Role of human placental apical membrane transporters in the efflux of glyburide, rosiglitazone, and metformin

Protein kinase C (PKC) isozymes, when activated, are translocated to particulate membrane fractions for transport to the apical membrane surface in a variety of cell types. Evidence of PKC translocation was demonstrated in human megakaryoblastic leukemic cells, and in cardiac myocytes and fibroblasts, using FTTC immunofluorescent antibody labeling techniques. Recently, we reported immunogold localizations of PKC subtypes I and II in toad urinary bladder epithelia, following 60 min stimulation with Mezerein (MZ), a PKC activator, or antidiuretic hormone (ADH). Localization of isozyme subtypes I and n was carried out in separate grids using specific monoclonal antibodies with subsequent labeling with 20nm protein A-gold probes. Each PKC subtype was found to be distributed singularly and in discrete isolated patches in the cytosol as well as in the apical membrane domains. To determine if the PKC isozymes co-localized within the cell, a double immunogold labeling technique using single grids was utilized.

Download Full-text

Agonist-induced polarized trafficking and surface expression of the adenosine 2b receptor in intestinal epithelial cells: role of SNARE proteins

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00164.2004 ◽

2004 ◽

Vol 287 (5) ◽

pp. G1100-G1107 ◽

Cited By ~ 31

Author(s):

Lixin Wang ◽

Vasantha Kolachala ◽

Baljit Walia ◽

Srividya Balasubramanian ◽

Randy A. Hall ◽

...

Keyword(s):

Adenosine Receptor ◽

Intestinal Inflammation ◽

Apical Membrane ◽

Snare Proteins ◽

Cell Fractionation ◽

Camp Synthesis ◽

Agonist Stimulation ◽

A2b Receptor ◽

In Cells

Adenosine, acting through the A2b receptor, induces vectorial chloride and IL-6 secretion in intestinal epithelia and may play an important role in intestinal inflammation. We have previously shown that apical or basolateral adenosine receptor stimulation results in the recruitment of the A2b receptor to the plasma membrane. In this study, we examined domain specificity of recruitment and the role of soluble N-ethylmaleimide (NEM) attachment receptor (SNARE) proteins in the agonist-mediated recruitment of the A2b receptor to the membrane. The colonic epithelial cell line T84 was used because it only expresses the A2b-subtype adenosine receptor. Cell fractionation, biotinylation, and electron microscopic studies showed that the A2b receptor is intracellular at rest and that apical or basolateral adenosine stimulation resulted in the recruitment of the receptor to the apical membrane. Upon agonist stimulation, the A2b receptor is enriched in the vesicle fraction containing vesicle-associated membrane protein (VAMP)-2. Furthermore, in cells stimulated with apical or basolateral adenosine, we demonstrate a complex consisting of VAMP-2, soluble NEM-sensitive factor attachment protein (SNAP)-23, and A2b receptor that is coimmunoprecipitated in cells stimulated with adenosine within 5 min and is no longer detected within 15 min. Inhibition of trafficking with NEM or nocodazole inhibits cAMP synthesis induced by apical or basolateral adenosine by 98 and 90%, respectively. cAMP synthesis induced by foskolin was not affected, suggesting that generalized signaling is not affected under these conditions. Collectively, our data suggest that 1) the A2b receptor is intracellular at rest; 2) apical or basolateral agonist stimulation induces recruitment of the A2b receptor to the apical membrane; 3) the SNARE proteins, VAMP-2 and SNAP-23, participate in the recruitment of the A2b receptor; and 4) the SNARE-mediated recruitment of the A2b receptor may be required for its signaling.

Download Full-text

Bioavailability of Flavonoids: The Role of Cell Membrane Transporters

Polyphenols: Mechanisms of Action in Human Health and Disease ◽

10.1016/b978-0-12-813006-3.00022-2 ◽

2018 ◽

pp. 295-320 ◽

Cited By ~ 1

Author(s):

Jovana Čvorović ◽

Lovro Ziberna ◽

Stefano Fornasaro ◽

Federica Tramer ◽

Sabina Passamonti

Keyword(s):

Cell Membrane ◽

Membrane Transporters

Download Full-text

Role of SNAP-23 in trafficking of H+-ATPase in cultured inner medullary collecting duct cells

