particles in isolated chloroplasts are compared with those reported for particles of plasma membranes.

scholarly journals Evidence that binding of CTP:phosphocholine cytidylyltransferase to membranes in rat hepatocytes is modulated by the ratio of bilayer- to non-bilayer-forming lipids

1993 ◽  
Vol 291 (2) ◽  
pp. 419-427 ◽  
Author(s):  
H Jamil ◽  
G M Hatch ◽  
D E Vance
Keyword(s):  
Phospholipase A2 ◽  
Phospholipase C ◽  
Cultured Cells ◽  
Normal Values ◽  
Common Factor ◽  
Rat Hepatocytes ◽  
Cellular Membranes ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Cellular Concentration

The mechanism by which phospholipase C (PLC) digestion of cultured cells mediates binding of CTP:phosphocholine cytidylyltransferase to cellular membranes was investigated. Incubation of choline-depleted rat hepatocytes with PLC caused a translocation of enzyme from cytosol to membranes concomitant with a decrease in the concentration of phosphatidylcholine with no effect on the concentration of other phospholipids. Removal of PLC and supplementation with choline restored the amount of phosphatidylcholine in the cells and translocated cytidylyltransferase to the cytosol. However, when phosphatidylcholine levels were decreased by incubation with phospholipase A2 (PLA2), there was no significant redistribution of cytidylyltransferase activity. With PLA2 the concentration of phosphatidylethanolamine, as well as of phosphatidylcholine, was significantly decreased. Since PLC, but not phospholipase A2, raised the cellular concentration of diacylglycerol, possibly diacylglycerol mediated the binding of cytidylyltransferase to membranes. This possibility was examined, but is unlikely, since addition of lysophosphatidylcholine to PLC-treated cells restored the concentration of phosphatidylcholine and released cytidylyltransferase into the cytosol, but did not lower diacylglycerol levels to normal values. Studies in vitro, incubations of cells with choline analogues and a survey of the literature suggested that the over-riding common factor in regulation of cytidylyltransferase binding to membranes may be the ratio of bilayer to non-bilayer lipids in that membrane.

scholarly journals Maintenance of structural and functional characteristics of skeletal-muscle mitochondria and sarcoplasmic-reticular membranes after chronic ethanol treatment

1991 ◽  
Vol 274 (2) ◽  
pp. 565-573 ◽  
Author(s):  
F Cardellach ◽  
T F Taraschi ◽  
J S Ellingson ◽  
C D Stubbs ◽  
E Rubin ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Order Parameter ◽  
Chronic Ethanol ◽  
Muscle Mitochondria ◽  
Skeletal Muscle Mitochondria ◽  
Ethanol Feeding

The effect of long-term ethanol intake on the structural and functional characteristics of rat skeletal-muscle mitochondria and sarcoplasmic reticulum was investigated. Functionally, skeletal-muscle mitochondria were characterized by a high respiratory control index and ADP/O ratio and a high State-3 respiration rate with different substrates. These parameters were not significantly different in preparations from control and ethanol-fed rats, except for a small increase in the rate of oxidation of alpha-oxoglutarate/malate in the latter. In submitochondrial particles from the two groups of animals there was no significant difference in cytochrome content, ATPase activity or the activity of respiratory-chain complexes. Mitochondrial membranes from untreated and ethanol-fed rats showed no difference in the baseline e.s.r. order parameter, and both preparations were equally sensitive to disordering by ethanol in vitro. Similarly, sarcoplasmic-reticulum preparations were not significantly affected by long-term ethanol feeding with respect to Ca2(+)-ATPase activity or in baseline order parameter and susceptibility to membrane disordering by ethanol in vitro. These membranes were also equally sensitive to degradation by exogenous phospholipase A2. Ethanol feeding did not alter the class composition of mitochondrial or sarcoplasmic-reticulum membrane phospholipids, nor the acyl composition of individual phospholipid classes. Specifically, the changes in acyl composition that characteristically occur in liver microsomal phosphatidylinositol and liver mitochondrial cardiolipin were not observed in the corresponding phospholipids from skeletal-muscle membranes. In experiments where membrane preparations from liver and skeletal muscle from the same ethanol-fed animals were compared, the liver membranes developed membrane tolerance, with the muscle membranes retaining normal sensitivity to disordering effects by ethanol. It is concluded that: (a) different tissues from the same animals differ in their susceptibility to ethanol; (b) the tissue-specific lack of development of membrane tolerance correlates with a lack of chemical changes in the phospholipids and with a retention of normal function of mitochondria and sarcoplasmic reticulum; (c) effects of chronic ethanol intake on muscle function are not due to a defect in the mitochondrial energy supply.

