727 Effect of Phosphatidylcholine Structure on the Adenylate Cyclase Activity of a Murine Fibroblast Cell Line Lido Calorini, Gabriele Mugnai, Antonella Mannini and Salvatore Ruggieri* Institute of General Pathology, University of F~orence, 50134 Florence, Italy ~Ib determine which structural characteristics of membrane phosphollpids influence adenylate cyclase activity, we mea- sured basal and sodium fluoride- or forskolin~stimulated activity in a routine fibroblast cell llne, L~, Balb/c3T3 cells grown in media supplemented with fetal calf serum (FCS), lipid.depleted FCS (LD-FCS) or LD-FCS complexed with different phosphatidylchollne (PC) molecular species. Cells grown in the presence of L1~FCS showed a substantial decrease in their basal and NaF~timulated adenylate cy- clase activities; however, their forskolin~stimulated activity was not altered, suggesting that the enzyme's catalytic site is not affected by changes in membrane lipids. Media sup- plemented with different LD-FCS/PC complexes were shown to prevent the LI~FC~mediated reduction of basal and NaF~stimulated adenylate cyclase activity to different extents. Addition of cis-9-16:11cis-9-16:l, cis-9-18:11cis-9-18:l or cis-9-18:11cis-9,12-18:2 sn-glycerophosphocholine (GPC) completely restored adenylate cyclase activity, while c/s- 11-18:1/c/~11-18:1 GPC was not effective and only a partial recovery was observed with 16:0/16:0, 16:0/c/~18:1 and trans.9-18:1 GPC. Considering the structural features of these seven PC molecular species, the findings suggest that an optimal lipid environment is conferred to the en- zyme by the presence of two c/s double bonds, each located in A9 position of the PC acyl chains. The limited effect of cis-9-16:llcis-9-18:1 GPC and cis-9-18:11cis-9-16:l GPC sug- gests that an equal length of the terminal hydrocarbon chains extending beyond the A9 double bonds is also im- portant. Moreover, complete restoration of adenylate cy- clase activity in cells exposed to 16.~/c/s~9,12-18:2 GPC sug- gests that two c/s-9,12 double bonds located on the same chain are as effective as two c/s-9 double bonds each located on two different chains of PC. As the four double bonds of 16:0/c/s-5,8,11,14-20:4 GPC had no effect, a mere increase in the number of double bonds seems insufficient to build an optimal lipid microenvironment for the enzym~ Lipids 28, 727-730 (1993). Adenylate cyclase [ATP pyrophosphate lyase (cyclizing), EC 4.6.1.1] is a membrane-associated enzyme which, by produc- ing the second messenger cAMP, affects various cellular physiological activities (1). The dependence of adenylate cyclase on lipids has been shown by a number of studies, including dietary manipulation (2-11), treatment with phos- pholipases (12-17), and incorporation into cell membranes of exogenous fatty acids (18-23), intact phospholipid mole- cules (24-32) or variations in phospholipid polar head groups (19,20,33). We feel that, under physiological conditions, the activity of adenylate cyclase may well be affected by the phospholipid molecular species present in the membranes (34). This relationship, has only been explored in a few in- *To whom correspondence should be addressed at the Institute of General Pathology, Viale G.B. Morgagui 50, 50134 Florence, Italy. Abbreviations: DMEM, Dulbecco's modified Minimal Essential Medium; FCS, fetal calf serum; GPC, sn-glycerm3-phosphocholine; HPLC, high-performance liquid chromatography; LD-FCS, lipid- depletedfetal calfserum; PC, phosphatidylcholine;PMV,plasma mem- brane vesicles; R-FCS, reconstituted fetal calf serum. stances (28-32), and only for a limited number of molecular species. In the present study we determined to what extent phos- phatidylcholines (PC) with a defined acyl chain composition affect the reduction of the adenylate cyclase activity which occurs in cell cultures grown in media supplemented with lipid-free serum (22). Adenylate cyclase activity was mea- sured in a murine fibroblast cell line La, Balb/c3T3 cells grown in media supplemented with fetal calf serum (FCS), lipid<lepleted FCS (LD-FCS), or in LD-FCS complexed with a series of PC which differed in the length of their acyl chains and in the number, location and configuration of their double bonds. We measured the basal activity of adenylate cyclase as well as the activity stimulated by sodium fluoride or forskolin to explore the G, protein and the catalytic unit of the enzyme respectively (35). Using this protocol we determined which structural features of the phospholipids are important to restore the activities of the regulatory and/or catalytic unit of the adenylate cyclase complex. MATERIALS AND METHODS Synthesis of PC Symmetric PC, dipalmitoyl (16:0/16:0), dipalmitoleoyl (cis-9-16:llcis-9-16:l ), dioleoyl (cis-9-18:llcis- 9-18:1), divaccenoyl (cis-11-18:llcis-11-18:1) and dielaidoyl (trans-9-18:lltrans-9-18:l) s n-glycero-3-phosphocholines (GPC) were synthesized by reaction of fatty acid anhy- drides with the cadmium chloride/GPC complex in the presence of 4-pyrrolidinopyridine as catalyst (36). Asym- metric PC molecules, namely 1-palmitoyl-2-oleoyl (16:01cis- 9-18:1), 1-palmitoleoyl-2-oleoyl (cis-9-16:licis-9-18:l), 1- oleoyl-2-palmitoleoyl (cis-9-18:1/cis-9-16:1), 1-palmitoyl-2- linoleoyl (16:01cis-9,12-18:2) and 1-oleoyl-2-linoleoyl (cis-9- 18:11cis-9,12-18:2) GPC, were synthesized by submitting the respective symmetric PC (16:0/16:0, cis-9-16:llcis-9- 16:1, cis-9-18:llcis-9-18:l GPC) to phospholipase A= hy- drolysis, followed by acylation of the 1-acyl-GPC in posi- tion 2 with the appropriate fatty acid anhydrides, using 4-pyrrolidinopyridine as catalyst (36). 1-Palmitoyl-2-ara- chidonoyl (16:01cis-5,8,11,14-20:4) GPC was supplied by Avanti Polar Lipids (Alabaster, AL). All PC used in this study were free of hydroperoxides and hydrolysis products, as was shown by high-performance liquid chromato- graphic (HPLC) analysis (37,38). Preparation of LD-FCS, reconstituted FCS and LD- FCS/PC complexes. LD-FCS was prepared by successive extractions of FCS with an ethanol/diethyl ether (3:1, vol]vol) mixture at -25 ~ (39). Lipids extracted from FCS were solubilized in a small volume of chloroform and mixed with dry LD-FCS in order to obtain the reconsti- tuted FCS (R-FCS). Individual PC (200 ;~g per mL of serum) together with cholesterol (100 ~g per mL of serum) were also solubilized in a small volume of chloroform and mixed with dry LD-FCS in order to obtain the various LD- FCS/PC complexes. The dry LD-FCS, R-FCS and LD- FCS/PC complexes were solubilized with distilled water under sonication and brought to the original volume of FCS that had been submitted to lipid extraction. The con- centrations of each PC molecular species and cholesterol Copyright 9 1993 by the American Oil Chemists' Society LIPIDS, Vol. 28, no. 8 (1993)