- Email: [email protected]
Manipulation of lipid composition of rat heart myocytes aged in culture and its effect on α1-adrenoceptor stimulation
Biochimica et Biophysica Acta 1348 Ž1997. 339–345
Manipulation of lipid composition of rat heart myocytes aged in culture and its effect on a 1-adrenoceptor stimulation Alessandra Bordoni a , Antonello Lorenzini a , David F. Horrobin b, PierLuigi Biagi a , Silvana Hrelia a,) a
Department of Biochemistry ‘G. Moruzzi’, UniÕersity of Bologna, Via Irnerio 48, 40126 Bologna, Italy b Efamol Research Institute, KentÕille, Canada Received 28 February 1997; revised 22 April 1997; accepted 28 April 1997
Abstract The fatty acid composition of the phosphoinositides was evaluated in cultured neonatal rat cardiomyocytes during the aging-like process in vitro, comparing data obtained from control and g-linolenic acid supplemented cardiomyocytes. The response to a 1 stimulation was evaluated in both control and supplemented cells to verify the relationship between the alterations of the phosphoinositide fatty acid composition concomitant to culture aging and the cell response to exogenous stimuli. Arachidonate level decreased as a function of age in all the phosphoinositides, which appeared to be more saturated as cells aged in culture. Inositol phosphate production in response to a 1 stimulation decreased as cells aged in culture. Supplementation of culture medium with g-linolenic acid caused significant modifications in the fatty acid pattern of the phosphoinositides, which appeared less saturated than the corresponding fractions isolated from unsupplemented cells during the aging-like process. The modifications induced by the supplementation in the phosphoinositide fatty acid composition prevented the age-related reduction of inositol phosphate production upon stimulation. These results clearly indicate a major role for the lipid composition in determining the response to a 1 stimulation, suggesting a nutritional approach to overcome some of the impairments of molecular events related to the process of aging. q 1997 Elsevier Science B.V. Keywords: Cardiomyocyte; Aging; a 1-Adrenoceptor; g-Linolenic acid; Phosphoinositide; Inositol phosphate
1. Introduction The cultured myocardial cell model offers several important advantages for biochemical studies. First, it employs pure myocardial cells that beat synchronously; second, quantitative recovery of radiolabeled metabolites is possible by extracting the my-
)
Corresponding author. Fax: q39 51 351208 or q39 51 244416; E-mail: [email protected]
ocardial cells; third, cultured cells have a higher myocardial cell content than intact heart; and fourth, the isolated myocardial cells may be exposed to constant concentrations of exogenous molecules provided by the medium. Cultured neonatal cardiomyocytes have a number of cell surface receptors which are coupled to different signaling pathways w1x. In particular, the stimulation of the a 1-adrenoceptors by the addition of different agonists to the medium activates the inositol cycle. Receptor stimulated phosphoinositide ŽPtdIns.
0005-2760r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 7 . 0 0 0 7 2 - 6
340
A. Bordoni et al.r Biochimica et Biophysica Acta 1348 (1997) 339–345
hydrolysis gives rise to two products, diacylglycerol, an activator of protein kinase C w2x, and a family of inositol phosphates ŽIPn. involved in the control of intracellular calcium w3x. PtdIns are minor phospholipids which exist in several states of phosphorylation: phosphatidylinositol ŽPI., phosphatidylinositol phosphate Ž PIP., and phosphatidylinositol bisphosphate ŽPIP2 . , which are metabolically interrelated and turn over very rapidly. Each of the PtdIns is the product of a distinct fatty acid selection mechanism w4x, and it has been shown that the different PtdIns species from rat myocardium w5,6x and brain w7x have distinctive fatty acid patterns, PIP and PIP2 being more saturated than the corresponding PI. It was previously demonstrated that the cultured myocytes undergo changes in the lipid composition as a function of culture age w8,9x. Since the control of both the biochemical and physiologic responses of the cells is receptor-mediated, it is of special interest to examine whether age-dependent alterations occur in the properties of a 1-adrenoceptors of myocyte membrane coupled with PtdIns metabolism. One of the main age-dependent alterations in the lipid composition of the myocyte cultures is an increase in linoleic and a decrease in arachidonic acid content w10x. These modifications could be correlated to a decrease in D6-desaturase activity as the cells age in culture. Such a decrease was demonstrated in heart microsomes from old rats w11x, and should be corrected by the administration of g-linolenic acid ŽGLA., the direct product of the desaturation reaction on linoleic acid, as previously demonstrated in liver microsomes of aged rats w12x. In this study we have supplemented the medium of neonatal rat cardiomyocytes with GLA, and examined the fatty acid composition of the three PtdIns classes during the aging-like process occurring in vitro, comparing data obtained from GLA supplemented cardiomyocytes with data obtained from unsupplemented cells. Furthermore, we have evaluated the cardiomyocyte response to the a 1-agonist phenylephrine ŽPhE. in young and old cultures of both control and GLA supplemented cells, in order to verify the relationship between the effects of the culture’s age and of the concomitant alteration of the PtdIns fatty acid composition and the cell response to exogenous stimuli.
