scholarly journals Sphingolipid Transport to the Apical Plasma Membrane Domain in Human Hepatoma Cells Is Controlled by PKC and PKA Activity: A Correlation with Cell Polarity in HepG2 Cells

1997 ◽  
Vol 138 (2) ◽  
pp. 307-321 ◽  
Author(s):  
Mirjam M.P. Zegers ◽  
Dick Hoekstra
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Cell Polarity ◽  
Kinase Activity ◽  
Apical Membrane ◽  
Lipid Transport ◽  
Apical Plasma Membrane ◽  
Membrane Domain ◽  
Plasma Membrane Domain ◽  
Stimulation Of

The regulation of sphingolipid transport to the bile canalicular apical membrane in the well differentiated HepG2 hepatoma cells was studied. By employing fluorescent lipid analogs, trafficking in a transcytosis-dependent pathway and a transcytosis-independent (‘direct') route between the trans-Golgi network and the apical membrane were examined. The two lipid transport routes were shown to operate independently, and both were regulated by kinase activity. The kinase inhibitor staurosporine inhibited the direct lipid transport route but slightly stimulated the transcytosis-dependent route. The protein kinase C (PKC) activator phorbol-12 myristate-13 acetate (PMA) inhibited apical lipid transport via both transport routes, while a specific inhibitor of this kinase stimulated apical lipid transport. Activation of protein kinase A (PKA) had opposing effects, in that a stimulation of apical lipid transport via both transport routes was seen. Interestingly, the regulatory effects of either kinase activity in sphingolipid transport correlated with changes in cell polarity. Stimulation of PKC activity resulted in a disappearance of the bile canalicular structures, as evidenced by the redistribution of several apical markers upon PMA treatment, which was accompanied by an inhibition of apical sphingolipid transport. By contrast, activation of PKA resulted in an increase in the number and size of bile canaliculi and a concomitant enhancement of apical sphingolipid transport. Taken together, our data indicate that apical membrane-directed sphingolipid transport in HepG2 cells is regulated by kinases, which could play a role in the biogenesis of the apical plasma membrane domain.

scholarly journals Expression, Distribution, and Activity of Na+,K+-ATPase in Normal and Cholestatic Rat Liver

1998 ◽  
Vol 46 (3) ◽  
pp. 405-410 ◽  
Author(s):  
Lukas Landmann ◽  
Sabine Angermüller ◽  
Christoph Rahner ◽  
Bruno Stieger
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Driving Force ◽  
Bile Secretion ◽  
Transport Systems ◽  
Apical Plasma Membrane ◽  
Membrane Domain ◽  
Expression Domain ◽  
Duct Ligation ◽  
Plasma Membrane Domain

Hepatocellular Na+,K+-ATPase is an important driving force for bile secretion and has been localized to the basolateral plasma membrane domain. Cholestasis or impaired bile flow is known to modulate the expression, domain specificity, and activity of various transport systems involved in bile secretion. This study examined Na+,K+-ATPase after ethinylestradiol (EE) treatment and after bile duct ligation (BDL), two rat models of cholestasis. It applied quantitative immunoblotting, biochemical and cytochemical determination of enzyme activity, and immunocytochemistry to the same livers. The data showed a good correlation among the results of the different methods. Neither EE nor BDL induced alterations in the subcellular distribution of Na+,K+-ATPase, which was found in the basolateral but not in the canalicular (apical) plasma membrane domain. Protein expression and enzyme activity showed a small (~10%) decrease after EE treatment and a similar increase after BDL. These modest changes could not be detected by immunofluorescence, immuno EM, or cytochemistry. The data, therefore, demonstrate that Na+,K+-ATPase is only slightly affected by EE and BDL. They suggest that other components of the bile secretory apparatus that take effect downstream of the primary basolateral driving force may play a more prominent role in the pathogenesis of cholestasis.

