Phospholipid degradation in hypoxic/reoxygenated cardiomyocytes in response to phospholipase C from Bacillus cereus

Forsdahl, Kirsti; Larsen, Terjes S

doi:10.1016/0022-2828(95)90039-x

Cited by 4 publications

(2 citation statements)

References 28 publications

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“…Estimation of FFA i from Total Intracellular FFA and Total Intracellular Lipid-FFA i was estimated from the concentration of total FFA transferred to the cells, as determined by uptake (Equation 2) and the concentration of total intracellular lipid, as determined by Forsdahl and Larsen (23). The estimate assumes, as in Ref, 24, that FFA i is in equilibrium with total cellular FFA, which leads to,…”

Section: Methodsmentioning

confidence: 99%

Fatty Acid (FFA) Transport in Cardiomyocytes Revealed by Imaging Unbound FFA Is Mediated by an FFA Pump Modulated by the CD36 Protein

Carley

Kleinfeld

2011

Journal of Biological Chemistry

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Regulation of FFA 3 transport across the cardiomyocyte membrane is essential for cardiovascular health and dysregulation may result in myocardial lipotoxicity (1). However, the mechanisms governing FFA transport across cell membranes are not well understood (2, 3). Proposed mechanisms include rapid flip-flop through the lipid phase of the plasma membrane (4) or a protein-mediated mechanism (2, 3, 5). Proteins reported to be correlated with FFA transport in cardiac or muscle cells include FAT/CD36, FABPpm, and FATP4 (6 -10).In previous studies of FFA transport we found virtually identical characteristics in adipocytes and preadipocytes, suggesting involvement of an unknown protein because FAT/ CD36, FABPpm, and FATP4 are expressed poorly or not at all in preadipocytes (2, 11-13). Transport in these studies was observed by imaging the concentration of intracellular unbound FFA (FFA i ) (11,12,14). This allowed us to directly monitor the movement of unbound FFA (FFA u ) from the extracellular medium and into the cytosol.Because FFA transport plays a vital role in the heart and because our adipocyte/preadipocyte results raise the possibility that novel FFA transport mechanisms might exist in other tissues, we have investigated FFA transport in cardiomyocytes isolated from wild type (WT) adult C57BL/6 mice and FAT/ CD36 Ϫ/Ϫ mice on the C57BL/6 background. We used several methods to monitor FFA transport in this study, so that key findings were confirmed independently of a specific method. These methods included 1) quantitative imaging of FFA i using a sensor of FFA, ADIFAB (acrylodan-labeled rat intestinal fatty acid-binding protein), 2) using the pH indicator BCECF-AM (2Ј,7Ј-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester) to monitor FFA influx and efflux, and 3) a new method to determine FFA uptake by monitoring the extracellular unbound FFA concentration (FFA o ) using ADIFAB in the extracellular medium.This study provides the first measurements of FFA i in cardiac cells and the first direct measurements of FFA influx and efflux across the cardiac plasma membrane. The results suggest that FFAs are pumped into cardiomyocytes and that FAT/CD36 interacts with the transporter/pump so that it significantly modifies the rate of efflux and, to a lesser extent, the rate of influx. The results significantly alter current views of the mechanisms of FFA transport in cardiac myocytes and may provide insights for understanding the role of FFA in cardiac dysregulation. EXPERIMENTAL PROCEDURES

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Section: Methodsmentioning

confidence: 99%

Fatty Acid (FFA) Transport in Cardiomyocytes Revealed by Imaging Unbound FFA Is Mediated by an FFA Pump Modulated by the CD36 Protein

Carley

Kleinfeld

2011

Journal of Biological Chemistry

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“…cereus, was chosen because of its antigenic similarity to a mammalian PC-preferring PLC. , This monomeric protein, with a molecular weight of 28.4 kDa, contains three tightly bound Zn 2+ cations essential to the enzyme activity (see the Supporting information, Figure S1). Although a large number of investigations have been conducted, for example, BC-PLC has been employed to study the mechanisms of oncogenesis and membrane degradation, , the mechanism of PLC-catalyzed lipid metabolism still remains elusive. Current findings indicate that the purified PLC only catalyzes the outer leaflet phospholipids .…”