2. Materials and methods 2.1. Chemicals Ham F10 was obtained from Gibco ŽScotland., horse serum, fetal calf serum and trypsin were from Boehringer Ž Mannheim, Germany. . PtdIns standards, rhodamine G and phenylephrine were purchased from Sigma ŽSt. Louis, MO. ; Dowex AG 1X8 was from BioRad Laboratories Ž Richmond, CA. ; 2-w 3 Hx myo-inositol Ž24.4 Cirmmol. was obtained from NEN Products ŽBoston, MA.. All chemicals and solvents were of analytical grade. 2.2. Cell cultures Cardiomyocytes were isolated from the ventricles of 2–4-day-old Wistar rats according to Yagev et al. w13x and grown in nutrient Ham F10 supplemented with 10% Žvrv. horse serum, 10% Žvrv. fetal calf serum Žcontrol group. and 60 m M free GLA Ž18:3Ž n–6.. ŽGLA treated group., as previously described w5,14x. To avoid possible interferences due to differences in serum composition, the same kind of sera were used for all the experiments, so that all cell cultures received the same kind and amount of nutrients, hormones and growth factors. Since GLA was dissolved in ethanol, control cells received the same ethanol concentration Ž 0.04% vrv. . GLA content of sera was negligible, as detected by gas chromatographic analysis Ždata not shown. . The cells were incubated at 378C, 5% CO 2 , 95% humidity, with a medium change every 48 h. 2.3. Morphology Changes in the morphology of cells in monolayers during the course of their aging in culture and after treatment with GLA were followed by phase contrast light microscopy ŽZeiss, Germany.. 2.4. Determination of PtdIns fatty acid pattern At days 8, 10, 15 and 17 of culture, some plates of both control and GLA treated cardiomyocytes were scraped off in ice cold methanolrHCl Ž100:1 vrv., and lipids were extracted according to Bligh and Dyer w15x. The PtdIns fractions were separated from other lipids by thin layer chromatography Ž TLC. using silica gel G high performance TLC plates
A. Bordoni et al.r Biochimica et Biophysica Acta 1348 (1997) 339–345
previously impregnated with 1% Žwrv. potassium oxalate in methanolrwater Ž2:3 vrv. and activated Ž15 min, 1008C.. The developing system was chloroform r acetoner m ethanolr acetic acidr w ater Ž40:15:13:12:8 vrv., as reported by Jolles et al. w16x. Spots of PI, PIP and PIP2 were visualized under ultraviolet light by spraying with rhodamine G Ž 0.01% wrv in methylene chloride., identified by comparison with co-chromatographed standards and scraped off. In the TLC developing system, butylated hydroxytoluene Ž0.02% wrv. was added to avoid lipid peroxidation, and preliminary experiments indicate that no lipid peroxidation occurred during the whole procedure of PtdIns separation and identification. The recovery of each fraction was higher than 90% as measured with authentic standards. All the samples were methyl esterified according to Stoffel et al. w17x and gas chromatographed on a C. Erba mod. HRGC 5160 gas chromatograph Ž Rodano, Milan, Italy. equipped with a capillary column, as previously described w5,18x. The blanks due to the TLC plates were subtracted. 2.5. Determination of PtdIns breakdown At days 7, 9, 14 and 16 of culture some plates of both control and GLA treated cardiomyocytes were radiolabeled with 2-w 3 Hx myo-inositol Ž1 m Cirml. for 24 h. At days 8, 10, 15 and 17 of culture the radiolabeled cardiomyocytes were stimulated with 30 m M PhE for 10 min in the presence of 10 mM LiCl, as previously reported w19x. Stimulations were stopped by rinsing with cold buffer; cells were scraped off in cold methanolrHCl Ž100:1 vrv. and the cellular lipid fraction separated according to Bligh and Dyer w15x. The aqueous upper phase derived from the Bligh and Dyer separation was used to evaluate the total IPn production at the maximal PhE stimulation, as reported in Ref. w19x. All data are expressed as means" S.D. of five different cell cultures, and statistical analysis was performed using Student’s t-test and one-way analysis of variance. 3. Results In order to examine alterations in the response to a 1-adrenergic stimulation during the process of aging
341
in culture, we investigated the PtdIns fatty acid composition and the PtdIns hydrolysis in younger Ž 8-dayold. and older Ž10-, 15- and 17-day-old. cardiomyocyte cultures following PhE stimulation. In our experimental conditions, after 5–8 days the cells have already recovered from the culture preparation procedure, appearing confluent with tight contacts between the cells. The myocytes after 5–8 days in culture are fully developed as functional myocytes which have completed their mitosis w9x. As suggested by Moscona-Amir et al. w9x, the major changes in cardiomyocyte lipid composition, enzyme levels, and beating rate are found to occur between 8 and 15 days in culture. In Table 1 is reported the fatty acid composition of the three PtdIns classes isolated from control and GLA treated cardiomyocytes after 8 days in culture. Independent of GLA supplementation, the fatty acid compositions of PIP and PIP2 did not resemble the composition of PI, appearing much more saturated, as previously demonstrated w5,6x. After 8 days in culture, GLA supplementation caused an increase in AA content in all the PtdIns classes; this increase Žabout 50%. was highly significant in PIP and PIP2 compared to control cells. In PI, the main change in response to GLA was a rise in 20:3Ž n–6.: this did not occur in PIP and PIP2 . Fig. 1 shows the modifications in the saturated ŽSFA. and polyunsaturated Ž PUFA. fatty acid contents of PI, PIP, and PIP2 from control and GLA treated cardiomyocytes as a function of the culture age. The SFA content increased and the PUFA content decreased with culture age in all the PtdIns classes in both control and GLA treated cells Ž P 0.001 in all conditions., but GLA treated cardiomyocytes appeared always less saturated than the corresponding untreated cells. Among the PUFAs, the major changes were detected in the AA content. Fig. 2 represents the agedependent modifications which occurred in the GLA and AA contents in the three PtdIns classes isolated from control and GLA treated cardiomyocytes. GLA relative molar content was always very low and did not significantly change during the culture period in the PI and PIP fractions either in control or in GLA treated cardiomyocytes, while a significant decrease was detected in the PIP2 fraction Ž P - 0.01 in control cells and P - 0.001 in GLA treated cells.. The car-
A. Bordoni et al.r Biochimica et Biophysica Acta 1348 (1997) 339–345
342
Table 1 Fatty acid composition Žmolr100 mol. of the three different PtdIns classes in both control and GLAtreated cardiomyocytes after 8 days in culture PI Control 14:0 15:0 16:0 16:1 17:0 18:0 18:1 18:2Ž n–6. 18:3Ž n–6. 20:3Ž n–6. 20:4Ž n–6. 22:6Ž n–3.
2.45 " 0.55 1.64 " 0.30 22.05 " 0.78 3.39 " 0.92 1.22 " 0.38 22.25 " 0.95 17.81 " 1.38 7.62 " 0.10 0.79 " 0.21 1.01 " 0.23 19.73 " 0.03 tr
PIP GLA treated 1.56 " 1.24 1.07 " 0.49 21.06 " 0.16 3.16 " 0.41 1.25 " 0.10 23.12 " 0.33 17.39 " 1.15 4.98 " 0.07 0.87 " 0.31 3.97 " 1.21 20.65 " 1.27 tr
Control
)
)
2.92 " 0.28 1.60 " 0.04 27.47 " 0.28 2.35 " 0.06 0.64 " 0.01 26.59 " 0.10 16.49 " 0.28 4.99 " 0.72 2.60 " 0.06 tr 11.17 " 0.90 2.83 " 0.16
PIP2 GLA treated )
0.38 " 0.28 0.59 " 0.04 ) 24.77 " 1.488 1.29 " 0.588 2.41 " 0.65 ) 25.28 " 0.14 ) 20.71 " 0.30 ) 5.14 " 0.16 3.03 " 0.59 tr 15.28 " 0.01 ) tr
Control
GLA treated
2.64 " 0.18 1.87 " 0.28 31.99 " 0.75 3.21 " 0.60 1.40 " 0.56 25.52 " 0.31 16.60 " 0.33 4.40 " 0.43 1.66 " 0.06 tr 8.47 " 0.24 1.07 " 0.10
2.82 " 0.13 1.76 " 0.10 31.09 " 0.94 3.66 " 0.06 2.12 " 0.29 23.06 " 0.44 16.76 " 1.21 2.87 " 0.33 2.59 " 0.01 tr 13.63 " 0.47 1.07 " 0.11
§ )
) )
)
The fatty acid composition analysis Žas methyl esters. was performed as described in Section 2. Data are means" S.D. of five different cell cultures. Statistical analysis was by Student’s t-test: § P - 0.05, 8 P - 0.01, ) P - 0.001.
