BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 248, 679–683 (1998) ARTICLE NO. RC989031 High Micromolar Ca 2/ beneath the Plasma Membrane in Stimulated Neutrophils Eryl V. Davies and Maurice B. Hallett Molecular Signalling Group, University Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff, CF4 4XN, United Kingdom Received June 2, 1998 Although the concentration of free Ca 2/ can be Ca 2/ near the inner face of the plasma membrane, as readily measured in the bulk cytosol (4), measurement reported by the membrane associated fluorescent Ca 2/ at the important sub-plasma membrane is more diffi- probe FFP-18, was higher than the bulk cytosolic free cult. Cell-lines, transfection to express the Ca 2/ -acti- Ca 2/ concentration both in resting neutrophils and in vated photoprotein aequorin engineered to target to the response to f-met-leu-phe. Influx caused Ca 2/ close to plasma membrane, has been used to demonstrate high the plasma membrane to rise more rapidly than the Ca 2/ beneath the plasma membrane (5, 6). However, bulk cytosolic free Ca 2/ and to reach a peak concentra- this approach is unsuitable in neutrophils, as these tion of at least 30 mM. This zone of high Ca 2/ was locali- cells have little ER and do not have significant protein sed to just beneath the plasma membrane and did not synthesis capacity (7). To our knowledge, only one pre- extend more than 0.1 mm into the cell, as it was unde- vious attempt has been made to quantify the submem- tected by the bulk cytosolic free Ca 2/ probes magfura2 brane Ca 2/ concentration in neutrophil. Total internal and fura2. From these data, reconstruction of the dis- reflection fluorescence of fluo3 (8) was used to excite tribution of Ca 2/ within the neutrophil showed that fluo3 within the neutrophils only within 0.1 mm of the the high Ca 2/ signal at the cell cortex rapidly subsided surface and thus provide a measure of near membrane to give a uniform free Ca 2/ across the cell.  1998 Academic Press Ca 2/ in this zone. This technique reported a peak fluo- Key Words: Ca 2/ signalling; neutrophils; FFP-18; rescence increase of the probe in this region only 20- membrane Ca 2/ . 60% higher than the bulk cytosolic free Ca 2/ . This would have result from a Ca 2/ concentration signifi- cantly below 30-100 mM Ca 2/ . In view of this discrep- ancy, it is thus important to determine in neutrophils whether the Ca 2/ change in the crucial sub-membrane It is well established that changes in cytosolic free Ca 2/ are responsible for triggering a number of re- zone, are only marginally higher than the bulk cytosolic free Ca 2/ measurements or whether Ca 2/ in this zone sponses in neutrophils (1). However, localisation of the Ca 2/ signal to specific locations within the cell may reaches the high micromolar concentrations required for exocytosis. also be important. For example, the crucial zone for triggering some cell surface-related responses in neu- In this paper, we have used FFP-18, an analogue of fura2 with a C18 fatty acid tail (9). It has been micro- trophils, such as pseudopod formation, oxidase activa- tion and (in cytochalasin B treated cells) exocytosis, injected into smooth muscle cells (10, 11), and endothe- lial cells (12) where it incorporates into membranes may be immediately beneath the plasma membrane. It has been proposed that in neutrophils, the cytosolic within the cell, including the plasma membrane (10- 12). We have previously used the acetoxymethyl ester free Ca 2/ at the site of exocytosis immediately under the plasma membrane may be considerably higher of FFP-18 (FFP-18-AM) to incorporate FFP-18 only the plasma membrane of neutrophils (4, 13). Exogenously than the bulk cytosolic free Ca 2/ . Exocytosis is trig- gered in neutrophils when the bulk cytosolic free Ca 2/ added FFP-18-AM initially incorporates into the outer leaflet of the plasma membrane, but as molecules reori- is above about 250 nM, yet in permeabilised neutro- phils (2) or, as shown recently by Nu ¨ ße et al. (3) in ent to face the inner surface by slow ‘‘flip-flop’’ diffusion cytosolic esterases cleave the AM ester to generate the internally perfused neutrophils, Ca 2/ concentrations in excess of 30-100 mM. are required to trigger exocytosis. Ca 2/ sensing FFP-18 on the inner face. As the hydro- philic head prevents rapid diffusion back across the This raises the question of sub-membrane Ca 2/ concen- tration during physiological stimulation. membrane, and the flow of lipid from the plasma mem- 0006-291X/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved. 679