Section: Introductionmentioning

confidence: 99%

Exploring Interfacial Hydrolysis of Artificial Neutral Lipid Monolayer and Bilayer Catalyzed by Phospholipase C

et al. 2022

Langmuir

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Phospholipase C (PLC) represents an important type of enzymes with the feature of hydrolyzing phospholipids at the position of the glycerophosphate bond, among which PLC extracted from Bacillus cereus (BC-PLC) has been extensively studied owing to its similarity to hitherto poorly characterized mammalian analogues. This study focuses on investigating the interfacial hydrolysis mechanism of phosphatidylcholine (PC) monolayer and bilayer membranes catalyzed by BC-PLC using sum frequency generation vibrational spectroscopy (SFG-VS) and laser scanning confocal microscopy (LSCM). We found that, upon interfacial hydrolysis, BC-PLC was adsorbed onto the lipid interface and catalyzed the lipolysis with no net orientation, as evidenced by the silent amide I band, indicating that ordered PLC alignment was not a prerequisite for the enzyme activity, which is very different from what we have reported for phospholipase A1 (PLA1) and phospholipase A2 (PLA2) [KaiS. Kai, S. Phys. Chem. Chem. Phys.2018206367; WangF. Wang, F. Langmuir2019351283112838; ZhangF. Zhang, F. Langmuir20203629462953]. For the PC monolayer, one of the two hydrolysates, phosphocholine, desorbed from the interface into the aqueous phase, while the other one, diacylglycerol (DG), stayed well packed with high order at the interface. For the PC bilayer, phosphocholine dispersed into the aqueous phase too, similar to the monolayer case; however, DG, presumably formed clusters with the unreacted lipid substrates and desorbed from the interface. With respect to both the monolayer and bilayer cases, mechanistic schematics were presented to illustrate the different interfacial hydrolysis processes. Therefore, this model experimental study in vitro provides significant molecular-level insights and contributes necessary knowledge to reveal the lipolysis kinetics with respect to PLC and lipid membranes with monolayer and bilayer structures.

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Phosphatidylcholine-Preferring Phospholipase C from B. cereus. Function, Structure, and Mechanism

Hergenrother

Martin

2000

Topics in Current Chemistry

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The PLC class of enzymes has been studied extensively over the past 15-20 years because of their involvement in signaling pathways in which extracellular messages are delivered to the cell to induce a response. Of the PLC isoenzymes, the PI-PLCs have perhaps been examined in the greatest detail because of their key role in initiating cellular response by hydrolyzing the phosphodiester bond of phosphatidylinositols and their phosphorylated derivatives to release the second messengers IP 3 and DAG. However, the extended release of DAG that is critical to maintaining the stimulatory response arises from hydrolysis of the more abundant phosphatidylcholine by PC-PLC or by PLD followed by phosphatidic acid phosphatase. Because no eukaryotic PC-PLC has been cloned or isolated in pure form, the phosphatidylcholine-preferring PLC from B. cereus (PLC Bc ) has emerged as a focal point for investigation and as a putative model for mammalian PC-PLCs. The similarity of the active site of PLC Bc with other phosphoryl transfer enzymes has also served as a stimulus for mechanistic studies. The present account details recent studies of this important member of the PLC superfamily of enzymes.

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Phospholipid degradation in hypoxic/reoxygenated cardiomyocytes in response to phospholipase C from Bacillus cereus

Cited by 4 publications

References 28 publications

Fatty Acid (FFA) Transport in Cardiomyocytes Revealed by Imaging Unbound FFA Is Mediated by an FFA Pump Modulated by the CD36 Protein

Fatty Acid (FFA) Transport in Cardiomyocytes Revealed by Imaging Unbound FFA Is Mediated by an FFA Pump Modulated by the CD36 Protein

Exploring Interfacial Hydrolysis of Artificial Neutral Lipid Monolayer and Bilayer Catalyzed by Phospholipase C

Phosphatidylcholine-Preferring Phospholipase C from B. cereus. Function, Structure, and Mechanism

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