AJP Cell Physiology ◽

10.1152/ajpcell.2001.280.4.c775 ◽

2001 ◽

Vol 280 (4) ◽

pp. C775-C781 ◽

Cited By ~ 36

Author(s):

Abhijit Banerjee ◽

Guangmu Li ◽

Edward A. Alexander ◽

John H. Schwartz

Keyword(s):

Botulinum Toxin ◽

Apical Membrane ◽

Collecting Duct ◽

Snare Complex ◽

Attachment Protein ◽

Regulated Exocytosis ◽

Inner Medullary Collecting Duct ◽

Sensitive Factor ◽

Medullary Collecting Duct

The trafficking of H+-ATPase vesicles to the apical membrane of inner medullary collecting duct (IMCD) cells utilizes a mechanism similar to that described in neurosecretory cells involving soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) proteins. Regulated exocytosis of these vesicles is associated with the formation of SNARE complexes. Clostridial neurotoxins that specifically cleave the target (t-) SNARE, syntaxin-1, or the vesicle SNARE, vesicle-associated membrane protein-2, reduce SNARE complex formation, H+-ATPase translocation to the apical membrane, and inhibit H+ secretion. The purpose of these experiments was to characterize the physiological role of a second t-SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-23, a homologue of the neuronal SNAP-25, in regulated exocytosis of H+-ATPase vesicles. Our experiments document that 25–50 nM botulinum toxin (Bot) A or E cleaves rat SNAP-23 and thereby reduces immunodetectable and35S-labeled SNAP-23 by >60% within 60 min. Addition of 25 nM BotE to IMCD homogenates reduces the amount of the 20 S-like SNARE complex that can be immunoprecipitated from the homogenate. Treatment of intact IMCD monolayers with BotE reduces the amount of H+-ATPase translocated to the apical membrane by 52 ± 2% of control and reduces the rate of H+ secretion by 77 ± 3% after acute cell acidification. We conclude that SNAP-23 is a substrate for botulinum toxin proteolysis and has a critical role in the regulation of H+-ATPase exocytosis and H+ secretion in these renal epithelial cells.

Download Full-text

Effect of basolateral or apical hyposmolarity on apical maxi K channels of everted rat collecting tubule

AJP Renal Physiology ◽

10.1152/ajprenal.1995.268.4.f569 ◽

1995 ◽

Vol 268 (4) ◽

pp. F569-F580 ◽

Cited By ~ 14

Author(s):

L. C. Stoner ◽

G. E. Morley

Keyword(s):

Apical Membrane ◽

Physiological Role ◽

Reversal Potential ◽

K Channel ◽

Apical Surface ◽

Open Probability ◽

Volume Regulatory Decrease ◽

Collecting Tubules ◽

High Conductance

We are able to evert and perfuse rat cortical collecting tubules (CCT) at 37 degrees C. Patch-clamp techniques were used to study high-conductance potassium channels (maxi K) on the apical membrane. Under control conditions (150 mM Na+ and 5 mM K+ in pipette and bathing solutions), the slope conductance averaged 109.8 +/- 6.6 pS (12 channels), and reversal potential (expressed as pipette voltage) was +26.3 +/- 2.4 mV. The percent of time the channel spends in the open state and unitary current when voltage was clamped to 0 mV were 1.4 +/- 0.7% and 3.12 +/- 0.42 pA, respectively. In six patches voltage clamped to 0 mV, the isosmotic solution perfused through the everted tubule (basolateral surface) was exchanged for one made 70 mosmol/kgH2O hyposmotic to the control saline. Open probability increased from 0.019 to 0.258, an increase of 0.239 +/- 0.065 (P ' 0.005). In four patches where a maxi K channel was evident, no increase in open probability was observed when a hyposmotic saline was placed on the apical surface. However, when vasopressin was present on the basolateral surface, apical application of hyposmotic saline resulted in a series of bursts of channel activity. The average increase in open probability during bursts was (0.055 +/- 0.017, P < 0.005). We conclude that one function of the maxi K channel located in the apical membrane of the rat CCT may be to release intracellular solute (potassium) during a volume regulatory decrease induced by placing a dilute solution on the basolateral surface or when the apical osmolarity is reduced in the presence of vasopressin. These data are consistent with the hypothesis that the physiological role of the channel is to regulate cell volume during water reabsorption.