diomyocyte cultures underwent major age-dependent changes in the AA relative molar content. In control cells, AA content decreased with culture age, particularly in the PI fraction in which the content of this fatty acid decreased almost linearly following aging in culture. Our approach was to treat the aging cultures with GLA in order to prevent age-related changes in the cellular lipid composition. In fact,
GLA supplementation was able to completely restore AA levels; in all the PtdIns fractions AA levels increased as the culture aged in vitro and appeared even higher than in young unsupplemented cells. Incubation of cardiomyocytes with w 3 Hxinositol led to labeling in the PtdIns, allowing the determination of both basal and PhE stimulated release of IPn. In our experimental conditions, in the presence of LiCl
Fig. 1. Modifications in SFA and PUFA content of the PtdIns classes obtained from control and GLA treated cardiomyocytes as a function of culture age. Open circles: SFAs, control; filled circles: SFAs, GLA treated; open squares: PUFAs, control; filled squares: PUFAs, GLA treated. The three PtdIns classes were separeted by T.L.C. and the SFA and PUFA content was measured by gas chromatographic analysis as reported in Section 2. Data are means" S.D. of 5 different cultures. Statistical analysis was by the one way analysis of variance comparing in each PtdIns class both the SFA and PUFA content in the different days of culture; control cardiomyocytes P - 0.001 in all cases; GLA treated cardiomyocytes P - 0.001 in all cases.
A. Bordoni et al.r Biochimica et Biophysica Acta 1348 (1997) 339–345
343
Fig. 2. Modifications in GLA and AA relative molar content of the PtdIns classes obtained from control and GLA treated cardiomyocytes as a function of culture age. Open circles: GLA, control; filled circles: GLA, GLA treated; open squares: AA, control; filled squares: AA, GLA treated. The three PtdIns classes were separeted by T.L.C. and the GLA and AA content was measured by gas chromatographic analysis as reported in Methods. Data are means" S.D. of 5 different cultures. Statistical analysis was by the one way analysis of variance comparing in each PtdIns class both the GLA and AA content in the different days of culture. PI fraction: control, AA P - 0.001, GLA treated, AA P - 0.001; PIP fraction: control, AA P - 0.001, GLA treated, AA P - 0.01; PIP2 fraction: control, GLA P - 0.01 and AA P - 0.001, GLA treated, GLA P - 0.001 and AA P - 0.001.
and after 10 min PhE stimulation, a significant accumulation of IPn was detected in both control and GLA treated cells in comparison to unstimulated cells. In Fig. 3 the IPn release following PhE stimulation is reported as a function of days of culture for
Fig. 3. Inositol phosphate release following stimulation with phenylephrine. w 3 Hx-inositol labeled cardiomyocytes were stimulated with 30 m M PhE for 10 min at 378C in the presence of 10 mM LiCl. Basal hydrolysis was determined in the absence of PhE: control cells 2160"190 cpm; GLA treated cells 2095"205 cpm independent of the day of culture. Fold stimulation is calculated as stimulatedrunstimulated IPn release. Data are means"S.D. of 5 different cultures. Statistical analysis was by the one way analysis of variance comparing IPn release in function of the culture age in the same group Žcontrol P - 0.001; GLA treated n.s.., and by the Student’s t test comparing GLA treated cells with control cells at each day of culture Žday 15 P - 0.05; day 17 P - 0.001..
the two groups of cardiomyocytes. Basal hydrolysis was determined in the absence of PhE and was similar in the two groups independent of the day of culture Ž2160 " 190 cpm for control cells and 2095 " 205 cpm in GLA treated cells.. IPn accumulation, consequent to receptor mediated PtdIns hydrolysis, is presented as fold stimulation calculated as stimulatedrunstimulated IPn release. In control cells, aging of cultures was accompanied by a marked reduction in the agonist-induced stimulation of PtdIns hydrolysis; in 8-day-old cultures, a threefold stimulation was detected in comparison to a less than twofold stimulation in the 17-day-old cultures Ž P - 0.001.. Treatment of cultures with GLA restored the age-dependent impairment in PtdIns hydrolysis in response to PhE, and no significant changes in IPn production were observed during the aging of cultures, although there was a small fall. 4. Discussion The effects of the lipid derived physical properties of membrane on its biological functions are well recognized, and the modification of the fatty acid residues may have profound effects on membrane functions. Primary cultures of beating, newborn, rat heart myocytes represent a suitable system to study the relationship between lipid composition and vari-
344
A. Bordoni et al.r Biochimica et Biophysica Acta 1348 (1997) 339–345
ous cellular functions. As these cells stop dividing after 5–8 days in culture and can be grown for long periods of time, the system can also be applied to study aging-like processes that occur in culture. Several groups have used cultures of heart cells to investigate age-related changes in the biological properties of specific receptors, such as cardiac muscarinic receptors in chick embryo w20x, and muscarinic w9x and endothelin receptors w21x in rat heart myocytes. It has been demonstrated that the aging of the cells in culture is accompanied by alterations in the biochemical responses toward specific agonists w9,20,21x. These alterations, together with modifications in the level of membrane and cytoplasmic enzymes, in the lateral mobility and organization of plasma membrane lipids and proteins, and in the beating rate of cultured myocytes are coupled to changes in the lipid composition as a function of culture age w8x. It has been demonstrated that changes in lipid composition in myocardial cells aged in culture parallel those reported upon aging in the heart of the whole animal w10x. Thus, basic aging-related processes in the cultured cell model system may also be relevant to aging in the whole animal. In this study we evaluated the changes in PtdIns fatty acid composition of neonatal rat cardiomyocytes as a function of culture age. Arachidonate level decreased as a function of age in all the three PtdIns classes, and the PtdIns fractions appeared to be more saturated as cells aged in culture. Since the control of both the biochemical and physiologic responses of the cells is receptor-mediated, it was of special interest to examine whether age-dependent alterations occur in the properties of specific myocyte membrane receptors. a 1-Adrenoceptors have an important role in the control of cardiac function and, biochemically, they are coupled with the PtdIns signaling pathway. We examined the response to a 1-adrenoceptor stimulation, evaluated as IPn production, in young vs. aged cultures of rat cardiomyocytes, and demonstrated that aging of cells in culture is accompanied by a significant decrease in IPn production. The age-dependent alterations in the response of a 1-adrenoceptor stimulation occurred concomitantly with changes in the PtdIns fatty acid composition, i.e., an increase in the SFA content and a decrease in the PUFA, and in particular AA, content.
