18.5
Biochim~ca et Bju~~y~~caActa, 1124 (1992) 185-189 0 1992 Elsevier Science Publishers B.V. All rights resewed 0005-27~0/92/$05.00
BBALIP 53851
Correlation between intracellular CAMP levels and phospholipids of Microsporum gypseum Anu Bindra and G.K. Khuller department
ofBi~hemist~,
Postgraduate Institute
of Medical Education and Research, Cha~d~~arh Lindsay
(Received 14 August 1991)
Key words: Atropine; Adenylate cyclase; Intracellular cyclic-AMP; Phospholipid; (M. gypseum)
Atropine, a modulator of CAMP has been used to examine the relationship between phospholipids and intracellular levels of CAMP in Microsporum gypseum. A decreased phospholipid content was observed in atropine grown cells as a result of reduced levels of intracellular CAMP. This decline was caused by the inhibitory effect of atropine on adenylate cyclase. Lowered phospholipid content was supported by decreased [14Clacetate incorporation as well as reduced activities of key enzymes of phospholipid biosynthesis. In vitro supplementation of atropine in control cells also caused inhibition in lipid synthesis indicating similar effects of atropine and its meta~lites. These results in conjunction with our previous report, in which enhanced levels of CAMP resulted in increased phospholipid synthesis, suggest a direct correlation between phospholipid biosynthesis and intracellular levels of CAMP in M. gypseum.
Introduction
Cyclic adenosine monophosphate (CAMP) participates in many types of cellular processes in both prokaryotic and eukaryotic organisms. Usually cAMP levels change in response to the signals acting on the cell membrane, but no such signal has been identified in fungi. Therefore, CAMP function in fungi is determined only by its endogenous CAMP levels. These levels depend upon the balance between the activities of adenylate cyclase and phosphodiesterase. Out of these two enzymes, adenylate cyclase, which synthesizes cyclic AMP is thought to be a major control point of CAMP metabolism. It is a membrane bound enzyme and is known to be sensitive to its membrane lipid environment 111. Perturbations of adenylate cyclaselipid interactions can alter the activity of the enzyme [2]. But whether changes in this enzyme activity effect the lipid composition or not is not known. Various workers used CAMP analogues and phosphodiesterase inhibitors to modulate CAMP levels in mammalian cells [3,41. In our earlier communications [5,6], we reported that increased CAMP levels enhanced the lipid biosyn-
Correspondence: G.K. Khuller, Dept. Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, India.
thesis in Microsporum gypseum. However, no information is available whether a direct correlation between cAMP and lipid biosynthesis exists in this fungi or not. Therefore, a study has been initiated with atropine IC,,H,,NO,), an inhibitor of adenylate cyclase, which is known to decrease the CAMP levels. In this communication, we report that lower levels of intracellular CAMP results in decreased synthesis of lipids in M. gypseum.
Materials
and Methods
Radiolabelled {‘4C]acetate (spec. act. 56.2 mCi/ mmol), (‘4Clglycerol-3-phosphate (spec. act. 4.75 mCi/mmol), E3H]ATP (13.5 Ci/mmoI) were obtained from BARC, Bombay. 13HlGlycerol (spec. act. 2.5 Ci/mmol), [ 3Hladenosine 3’-5’-monophosphate were purchased from Amersham (U.K.). Atropine, acetyl coenzyme A and dibutyryl CAMP were obtained from Sigma (St. Louis, U.S.A.). Other chemicals used were of analytical grade. Growth of the culture M. gypseum NCPF 412 obtained from the Mycologi-
cal Reference Laboratory (School of Hygiene and Tropical Medicine, London) was grown as shake culture in Sabouraud’s broth (4% glucose, 1% peptone,
186
pH 5.4-5.6) at 27°C. Celis were harvested in the mid log phase (4 days) and processed for different studies. Pulse and chase studies
[‘4ClAcetate was used as radiolabelled precursor for these studies. To study in vitro uptake of [14C]acetate into lipids, log phase cells were harvested, washed and resuspended in Kreb’s Ringer Buffer (pH 5.4). The cell suspensions were incubated with different concentration of atropine alongwith labelled acetate for 1 h at 37°C with shaking. After 1 h, 10% TCA was added to stop the reaction. Cells were harvested, washed with normal saline, dried and processed for lipid extraction. For in vivo studies, cells were grown in the presence of atropine at its optimum cont. Cells were harvested and [ ‘“Clacetate pulse was given for different time intervals ranging from 10 to 60 min. After appropriate incubation, cells were filtered and lipids were extracted. To study the turnover of phospholipids, the labelled cells were harvested, washed and resuspended in fresh Kreb’s Ringer Buffer. The aliquots were taken at different time intervals ranging from 0 to 7 h. Phospholipids were extracted from dried cells and radioactivity incorporated was measured. ~u~nt~tatio~ of lipids