Distribution of sodium transporters and aquaporin-1 in the human choroid plexus

2006 ◽  
Vol 291 (1) ◽  
pp. C59-C67 ◽  
Author(s):  
Jeppe Praetorius ◽  
Søren Nielsen
Keyword(s):  
Plasma Membrane ◽  
Choroid Plexus ◽  
Water Channel ◽  
Apical Plasma Membrane ◽  
Membrane Domain ◽  
Choroid Plexus Epithelium ◽  
Human Choroid Plexus ◽  
Fluid Production ◽  
Species Pattern ◽  
Plasma Membrane Domain

The choroid plexus epithelium secretes electrolytes and fluid in the brain ventricular lumen at high rates. Several channels and ion carriers have been identified as likely mediators of this transport in rodent choroid plexus. This study aimed to map several of these proteins to the human choroid plexus. Immunoperoxidase-histochemistry was employed to determine the cellular and subcellular localization of the proteins. The water channel, aquaporin (AQP) 1, was predominantly situated in the apical plasma membrane domain, although distinct basolateral and endothelial immunoreactivity was also observed. The Na+-K+-ATPase α1-subunit was exclusively localized apically in the human choroid plexus epithelial cells. Immunoreactivity for the Na+-K+-2Cl− cotransporter, NKCC1, was likewise confined to the apical plasma membrane domain of the epithelium. The Cl−/HCO3− exchanger, AE2, was localized basolaterally, as was the Na+-dependent Cl−/HCO3− exchanger, NCBE, and the electroneutral Na+-HCO3− cotransporter, NBCn1. No immunoreactivity was found toward the Na+-dependent acid/base transporters NHE1 or NBCe2. Hence, the human choroid plexus epithelium displays an almost identical distribution pattern of water channels and Na+ transporters as the rat and mouse choroid plexus. This general cross species pattern suggests central roles for these transporters in choroid plexus functions such as cerebrospinal fluid production.

Luminal l-alanine stimulates exocytosis at the K+-conductive apical membrane ofAplysia enterocytes

1998 ◽  
Vol 275 (5) ◽  
pp. C1300-C1312 ◽  
Author(s):  
Jerod Denton ◽  
Derek Boahene ◽  
William M. Moran
Keyword(s):  
Plasma Membrane ◽  
Apical Membrane ◽  
Fluid Phase ◽  
Electric Circuit ◽  
Apical Plasma Membrane ◽  
Luminal Surface ◽  
In Series ◽  
Microelectrode Techniques ◽  
Stimulation Of ◽  
Sustained Increase

In Aplysia intestine, stimulation of Na+ absorption with luminal alanine increases apical membrane K+ conductance ( G K,a), which presumably regulates enterocyte volume during stimulated Na+ absorption. However, the mechanism responsible for the sustained increase in plasma membrane K+ conductance is not known for any nutrient-absorbing epithelium. In the present study, we have begun to test the hypothesis that the alanine-induced increase in G K,a in Aplysia enterocytes results from exocytic insertion of K+ channels into the apical membrane. We used the fluid-phase marker horseradish peroxidase to assess the effect of alanine on apical membrane exocytosis and conventional microelectrode techniques to assess the effect of alanine on fractional capacitance of the apical membrane (f C a). Luminal alanine significantly increased apical membrane exocytosis from 1.04 ± 0.30 to 1.39 ± 0.38 ng ⋅ min−1 ⋅ cm−2. To measure f C a, we modeled the Aplysia enterocyte as a double resistance-capacitance (RC) electric circuit arranged in series. Several criteria were tested to confirm application of the model to the enterocytes, and all satisfied the model. When added to the luminal surface, alanine significantly increased f C a from 0.27 ± 0.02 to 0.33 ± 0.04 ( n = 10) after 4 min. There are two possible explanations for our findings: 1) the increase in exocytosis, which adds membrane to the apical plasma membrane, prevents plasma membrane fracture, and 2) the increase in exocytosis delivers K+ channels to the apical membrane by exocytic insertion. After the alanine-induced depolarization of apical membrane potential ( V a), there is a strong correlation ( r = 0.96) between repolarization of V a, which reflects the increase in G K,a, and increase in f C a. This correlation supports the exocytic insertion hypothesis for activation of G K,a.