It has been suggested that the manipulation of the lipid composition of the cultured cells may restore biochemical and physiological responses damaged by the process of aging w8,21x. Treatment with phosphatidylcholine liposomes has been shown to prevent the age-dependent changes in the response of the rat heart myocytes to the stimulation of muscarinic receptors w9x. The decreased activity of the D6-desaturase enzyme ŽD6D. , which converts linoleic acid to GLA, catalyzing the rate limiting step of the biochemical pathway leading to AA, is a peculiarity of the aging process in the liver w22x and in the heart of the whole animal w11x, and can be corrected by supplementation with GLA w12x. Cultured neonatal rat cardiomyocytes have been demonstrated to have a measurable D6D activity, which allows them to partially provide to their need of PUFA and, particularly, of AA w23x. Since arachidonate levels are important in the effectiveness of the cell responses to agonists which imply PtdIns hydrolysis, we decided to grow cardiomyocytes in a GLA enriched medium in order to prevent the age-related decline in AA level observed in the PtdIns fractions. Supplementation of culture medium with GLA caused significant modifications in the fatty acid pattern of the three PtdIns classes; in the GLA treated cardiomyocytes AA levels in the PI, PIP and PIP2 fractions of old cells were similar, or even higher, than in young cells, and the PtdIns fractions appeared less saturated as cells aged in culture than the corresponding fractions isolated from unsupplemented cells. If the changes occurring with aging are responsible for the altered response to a 1-adrenoceptor stimulation, their prevention should eliminate the aging-related changes in the response. In fact in GLA treated cardiomyocytes, the modifications induced in the PtdIns fatty acid composition prevented the age-related reduction of IPn production upon PhE stimulation. These results clearly indicate a major role for the lipid composition of cardiomyocytes in determining the response to a 1-adrenoceptor stimulation. The changes in the composition of the lipids may lead to altered localization or to changes in conformation of the a 1-adrenoceptors andror G proteins in the plasma membrane, thus having a direct effect on the coupling between the two systems. In addition, these compositional changes may be associated with the a 1-adren-
A. Bordoni et al.r Biochimica et Biophysica Acta 1348 (1997) 339–345
oceptor mediated activation of phospholipase C. Studies in rat brain w24x and GH 3 cells w25x have suggested that the arachidonate containing PIP2 is a preferred substrate for phospholipase C; thus, reduction in arachidonate could lead to attenuated IPn production. The prevention of the age-related changes in the AA levels of the PtdIns by supplementation with GLA restored the reduction in IPn production observed in old cultures. We can hypothesize that also the other product of phospholipase C hydrolysis of PtdIns, diacylglycerol, may respond to fatty acid modification, as demonstrated in cultures supplemented with docosahexaenoic acid w26x; diacylglycerols of different fatty acid composition do not equally activate protein kinase C w27,28x. In conclusion, fatty acid modifications may influence the mechanism of signal transduction by changing membrane structure, second messenger precursor pool, and second messenger generation. This suggests a nutritional approach to overcome some of the impairments of molecular events related to the process of aging. Acknowledgements We thank Christian Spano` for skilful technical assistance and Callanish Ltd. Ž Breasclete, Scotland. for the kind gift of GLA. This work was supported in part by grants from MURST 60% ŽItaly.. References w1x M. Ettaiche, P. Athias, M. Variot, J. Klepping, Can. J. Physiol. Pharmacol. 63 Ž1985. 1221–1227. w2x Y. Nishizuka, Nature 334 Ž1988. 661–665. w3x M.J. Berridge, R.F. Irvine, Nature 341 Ž1989. 197–205. w4x B.J. Holub, Nutr. Rev. 45 Ž1987. 65–71. w5x S. Hrelia, P.L. Biagi, J.M.J. Lamers, A. Bordoni, Cardioscience 3 Ž1992. 91–95.
345
w6x J.M.J. Lamers, D.H.W. Dekkers, N. Mesaeli, V. Panagia, H.A.A. van Heugten, Biochem. Biophys. Res. Commun. 191 Ž1993. 487–494. w7x J.C. Haycock, A.S. Evers, Biochim. Biophys. Acta 960 Ž1988. 54–60. w8x E. Yechiel, Y. Barenholz, J. Biol. Chem. 260 Ž1985. 9123– 9131. w9x E. Moscona-Amir, Y.I. Henis, E. Yechiel, Y. Barenholz, M. Sokolovsky, Biochemistry 25 Ž1986. 8118–8124. w10x C.G. Rogers, Lipids 9 Ž1974. 541–547. w11x J.A. Lopez Jimenez, A. Bordoni, S. Hrelia, C.A. Rossi, E. Turchetto, S. Zamora Navarro, P.L. Biagi, Biochem. Biophys. Res. Commun. 192 Ž1993. 1037–1041. w12x P.L. Biagi, A. Bordoni, S. Hrelia, M. Celadon, D.F. Horrobin, Biochim. Biophys. Acta 1083 Ž1991. 187–192. w13x S. Yagev, M. Heller, A. Pinson, In Vitro 20 Ž1984. 893–898. w14x A. Bordoni, P.L. Biagi, C.A. Rossi, S. Hrelia, Biochem. Biophys. Res. Commun. 174 Ž1991. 869–877. w15x E.G. Bligh, W.J. Dyer, Can. J. Biochem. Physiol. 37 Ž1959. 911–918. w16x J. Jolles, H. Zwiers, A. Dekker, A.A. Wirtz, W.H. Gispen, Biochem. J. 194 Ž1981. 283–291. w17x W. Stoffel, F. Chu, E.H. Ahrens Jr., Anal. Chem. 31 Ž1959. 307–311. w18x A. Bordoni, S. Hrelia, P.L. Biagi, B. Berra, Int. J. Cancer 50 Ž1992. 402–404. w19x A. Bordoni, P.L. Biagi, C.A. Rossi, S. Hrelia, Biochem. Biophys. Res. Commun. 198 Ž1994. 366–371. w20x N.M. Nathanson, J. Neurochem. 41 Ž1983. 1545–1549. w21x R. Galron, Y. Kloog, A. Bdolah, M. Sokolovsky, Eur. J. Pharmacol. 188 Ž1990. 85–88. w22x S. Hrelia, A. Bordoni, M. Celadon, E. Turchetto, P.L. Biagi, Biochem. Biophys. Res. Commun. 163 Ž1989. 348–355. w23x A. Bordoni, J.A. Lopez Jimenez, C. Spano, ` P.L. Biagi, D.F. Horrobin, S. Hrelia, Mol. Cell. Biochem. 157 Ž1996. 217– 222. w24x J.C. Haycock, A.S. Evers, Biochim. Biophys. Acta 960 Ž1988. 54–60. w25x D.T. Dudley, E. Macfarlane, A.A. Spector, Biochem. J. 216 Ž1987. 669–680. w26x A. Bordoni, P.L. Biagi, E. Turchetto, C.A. Rossi, S. Hrelia, Cardioscience 3 Ž1992. 251–255. w27x M. Go, K. Sekiguchi, H. Nomura, U. Kikkawa, Y. Nishizuka, Biochem. Biophys. Res. Commun. 144 Ž1987. 598–605. w28x S. Hrelia, P.L. Biagi, E. Turchetto, C.A. Rossi, A. Bordoni, Biochem. Biophys. Res. Commun. 183 Ž1992. 893–898.