Download Full-text

Role of NHE isoforms in mediating bicarbonate reabsorption along the nephron

AJP Renal Physiology ◽

10.1152/ajprenal.2001.281.6.f1117 ◽

2001 ◽

Vol 281 (6) ◽

pp. F1117-F1122 ◽

Cited By ~ 55

Author(s):

Tong Wang ◽

Max Hropot ◽

Peter S. Aronson ◽

Gerhard Giebisch

Keyword(s):

Proximal Tubule ◽

Functional Role ◽

Apical Membrane ◽

Rat Kidney ◽

Significant Inhibition ◽

Distal Convoluted Tubule ◽

Loop Of Henle ◽

Nhe Isoforms

This study assessed the functional role of Na+/H+ exchanger (NHE) isoforms NHE3 and NHE2 in the proximal tubule, loop of Henle, and distal convoluted tubule of the rat kidney by comparing sensitivity of transport to inhibition by Hoe-694 (an agent known to inhibit NHE2 but not NHE3) and S-3226 (an agent with much higher affinity for NHE3 than NHE2). Rates of transport of fluid ( J v) and HCO[Formula: see text]( J HCO3) were studied by in situ microperfusion. In the proximal tubule, addition of ethylisopropylamiloride or S-3226 significantly reduced J v and J HCO3, but addition of Hoe-694 caused no significant inhibition. In the loop of Henle, J HCO3 was also inhibited by S-3226 and not by Hoe-694, although much higher concentrations of S-3226 were required than what was necessary to inhibit transport in the proximal tubule. In contrast, in the distal convoluted tubule, J HCO3was inhibited by Hoe-694 but not by S-3226. These results are consistent with the conclusion that NHE2 rather than NHE3 is the predominant isoform responsible for apical membrane Na+/H+ exchange in the distal convoluted tubule, whereas NHE3 is the predominant apical isoform in the proximal tubule and possibly also in the loop of Henle.

Download Full-text

Role of intestinal sterol transporters Abcg5, Abcg8, and Npc1l1 in cholesterol absorption in mice: gender and age effects

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00172.2005 ◽

2006 ◽

Vol 290 (2) ◽

pp. G269-G276 ◽

Cited By ~ 54

Author(s):

Li-Ping Duan ◽

Helen H. Wang ◽

Akira Ohashi ◽

David Q.-H. Wang

Keyword(s):

Molecular Mechanisms ◽

Apical Membrane ◽

Cholesterol Absorption ◽

Absorption Efficiency ◽

Biliary Cholesterol ◽

Intestinal Cholesterol Absorption ◽

Gender And Age ◽

Multistep Process ◽

17Β Estradiol

Recent studies have indicated that intestinal cholesterol absorption is a multistep process, which is regulated by multiple genes at the enterocyte level. However, the molecular mechanisms whereby there are gender differences in intestinal cholesterol absorption efficiency and the efficiency of cholesterol absorption increases with age have not yet been fully understood. To explore whether aging increases cholesterol absorption via intestinal sterol transporters, we studied the higher cholesterol-absorbing C57L/J vs. the lower cholesterol-absorbing AKR/J mice at 8 (young adult), 36 (older adult), and 50 (aged) wk of age. To test the hypothesis that estrogen receptor (ER )α plays an important regulatory role in cholesterol absorption, we investigated the gonadectomized mice of both genders treated with 17β-estradiol-releasing pellets at 0, 3, or 6 μg/day and antiestrogenic ICI 182,780 at 125 μg/day. We found that hepatic outputs of biliary cholesterol were significantly increased with age and in response to high levels of estrogen. Aging significantly enhances cholesterol absorption by suppressing expression of the jejunal and ileal sterol efflux transporters [ATP-binding cassette ( Abc) g5 and Abcg8] and upregulating expression of the putative duodenal and jejunal sterol influx transporter Npc1l1. Estrogen significantly augmented cholesterol absorption mostly due to an upregulated expression of intestinal Npc1l1, Abcg5, and Abcg8 via the intestinal ERα pathway, which can be fully abolished by the antagonist. We conclude that ERα activated by estrogen and aging enhances cholesterol absorption by increasing biliary lipid output and mediating intestinal sterol transporters favoring influx of intraluminal cholesterol molecules across the apical membrane of the enterocyte.

Download Full-text