scholarly journals Trace Amine-Associated Receptor 1 Localization at the Apical Plasma Membrane Domain of Fisher Rat Thyroid Epithelial Cells Is Confined to Cilia

2015 ◽  
Vol 4 (1) ◽  
pp. 30-41 ◽  
Author(s):  
Joanna Szumska ◽  
Maria Qatato ◽  
Maren Rehders ◽  
Dagmar F�hrer ◽  
Heike Biebermann ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Apical Plasma Membrane ◽  
Membrane Domain ◽  
Trace Amine ◽  
Rat Thyroid ◽  
Plasma Membrane Domain

scholarly journals Segregation of Glucosylceramide and Sphingomyelin Occurs in the Apical to Basolateral Transcytotic Route in HepG2 Cells

1997 ◽  
Vol 137 (2) ◽  
pp. 347-357 ◽  
Author(s):  
Sven C.D. van IJzendoorn ◽  
Mirjam M.P. Zegers ◽  
Jan Willem Kok ◽  
Dick Hoekstra
Keyword(s):  
Plasma Membrane ◽  
Hepg2 Cells ◽  
Accurate Estimation ◽  
Apical Plasma Membrane ◽  
Membrane Domain ◽  
Dibutyryl Camp ◽  
Preferential Localization ◽  
Basolateral Plasma Membrane ◽  
Plasma Membrane Domain ◽  
Fluorescent Lipid

HepG2 cells are highly differentiated hepatoma cells that have retained an apical, bile canalicular (BC) plasma membrane polarity. We investigated the dynamics of two BC-associated sphingolipids, glucosylceramide (GlcCer) and sphingomyelin (SM). For this, the cells were labeled with fluorescent acyl chainlabeled 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- amino]hexanoic acid (C6-NBD) derivatives of either GlcCer (C6-NBD-GlcCer) or SM (C6-NBD-SM). The pool of the fluorescent lipid analogues present in the basolateral plasma membrane domain was subsequently depleted and the apically located C6-NBD-lipid was chased at 37°C. By using fluorescence microscopical analysis and a new assay that allows an accurate estimation of the fluorescent lipid pool in the apical membrane, qualitative and quantitative insight was obtained concerning kinetics, extent and (intra)cellular sites of the redistribution of apically located C6-NBD-GlcCer and C6-NBD-SM. It is demonstrated that both lipids display a preferential localization, C6-NBD-GlcCer in the apical and C6-NBD-SM in the basolateral area. Such a preference is expressed during transcytosis of both sphingolipids from the apical to the basolateral plasma membrane domain, a novel lipid trafficking route in HepG2 cells. Whereas the vast majority of the apically derived C6-NBD-SM was rapidly transcytosed to the basolateral surface, most of the apically internalized C6-NBD-GlcCer was efficiently redirected to the BC. The redirection of C6-NBD-GlcCer did not involve trafficking via the Golgi apparatus. Evidence is provided which suggests the involvement of vesicular compartments, located subjacent to the apical plasma membrane. Interestingly, the observed difference in preferential localization of C6-NBD-GlcCer and C6NBD-SM was perturbed by treatment of the cells with dibutyryl cAMP, a stable cAMP analogue. While the preferential apical localization of C6-NBD-GlcCer was amplified, dibutyryl cAMP-treatment caused apically retrieved C6-NBD-SM to be processed via a similar pathway as that of C6-NBD-GlcCer. The data unambiguously demonstrate that segregation of GlcCer and SM occurs in the reverse transcytotic route, i.e., during apical to basolateral transport, which results in the preferential localization of GlcCer and SM in the apical and basolateral region of the cells, respectively. A role for non-Golgi–related, sub-apical vesicular compartments in the sorting of GlcCer and SM is proposed.

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