Manipulation of lipid composition of rat heart myocytes aged in culture and its effect on a 1-adrenoceptor stimulation Alessandra Bordoni a , Antonello Lorenzini a , David F. Horrobin b, PierLuigi Biagi a , Silvana Hrelia a,) a
Department of Biochemistry ‘G. Moruzzi’, UniÕersity of Bologna, Via Irnerio 48, 40126 Bologna, Italy b Efamol Research Institute, KentÕille, Canada Received 28 February 1997; revised 22 April 1997; accepted 28 April 1997
Abstract The fatty acid composition of the phosphoinositides was evaluated in cultured neonatal rat cardiomyocytes during the aging-like process in vitro, comparing data obtained from control and g-linolenic acid supplemented cardiomyocytes. The response to a 1 stimulation was evaluated in both control and supplemented cells to verify the relationship between the alterations of the phosphoinositide fatty acid composition concomitant to culture aging and the cell response to exogenous stimuli. Arachidonate level decreased as a function of age in all the phosphoinositides, which appeared to be more saturated as cells aged in culture. Inositol phosphate production in response to a 1 stimulation decreased as cells aged in culture. Supplementation of culture medium with g-linolenic acid caused significant modifications in the fatty acid pattern of the phosphoinositides, which appeared less saturated than the corresponding fractions isolated from unsupplemented cells during the aging-like process. The modifications induced by the supplementation in the phosphoinositide fatty acid composition prevented the age-related reduction of inositol phosphate production upon stimulation. These results clearly indicate a major role for the lipid composition in determining the response to a 1 stimulation, suggesting a nutritional approach to overcome some of the impairments of molecular events related to the process of aging. q 1997 Elsevier Science B.V. Keywords: Cardiomyocyte; Aging; a 1-Adrenoceptor; g-Linolenic acid; Phosphoinositide; Inositol phosphate
1. Introduction The cultured myocardial cell model offers several important advantages for biochemical studies. First, it employs pure myocardial cells that beat synchronously; second, quantitative recovery of radiolabeled metabolites is possible by extracting the my-
)
Corresponding author. Fax: q39 51 351208 or q39 51 244416; E-mail: [email protected]
ocardial cells; third, cultured cells have a higher myocardial cell content than intact heart; and fourth, the isolated myocardial cells may be exposed to constant concentrations of exogenous molecules provided by the medium. Cultured neonatal cardiomyocytes have a number of cell surface receptors which are coupled to different signaling pathways w1x. In particular, the stimulation of the a 1-adrenoceptors by the addition of different agonists to the medium activates the inositol cycle. Receptor stimulated phosphoinositide ŽPtdIns.
0005-2760r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 7 . 0 0 0 7 2 - 6
340
A. Bordoni et al.r Biochimica et Biophysica Acta 1348 (1997) 339–345
hydrolysis gives rise to two products, diacylglycerol, an activator of protein kinase C w2x, and a family of inositol phosphates ŽIPn. involved in the control of intracellular calcium w3x. PtdIns are minor phospholipids which exist in several states of phosphorylation: phosphatidylinositol ŽPI., phosphatidylinositol phosphate Ž PIP., and phosphatidylinositol bisphosphate ŽPIP2 . , which are metabolically interrelated and turn over very rapidly. Each of the PtdIns is the product of a distinct fatty acid selection mechanism w4x, and it has been shown that the different PtdIns species from rat myocardium w5,6x and brain w7x have distinctive fatty acid patterns, PIP and PIP2 being more saturated than the corresponding PI. It was previously demonstrated that the cultured myocytes undergo changes in the lipid composition as a function of culture age w8,9x. Since the control of both the biochemical and physiologic responses of the cells is receptor-mediated, it is of special interest to examine whether age-dependent alterations occur in the properties of a 1-adrenoceptors of myocyte membrane coupled with PtdIns metabolism. One of the main age-dependent alterations in the lipid composition of the myocyte cultures is an increase in linoleic and a decrease in arachidonic acid content w10x. These modifications could be correlated to a decrease in D6-desaturase activity as the cells age in culture. Such a decrease was demonstrated in heart microsomes from old rats w11x, and should be corrected by the administration of g-linolenic acid ŽGLA., the direct product of the desaturation reaction on linoleic acid, as previously demonstrated in liver microsomes of aged rats w12x. In this study we have supplemented the medium of neonatal rat cardiomyocytes with GLA, and examined the fatty acid composition of the three PtdIns classes during the aging-like process occurring in vitro, comparing data obtained from GLA supplemented cardiomyocytes with data obtained from unsupplemented cells. Furthermore, we have evaluated the cardiomyocyte response to the a 1-agonist phenylephrine ŽPhE. in young and old cultures of both control and GLA supplemented cells, in order to verify the relationship between the effects of the culture’s age and of the concomitant alteration of the PtdIns fatty acid composition and the cell response to exogenous stimuli.