scholarly journals Effects of glucagon and Ca2+ on the metabolism of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in isolated rat hepatocytes and plasma membranes

1987 ◽  
Vol 241 (3) ◽  
pp. 835-845 ◽  
Author(s):  
D E Whipps ◽  
A E Armston ◽  
H J Pryor ◽  
A P Halestrap
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Rat Hepatocytes ◽  
Mitochondrial Fraction ◽  
Ruthenium Red ◽  
Isolated Rat Hepatocytes ◽  
Guanine Nucleotide Regulatory Protein ◽  
Phosphatidylinositol 4

Rat hepatocytes whose phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) had been labelled for 60 min with 32P were treated with glucagon for 10 min or phenylephrine for 2 min. Glucagon caused a 20% increase in PIP but no change in PIP2 whereas phenylephrine caused a similar increase in PIP but a 15% decrease in PIP2. Addition of both hormones together for 10 min produced a 40% increase in PIP. A crude liver mitochondrial fraction incubated with [32P]Pi and ADP incorporated label into PIP, PIP2 and phosphatidic acid. The PIP2 was shown to be in contaminating plasma membranes and PIP in both lysosomal and plasma-membrane contamination. A minor but definitely mitochondrial phospholipid, more polar than PIP2, was shown to be labelled with 32P both in vitro and in hepatocytes. The rate of 32P incorporation into PIP was faster in mitochondrial/plasma-membrane preparations from rats treated with glucagon or if 3 microM-Ca2+ and Ruthenium Red were present in the incubation buffer. Loss of 32P from membranes labelled in vitro was shown to be accompanied by formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate, and was faster in preparations from glucagon-treated rats or in the presence of 3 microM-Ca2+. It is concluded that glucagon stimulates both PIP2 phosphodiesterase and phosphatidylinositol kinase activities, as does the presence of 3 microM-Ca2+. The resulting formation of IP3 may be responsible for the observed release of intracellular Ca2+ stores. The roles of a guanine nucleotide regulatory protein and phosphorylation in mediating these effects are discussed.

Silencing of phospholipase C gamma 2 promotes proliferation of rat hepatocytes in vitro

2018 ◽  
Vol 119 (5) ◽  
pp. 4085-4096
Author(s):  
Xiaoguang Chen ◽  
Xuemin Zhu ◽  
Yumei Liu ◽  
Qiongxia Lv ◽  
Jun Ma
Keyword(s):  
Phospholipase C ◽  
Rat Hepatocytes ◽  
Phospholipase C Gamma

scholarly journals Immunohistochemical demonstration of tubulin and actin in rat hepatocytes in situ using a perfusion extraction-fixation procedure.

1985 ◽  
Vol 33 (12) ◽  
pp. 1197-1204 ◽  
Author(s):  
M Oyamada ◽  
M Mori
Keyword(s):  
Plasma Membranes ◽  
Rat Hepatocytes ◽  
Cytoskeletal Proteins ◽  
Immunohistochemical Method ◽  
Fixation Procedure ◽  
Nonionic Detergent ◽  
Cells Cultured ◽  
In Cells

There have been many studies on the localization by immunocytochemistry of cytoskeletal proteins in cells cultured in vitro. However, the distribution of cytoskeleton in cells in situ has yet to be elucidated. In the present study we developed an immunohistochemical method for visualizing tubulin and actin in rat hepatocytes in situ, using a perfusion extraction-fixation procedure, in which the liver was perfused through the portal vein with a nonionic detergent to make the plasma membranes permeable to soluble substances, followed by a fixative to preserve cytoskeletal structure. Using the immunogold and peroxidase-antiperoxidase (PAP) staining procedures, we found that in hepatocytes in situ, tubulin was localized in cytoplasmic filamentous networks and in spindle fibers, as in hepatocytes and other cells in vitro. On the other hand, the distribution of actin in hepatocytes in situ was considerably different from that in well-spread hepatocytes and other cells cultured in vitro. In hepatocytes in situ, actin did not form any stress fibers, but was distributed preferentially under the plasma membrane, especially around the bile canaliculi. The perfusion extraction-fixation procedure could be adapted to visualize cytoskeleton in other tissues.

scholarly journals A role of asialoglycoproteins for plasma-membrane-induced inhibition of the switching from α1 to β subtypes in adrenergic response during primary culture of rat hepatocytes

