Traditionally, phospholipase activation is monitored in static incubations by the intracellular accumulation of lipidic byproducts of phospholipid hydrolysis. Thus, for phospholipase C (PLC), diacylglycerol accumulation can be assessed, as can intracellular accumulation of phosphorylated derivatives of inositol. For PLD, phosphatidic acid is often assessed, or one of its transphosphatidylation products (e.g., phosphatidylethanol) is used. For PLA2 , the intracellular accumulation of arachidonic acid (and/or lysophospholipid) is quantified. However, these approaches have several limitations in terms of both specificity and sensitivity (Table 1 ). Table 1Caveats in Use of Static Incubations to Assess Intracellular Accumulation of Lipidic Metabolites of Phospholipid Hydrolysis
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1. Metabolites can be rapidly metabolized, interconverted, or re-esterified(10). |
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2. Radiolabeling of lipid pools with lipid-soluble precursors (e.g., fatty acids) has many interpretive problems. Also, metabolically “inactive” pools of phospholipid substrates may produce high “basal levels” of lipid byproducts, and obscure acute changes induced by agonists. |
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3. Accumulation in the medium of certain constituents (e.g., H+ , released hormones) may perturb physiologic changesseeTable 2 , item 7) |
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4. Important rapid and/or phasic changes can be easily missed (11 ). |
Table 2Caveats in Using Isotopic Efflux Studies as Indices of Phospholipid Hydrolysis
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1. Exchange mechanisms (e.g., base exchange) can also lead to efflux of label. |
a. Document reduction in substrate (phospholipid) bymassmeasurements. |
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b. Demonstrate that qualitatively similar patterns of efflux occur in the absence of “chase” (butseeitem 4,below). |
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2. Lack of isotopic equilibrium (i.e., short labeling period) may limit conclusions, especially if “compartmentation” exists; provision of chase (to prevent reincorporation of label) can disturb isotopic equilibrium. |
a. Document isotopic steady state, ideally in both lipidic and aqueous compartments. |
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b.Seesolution 1b,above. |
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c. Provide mass measurements of metabolite. |
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3. Labeling of endogenous substrate may be perturbed by experimental conditions or test agents |
a. Document that labeling is unaltered by test agents. |
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b. Confirm results using exogenous substrate. |
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c. Express results asfractionalefflux rates (i.e., in terms of total labeling in lipid extracts). |
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4. Released label may be reincorporated into substrate, or adhere to tubing. |
a. Provide “chase” (butseeitems 1 and 2,above). |
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b. Concomitantly assess intracellular metabolites prior to their metabolism or reincorporation (e.g.,IPs). |
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c. Block reincorporation(10). |
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d. Block adherence by adding albumin to perifusate (including during preincubation period), or flushing line with chase. |
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5. Leakiness of cell membrane (or adherence to cells of dpm from the labeling period) can cause physiologically irrelevant efflux. |
a. Document a concurrentrise(rather than adecline) in intracellular levels of the metabolite (i.e., in the aqueous phase of cell extracts)(12). |
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b. Document integrity of cell membranes. |
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c. Eliminate bubbles in perifusion line. |
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d. Wash cells extensively after labeling (until basal efflux rates are stable). |
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6. Unanticipated metabolites may contribute to efflux of radioactivity (see alsoitem 5,above). |
a. Identify metabolites in medium (anion or cation exchange chromatography; TLC system for aqueous samples)(2). |
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b. Block interconversion or breakdown of metabolites (e. g., using Li+ )(4,5) |
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c. Document stoichiometric intracellular >generation of lipidic byproduct (e.g., diglycerides, phosphatidic acid, lysophospholipid). |
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7. Constituents of perifusion medium (e.g., albumin[3], fatty acids) may influence results. In static incubations, compounds which might influence results (e.g.,H+ , insulin[11], phospholipid metabolites) may accumulate in the medium. |
a. Maximize test conditions for all variables. |
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b. Confirm results from static incubations with dynamic incubations (perifusions). |
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8. In neurosecretory cells, unincorporated label might accumulate in secretory granules or vesicles, and be cosecreted passively. |
a. Verify findings in the presence of a secretory inhitor (one not directly altering phospholipase activation). |