2. Materials and methods 2.1. Chemicals Ham F10 was obtained from Gibco ŽScotland., horse serum, fetal calf serum and trypsin were from Boehringer Ž Mannheim, Germany. . PtdIns standards, rhodamine G and phenylephrine were purchased from Sigma ŽSt. Louis, MO. ; Dowex AG 1X8 was from BioRad Laboratories Ž Richmond, CA. ; 2-w 3 Hx myo-inositol Ž24.4 Cirmmol. was obtained from NEN Products ŽBoston, MA.. All chemicals and solvents were of analytical grade. 2.2. Cell cultures Cardiomyocytes were isolated from the ventricles of 2–4-day-old Wistar rats according to Yagev et al. w13x and grown in nutrient Ham F10 supplemented with 10% Žvrv. horse serum, 10% Žvrv. fetal calf serum Žcontrol group. and 60 m M free GLA Ž18:3Ž n–6.. ŽGLA treated group., as previously described w5,14x. To avoid possible interferences due to differences in serum composition, the same kind of sera were used for all the experiments, so that all cell cultures received the same kind and amount of nutrients, hormones and growth factors. Since GLA was dissolved in ethanol, control cells received the same ethanol concentration Ž 0.04% vrv. . GLA content of sera was negligible, as detected by gas chromatographic analysis Ždata not shown. . The cells were incubated at 378C, 5% CO 2 , 95% humidity, with a medium change every 48 h. 2.3. Morphology Changes in the morphology of cells in monolayers during the course of their aging in culture and after treatment with GLA were followed by phase contrast light microscopy ŽZeiss, Germany.. 2.4. Determination of PtdIns fatty acid pattern At days 8, 10, 15 and 17 of culture, some plates of both control and GLA treated cardiomyocytes were scraped off in ice cold methanolrHCl Ž100:1 vrv., and lipids were extracted according to Bligh and Dyer w15x. The PtdIns fractions were separated from other lipids by thin layer chromatography Ž TLC. using silica gel G high performance TLC plates
A. Bordoni et al.r Biochimica et Biophysica Acta 1348 (1997) 339–345
previously impregnated with 1% Žwrv. potassium oxalate in methanolrwater Ž2:3 vrv. and activated Ž15 min, 1008C.. The developing system was chloroform r acetoner m ethanolr acetic acidr w ater Ž40:15:13:12:8 vrv., as reported by Jolles et al. w16x. Spots of PI, PIP and PIP2 were visualized under ultraviolet light by spraying with rhodamine G Ž 0.01% wrv in methylene chloride., identified by comparison with co-chromatographed standards and scraped off. In the TLC developing system, butylated hydroxytoluene Ž0.02% wrv. was added to avoid lipid peroxidation, and preliminary experiments indicate that no lipid peroxidation occurred during the whole procedure of PtdIns separation and identification. The recovery of each fraction was higher than 90% as measured with authentic standards. All the samples were methyl esterified according to Stoffel et al. w17x and gas chromatographed on a C. Erba mod. HRGC 5160 gas chromatograph Ž Rodano, Milan, Italy. equipped with a capillary column, as previously described w5,18x. The blanks due to the TLC plates were subtracted. 2.5. Determination of PtdIns breakdown At days 7, 9, 14 and 16 of culture some plates of both control and GLA treated cardiomyocytes were radiolabeled with 2-w 3 Hx myo-inositol Ž1 m Cirml. for 24 h. At days 8, 10, 15 and 17 of culture the radiolabeled cardiomyocytes were stimulated with 30 m M PhE for 10 min in the presence of 10 mM LiCl, as previously reported w19x. Stimulations were stopped by rinsing with cold buffer; cells were scraped off in cold methanolrHCl Ž100:1 vrv. and the cellular lipid fraction separated according to Bligh and Dyer w15x. The aqueous upper phase derived from the Bligh and Dyer separation was used to evaluate the total IPn production at the maximal PhE stimulation, as reported in Ref. w19x. All data are expressed as means" S.D. of five different cell cultures, and statistical analysis was performed using Student’s t-test and one-way analysis of variance. 3. Results In order to examine alterations in the response to a 1-adrenergic stimulation during the process of aging
341
in culture, we investigated the PtdIns fatty acid composition and the PtdIns hydrolysis in younger Ž 8-dayold. and older Ž10-, 15- and 17-day-old. cardiomyocyte cultures following PhE stimulation. In our experimental conditions, after 5–8 days the cells have already recovered from the culture preparation procedure, appearing confluent with tight contacts between the cells. The myocytes after 5–8 days in culture are fully developed as functional myocytes which have completed their mitosis w9x. As suggested by Moscona-Amir et al. w9x, the major changes in cardiomyocyte lipid composition, enzyme levels, and beating rate are found to occur between 8 and 15 days in culture. In Table 1 is reported the fatty acid composition of the three PtdIns classes isolated from control and GLA treated cardiomyocytes after 8 days in culture. Independent of GLA supplementation, the fatty acid compositions of PIP and PIP2 did not resemble the composition of PI, appearing much more saturated, as previously demonstrated w5,6x. After 8 days in culture, GLA supplementation caused an increase in AA content in all the PtdIns classes; this increase Žabout 50%. was highly significant in PIP and PIP2 compared to control cells. In PI, the main change in response to GLA was a rise in 20:3Ž n–6.: this did not occur in PIP and PIP2 . Fig. 1 shows the modifications in the saturated ŽSFA. and polyunsaturated Ž PUFA. fatty acid contents of PI, PIP, and PIP2 from control and GLA treated cardiomyocytes as a function of the culture age. The SFA content increased and the PUFA content decreased with culture age in all the PtdIns classes in both control and GLA treated cells Ž P 0.001 in all conditions., but GLA treated cardiomyocytes appeared always less saturated than the corresponding untreated cells. Among the PUFAs, the major changes were detected in the AA content. Fig. 2 represents the agedependent modifications which occurred in the GLA and AA contents in the three PtdIns classes isolated from control and GLA treated cardiomyocytes. GLA relative molar content was always very low and did not significantly change during the culture period in the PI and PIP fractions either in control or in GLA treated cardiomyocytes, while a significant decrease was detected in the PIP2 fraction Ž P - 0.01 in control cells and P - 0.001 in GLA treated cells.. The car-
A. Bordoni et al.r Biochimica et Biophysica Acta 1348 (1997) 339–345
342
Table 1 Fatty acid composition Žmolr100 mol. of the three different PtdIns classes in both control and GLAtreated cardiomyocytes after 8 days in culture PI Control 14:0 15:0 16:0 16:1 17:0 18:0 18:1 18:2Ž n–6. 18:3Ž n–6. 20:3Ž n–6. 20:4Ž n–6. 22:6Ž n–3.
2.45 " 0.55 1.64 " 0.30 22.05 " 0.78 3.39 " 0.92 1.22 " 0.38 22.25 " 0.95 17.81 " 1.38 7.62 " 0.10 0.79 " 0.21 1.01 " 0.23 19.73 " 0.03 tr
PIP GLA treated 1.56 " 1.24 1.07 " 0.49 21.06 " 0.16 3.16 " 0.41 1.25 " 0.10 23.12 " 0.33 17.39 " 1.15 4.98 " 0.07 0.87 " 0.31 3.97 " 1.21 20.65 " 1.27 tr
Control
)
)
2.92 " 0.28 1.60 " 0.04 27.47 " 0.28 2.35 " 0.06 0.64 " 0.01 26.59 " 0.10 16.49 " 0.28 4.99 " 0.72 2.60 " 0.06 tr 11.17 " 0.90 2.83 " 0.16
PIP2 GLA treated )
0.38 " 0.28 0.59 " 0.04 ) 24.77 " 1.488 1.29 " 0.588 2.41 " 0.65 ) 25.28 " 0.14 ) 20.71 " 0.30 ) 5.14 " 0.16 3.03 " 0.59 tr 15.28 " 0.01 ) tr
Control
GLA treated
2.64 " 0.18 1.87 " 0.28 31.99 " 0.75 3.21 " 0.60 1.40 " 0.56 25.52 " 0.31 16.60 " 0.33 4.40 " 0.43 1.66 " 0.06 tr 8.47 " 0.24 1.07 " 0.10
2.82 " 0.13 1.76 " 0.10 31.09 " 0.94 3.66 " 0.06 2.12 " 0.29 23.06 " 0.44 16.76 " 1.21 2.87 " 0.33 2.59 " 0.01 tr 13.63 " 0.47 1.07 " 0.11
§ )
) )
)
The fatty acid composition analysis Žas methyl esters. was performed as described in Section 2. Data are means" S.D. of five different cell cultures. Statistical analysis was by Student’s t-test: § P - 0.05, 8 P - 0.01, ) P - 0.001.