1996 ◽  
Vol 316 (3) ◽  
pp. 743-749 ◽  
Author(s):  
Yasuo KAJIYAMA ◽  
Yutaka SANAI ◽  
Michio UI
Keyword(s):  
Primary Culture ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Rat Hepatocytes ◽  
Prior Treatment ◽  
Adult Rats ◽  
Adult Rat ◽  
Liver Membranes ◽  
Young Rat

Adrenergic responses of rat hepatocytes were studied by measuring Ins(1,4,5)P3 (for the response via α1-subtype receptors) and cAMP (for β-subtype response) generation during brief incubation of cells with respective agonists. Hepatocytes from young rats with an age of 1 week displayed a very high β response without a significant α1 response. The β response decreased and the α1 response increased progressively as the age increased; the response was almost exclusively via α1 receptors in hepatocytes of adult rats 9 weeks or more old. The β response developed, again at the expense of the α1 response, in hepatocytes from adult rats during the primary culture at low cell densities [(1–2.5)×104 cells/cm2]. Such ‘α1 to β subtype switching’ of adrenergic responses in vitro was totally inhibited by adding plasma membranes prepared from adult rat liver into the low-cell-density culture, but not inhibited at all by membranes from young rat liver. The inhibitory effect of adult rat liver membranes was lost when the membranes had been exposed to endoglycosidase F or β-galactosidase but was not affected by prior treatment with sialidase. On the contrary, young rat liver membranes became inhibitory to ‘α1 to β subtype switching’ after prior treatment with sialidase. Thus glycoproteins with unsialylated galactosyl termini on the surface of adult rat hepatocytes are likely to function as a determinant of the relative development of α1/β subtypes of adrenergic responses; the β response is predominant in hepatocytes in the juvenile, presumably as a result of sialylation of the galactosyl termini of the functional glycoproteins.

Structural changes of spores of tree fungal pathogens after treatment with the designed antimicrobial peptide D2A21

2000 ◽  
Vol 78 (4) ◽  
pp. 462-471 ◽  
Author(s):  
D Rioux ◽  
V Jacobi ◽  
M Simard ◽  
R C Hamelin
Keyword(s):  
Plasma Membrane ◽  
Spore Germination ◽  
Fungal Pathogens ◽  
Plasma Membranes ◽  
Structural Changes ◽  
Cronartium Ribicola ◽  
Ophiostoma Ulmi ◽  
Nectria Galligena ◽  
In Vitro Effects

In vitro effects of the antimicrobial synthetic D2A21 peptide on the structure of spores of four fungal pathogens causing important tree diseases were examined by microscopy in parallel with tests to measure inhibition of spore germination. With light microscopy, the use of SYTOX®green stain indicated that the peptide rapidly altered the plasma membrane of conidia of three Ascomycetes: Gremmeniella abietina (Lagerberg) Morelet var. abietina Petrini et al., Ophiostoma ulmi (Buism.) Nannf., and Nectria galligena Bres. With basidiospores of Cronartium ribicola J.C. Fisch., a difference between control and treated spores was also found, but it was less pronounced than with conidia of the Ascomycetes. In transmission electron microscopy, untreated conidia showed typical cytoplasmic contents with the regular presence of mitochondria, ribosomes, and nuclei, at times accompanied by vacuoles of various sizes. At concentrations of the peptide inhibitory to spore germination, plasma membranes, as well as nuclear and mitochondrial envelopes, were either generally difficult to discern or were distorted and swollen. At more advanced stages, the cytoplasm of treated spores contained numerous vesicles and was in places more electron-dense than in controls. Cytoplasm leakage was also regularly observed. Present observations strongly suggest that the primary site of action of this peptide is located at the plasma membrane level.Key words: Cronartium ribicola, Gremmeniella abietina, Nectria galligena, Ophiostoma ulmi, D2A21, peptide.

scholarly journals Transverse organization of phospholipids across the bilayer of plasma-membrane subfractions of rat hepatocytes

1978 ◽  
Vol 174 (2) ◽  
pp. 563-567 ◽  
Author(s):  
J A Higgins ◽  
W H Evans
Keyword(s):  
Plasma Membrane ◽  
Phospholipase C ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Rat Hepatocytes ◽  
Functional Domains ◽  
Asymmetric Distribution

Phospholipase C treatment of vesicular subfractions of plasma membranes derived from the three functional domains of rat liver indicated that there is an asymmetric distribution of phospholipids across the bilayer of these membranes. The bile-canalicular and sinusoidal membranes were similar and different from the contiguous membrane.

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