diomyocyte cultures underwent major age-dependent changes in the AA relative molar content. In control cells, AA content decreased with culture age, particularly in the PI fraction in which the content of this fatty acid decreased almost linearly following aging in culture. Our approach was to treat the aging cultures with GLA in order to prevent age-related changes in the cellular lipid composition. In fact,
GLA supplementation was able to completely restore AA levels; in all the PtdIns fractions AA levels increased as the culture aged in vitro and appeared even higher than in young unsupplemented cells. Incubation of cardiomyocytes with w 3 Hxinositol led to labeling in the PtdIns, allowing the determination of both basal and PhE stimulated release of IPn. In our experimental conditions, in the presence of LiCl
Fig. 1. Modifications in SFA and PUFA content of the PtdIns classes obtained from control and GLA treated cardiomyocytes as a function of culture age. Open circles: SFAs, control; filled circles: SFAs, GLA treated; open squares: PUFAs, control; filled squares: PUFAs, GLA treated. The three PtdIns classes were separeted by T.L.C. and the SFA and PUFA content was measured by gas chromatographic analysis as reported in Section 2. Data are means" S.D. of 5 different cultures. Statistical analysis was by the one way analysis of variance comparing in each PtdIns class both the SFA and PUFA content in the different days of culture; control cardiomyocytes P - 0.001 in all cases; GLA treated cardiomyocytes P - 0.001 in all cases.
A. Bordoni et al.r Biochimica et Biophysica Acta 1348 (1997) 339–345
343
Fig. 2. Modifications in GLA and AA relative molar content of the PtdIns classes obtained from control and GLA treated cardiomyocytes as a function of culture age. Open circles: GLA, control; filled circles: GLA, GLA treated; open squares: AA, control; filled squares: AA, GLA treated. The three PtdIns classes were separeted by T.L.C. and the GLA and AA content was measured by gas chromatographic analysis as reported in Methods. Data are means" S.D. of 5 different cultures. Statistical analysis was by the one way analysis of variance comparing in each PtdIns class both the GLA and AA content in the different days of culture. PI fraction: control, AA P - 0.001, GLA treated, AA P - 0.001; PIP fraction: control, AA P - 0.001, GLA treated, AA P - 0.01; PIP2 fraction: control, GLA P - 0.01 and AA P - 0.001, GLA treated, GLA P - 0.001 and AA P - 0.001.
and after 10 min PhE stimulation, a significant accumulation of IPn was detected in both control and GLA treated cells in comparison to unstimulated cells. In Fig. 3 the IPn release following PhE stimulation is reported as a function of days of culture for
Fig. 3. Inositol phosphate release following stimulation with phenylephrine. w 3 Hx-inositol labeled cardiomyocytes were stimulated with 30 m M PhE for 10 min at 378C in the presence of 10 mM LiCl. Basal hydrolysis was determined in the absence of PhE: control cells 2160"190 cpm; GLA treated cells 2095"205 cpm independent of the day of culture. Fold stimulation is calculated as stimulatedrunstimulated IPn release. Data are means"S.D. of 5 different cultures. Statistical analysis was by the one way analysis of variance comparing IPn release in function of the culture age in the same group Žcontrol P - 0.001; GLA treated n.s.., and by the Student’s t test comparing GLA treated cells with control cells at each day of culture Žday 15 P - 0.05; day 17 P - 0.001..
the two groups of cardiomyocytes. Basal hydrolysis was determined in the absence of PhE and was similar in the two groups independent of the day of culture Ž2160 " 190 cpm for control cells and 2095 " 205 cpm in GLA treated cells.. IPn accumulation, consequent to receptor mediated PtdIns hydrolysis, is presented as fold stimulation calculated as stimulatedrunstimulated IPn release. In control cells, aging of cultures was accompanied by a marked reduction in the agonist-induced stimulation of PtdIns hydrolysis; in 8-day-old cultures, a threefold stimulation was detected in comparison to a less than twofold stimulation in the 17-day-old cultures Ž P - 0.001.. Treatment of cultures with GLA restored the age-dependent impairment in PtdIns hydrolysis in response to PhE, and no significant changes in IPn production were observed during the aging of cultures, although there was a small fall. 4. Discussion The effects of the lipid derived physical properties of membrane on its biological functions are well recognized, and the modification of the fatty acid residues may have profound effects on membrane functions. Primary cultures of beating, newborn, rat heart myocytes represent a suitable system to study the relationship between lipid composition and vari-
344
A. Bordoni et al.r Biochimica et Biophysica Acta 1348 (1997) 339–345
ous cellular functions. As these cells stop dividing after 5–8 days in culture and can be grown for long periods of time, the system can also be applied to study aging-like processes that occur in culture. Several groups have used cultures of heart cells to investigate age-related changes in the biological properties of specific receptors, such as cardiac muscarinic receptors in chick embryo w20x, and muscarinic w9x and endothelin receptors w21x in rat heart myocytes. It has been demonstrated that the aging of the cells in culture is accompanied by alterations in the biochemical responses toward specific agonists w9,20,21x. These alterations, together with modifications in the level of membrane and cytoplasmic enzymes, in the lateral mobility and organization of plasma membrane lipids and proteins, and in the beating rate of cultured myocytes are coupled to changes in the lipid composition as a function of culture age w8x. It has been demonstrated that changes in lipid composition in myocardial cells aged in culture parallel those reported upon aging in the heart of the whole animal w10x. Thus, basic aging-related processes in the cultured cell model system may also be relevant to aging in the whole animal. In this study we evaluated the changes in PtdIns fatty acid composition of neonatal rat cardiomyocytes as a function of culture age. Arachidonate level decreased as a function of age in all the three PtdIns classes, and the PtdIns fractions appeared to be more saturated as cells aged in culture. Since the control of both the biochemical and physiologic responses of the cells is receptor-mediated, it was of special interest to examine whether age-dependent alterations occur in the properties of specific myocyte membrane receptors. a 1-Adrenoceptors have an important role in the control of cardiac function and, biochemically, they are coupled with the PtdIns signaling pathway. We examined the response to a 1-adrenoceptor stimulation, evaluated as IPn production, in young vs. aged cultures of rat cardiomyocytes, and demonstrated that aging of cells in culture is accompanied by a significant decrease in IPn production. The age-dependent alterations in the response of a 1-adrenoceptor stimulation occurred concomitantly with changes in the PtdIns fatty acid composition, i.e., an increase in the SFA content and a decrease in the PUFA, and in particular AA, content.
It has been suggested that the manipulation of the lipid composition of the cultured cells may restore biochemical and physiological responses damaged by the process of aging w8,21x. Treatment with phosphatidylcholine liposomes has been shown to prevent the age-dependent changes in the response of the rat heart myocytes to the stimulation of muscarinic receptors w9x. The decreased activity of the D6-desaturase enzyme ŽD6D. , which converts linoleic acid to GLA, catalyzing the rate limiting step of the biochemical pathway leading to AA, is a peculiarity of the aging process in the liver w22x and in the heart of the whole animal w11x, and can be corrected by supplementation with GLA w12x. Cultured neonatal rat cardiomyocytes have been demonstrated to have a measurable D6D activity, which allows them to partially provide to their need of PUFA and, particularly, of AA w23x. Since arachidonate levels are important in the effectiveness of the cell responses to agonists which imply PtdIns hydrolysis, we decided to grow cardiomyocytes in a GLA enriched medium in order to prevent the age-related decline in AA level observed in the PtdIns fractions. Supplementation of culture medium with GLA caused significant modifications in the fatty acid pattern of the three PtdIns classes; in the GLA treated cardiomyocytes AA levels in the PI, PIP and PIP2 fractions of old cells were similar, or even higher, than in young cells, and the PtdIns fractions appeared less saturated as cells aged in culture than the corresponding fractions isolated from unsupplemented cells. If the changes occurring with aging are responsible for the altered response to a 1-adrenoceptor stimulation, their prevention should eliminate the aging-related changes in the response. In fact in GLA treated cardiomyocytes, the modifications induced in the PtdIns fatty acid composition prevented the age-related reduction of IPn production upon PhE stimulation. These results clearly indicate a major role for the lipid composition of cardiomyocytes in determining the response to a 1-adrenoceptor stimulation. The changes in the composition of the lipids may lead to altered localization or to changes in conformation of the a 1-adrenoceptors andror G proteins in the plasma membrane, thus having a direct effect on the coupling between the two systems. In addition, these compositional changes may be associated with the a 1-adren-
A. Bordoni et al.r Biochimica et Biophysica Acta 1348 (1997) 339–345
oceptor mediated activation of phospholipase C. Studies in rat brain w24x and GH 3 cells w25x have suggested that the arachidonate containing PIP2 is a preferred substrate for phospholipase C; thus, reduction in arachidonate could lead to attenuated IPn production. The prevention of the age-related changes in the AA levels of the PtdIns by supplementation with GLA restored the reduction in IPn production observed in old cultures. We can hypothesize that also the other product of phospholipase C hydrolysis of PtdIns, diacylglycerol, may respond to fatty acid modification, as demonstrated in cultures supplemented with docosahexaenoic acid w26x; diacylglycerols of different fatty acid composition do not equally activate protein kinase C w27,28x. In conclusion, fatty acid modifications may influence the mechanism of signal transduction by changing membrane structure, second messenger precursor pool, and second messenger generation. This suggests a nutritional approach to overcome some of the impairments of molecular events related to the process of aging. Acknowledgements We thank Christian Spano` for skilful technical assistance and Callanish Ltd. Ž Breasclete, Scotland. for the kind gift of GLA. This work was supported in part by grants from MURST 60% ŽItaly.. References w1x M. Ettaiche, P. Athias, M. Variot, J. Klepping, Can. J. Physiol. Pharmacol. 63 Ž1985. 1221–1227. w2x Y. Nishizuka, Nature 334 Ž1988. 661–665. w3x M.J. Berridge, R.F. Irvine, Nature 341 Ž1989. 197–205. w4x B.J. Holub, Nutr. Rev. 45 Ž1987. 65–71. w5x S. Hrelia, P.L. Biagi, J.M.J. Lamers, A. Bordoni, Cardioscience 3 Ž1992. 91–95.
345
w6x J.M.J. Lamers, D.H.W. Dekkers, N. Mesaeli, V. Panagia, H.A.A. van Heugten, Biochem. Biophys. Res. Commun. 191 Ž1993. 487–494. w7x J.C. Haycock, A.S. Evers, Biochim. Biophys. Acta 960 Ž1988. 54–60. w8x E. Yechiel, Y. Barenholz, J. Biol. Chem. 260 Ž1985. 9123– 9131. w9x E. Moscona-Amir, Y.I. Henis, E. Yechiel, Y. Barenholz, M. Sokolovsky, Biochemistry 25 Ž1986. 8118–8124. w10x C.G. Rogers, Lipids 9 Ž1974. 541–547. w11x J.A. Lopez Jimenez, A. Bordoni, S. Hrelia, C.A. Rossi, E. Turchetto, S. Zamora Navarro, P.L. Biagi, Biochem. Biophys. Res. Commun. 192 Ž1993. 1037–1041. w12x P.L. Biagi, A. Bordoni, S. Hrelia, M. Celadon, D.F. Horrobin, Biochim. Biophys. Acta 1083 Ž1991. 187–192. w13x S. Yagev, M. Heller, A. Pinson, In Vitro 20 Ž1984. 893–898. w14x A. Bordoni, P.L. Biagi, C.A. Rossi, S. Hrelia, Biochem. Biophys. Res. Commun. 174 Ž1991. 869–877. w15x E.G. Bligh, W.J. Dyer, Can. J. Biochem. Physiol. 37 Ž1959. 911–918. w16x J. Jolles, H. Zwiers, A. Dekker, A.A. Wirtz, W.H. Gispen, Biochem. J. 194 Ž1981. 283–291. w17x W. Stoffel, F. Chu, E.H. Ahrens Jr., Anal. Chem. 31 Ž1959. 307–311. w18x A. Bordoni, S. Hrelia, P.L. Biagi, B. Berra, Int. J. Cancer 50 Ž1992. 402–404. w19x A. Bordoni, P.L. Biagi, C.A. Rossi, S. Hrelia, Biochem. Biophys. Res. Commun. 198 Ž1994. 366–371. w20x N.M. Nathanson, J. Neurochem. 41 Ž1983. 1545–1549. w21x R. Galron, Y. Kloog, A. Bdolah, M. Sokolovsky, Eur. J. Pharmacol. 188 Ž1990. 85–88. w22x S. Hrelia, A. Bordoni, M. Celadon, E. Turchetto, P.L. Biagi, Biochem. Biophys. Res. Commun. 163 Ž1989. 348–355. w23x A. Bordoni, J.A. Lopez Jimenez, C. Spano, ` P.L. Biagi, D.F. Horrobin, S. Hrelia, Mol. Cell. Biochem. 157 Ž1996. 217– 222. w24x J.C. Haycock, A.S. Evers, Biochim. Biophys. Acta 960 Ž1988. 54–60. w25x D.T. Dudley, E. Macfarlane, A.A. Spector, Biochem. J. 216 Ž1987. 669–680. w26x A. Bordoni, P.L. Biagi, E. Turchetto, C.A. Rossi, S. Hrelia, Cardioscience 3 Ž1992. 251–255. w27x M. Go, K. Sekiguchi, H. Nomura, U. Kikkawa, Y. Nishizuka, Biochem. Biophys. Res. Commun. 144 Ž1987. 598–605. w28x S. Hrelia, P.L. Biagi, E. Turchetto, C.A. Rossi, A. Bordoni, Biochem. Biophys. Res. Commun. 183 Ž1992. 893–898.