Lipids were extracted by the method of Folch et al. [7]. Phospholipids were quantitated by the method of Marinetti [S]. Individual phospholipids were separated by single dimensional thin-layer chromatography in chloroform/methanol/7 M ammonia (65 : 25 : 4, v/v) and identified as detailed earIier [9]. Total phospholipids from pulse and chase studies were seperated by thin-layer chromatography in acetone. The respective spots were scraped off the plate and transferred into vials containing toluene-based scintillation fluid for radioactivity determination. Determination of intracellular CAMP leuels
Cells were harvested in their mid log phase. After light homogenization in the cold, the cells were boiled in 2% perchloric acid for 15 min. The homogenate was cooled and centrifuged at 10000 X g for 5 min at 0-4°C. The supernatant was collected and its pH was adjusted to 7.4-7.6 with NaOH. This extract was then passed through Dowex 1 X 8 ~2~-400 mesh, Cl - form) equilibrated with water. The column was washed with 10 ml of water to remove any unabsorbed material. cAMP was selectively eluted with 0.05 M HCl and was measured by the method of Takeda et al. [lo]. Enzyme assays
The activity of adenylate cyclase was measured by the method of Salomon et al. 1111with slight modification. The labelled CAMP was purified by ZnSO,Na,CO, precipitation instead of ZnSO,-BatOH), pre-
cipitation as described in the method. Phosphodiesterase was assayed by the method of Aboud and Burger [ 121. The activity of glycerol kinase was determined by the method of Kasinathan et al. [131. For the activity of acyltransferase, the assay system used was of Okuyama et al. [14] and filter disc method of Goldfine [ 151 as modified by Van den Bosch and Vogclos [ 161 was employed. The homogenate for adenylate cyclase, phosphodiesterase and acyltransferase was prepared by harvesting and sonicating the cells at 4°C at 40 mM Tris-HCl buffer (pH 7.51. For glycerol kinase, homogenate was prepared in 10 mM Tris-HCl buffer (pH 8.0) with 250 mM sucrose. The homogenate was centrifuged at 5000 x g for 20 min to remove cell debris and the supernatant was used for various enzyme assays. Results and Discussion
The roIe of CAMP in phospholipid biosynthesis of JW. gypseum was studied by using atropine, a known effector of CAMP levels. The studies were done by both in vitro and in vivo supplementing the atropine in order to study the primary and secondary effects on CAMP on lipids. During in vitro studies, [ ‘“Cjacetate incorporation into lipids was measured in the presence of different concentrations of atropine ranging from 1.0 to 8.0 mM supplied in the incubation mixture for optimum time (1 h). There was gradual decrease in the incorporation into total lipids and total phospholipids with increasing concentration of atropine (Table I>. At 2.0 mM, 50% decrease was noticed in the incorporation into total phospholipids. Hence, this concentration was used for further studies. This decrease in incorporation into phospholipids was supported by observations of Tertov et al. [17] and Kennerly et al. [18] in which methylxanthine compounds (known modulators of CAMP levels) inhibited the incorporation of [ ‘HIacetate and .12PO, into phospholipids of aortic cells and unstimulated mast cells, respectively. After determining the optimum concentration, atropine (2.0 mM) was supplied in the growth medium for in vivo studies. Cells grown in the presence of atropine exhibited decreased content of phospholipids as compared to non”supplemented cells (Table 111. Among individual phospholipids, maximum decrease was observed in lysophosphatidylcholine fraction (P < 0.01). A significant change was atso seen in PC and PE fractions (P < 0.05) while PS, PI and unknown fractions did not exhibit any change. In order to find out the reason for the maximum decrease in LPC levels, activity of phospholipase A (enzyme that converts PC into LPC) was examined. A 46% decrease (from 32.91 to 17.53 nmol/mg protein per h) in the activity of phospholipase A was seen in atropine grown cells thus
187 TABLE 1 Effect
of different concentrations of atropine on /“C]acetate incorporation into total lipids and total phospholipids of control cells
L.og phase ceils were harvested, washed and resuspended in Kreb’s Ringer buffer (pH 5.4). Cells were incubated with different concentrations of atropine (1.0-8.0 mM) and labelled with [l-‘4C]acetate for 1 h at 37°C with shaking. Cells were filtered and lipids were extracted. Phospholipids were separated from total lipids by TLC and radioactivity incorporated was determined. Values are mean + SD. of three independent batches. * P < 0.05; * * P < 0.01. Number in parenthesis depicts % age inhibition as compared to control. Atropine tmM) Control
1.o 2.0 4.0 6.0 X.0
Acetate incorporation (CPM/mg dry wt.) (total lipids)
(total phosphoiipids)
453Oi210 3257+206 * (28.10) 2902i326 * (35.93) 1832i_ 173 * * (59.55) 1043+ 129 * * (76.97) S92& 141 * * (86.93)
1950+ 170 1349k 130 * 9175 88 * 607% 75 ** 458 + 5X * * 350:_ 42 **
confirming the decreased content of LPC in these cells. Although no information is available regarding the dependence of phospholipase on CAMP in microorganisms but phospholipase A was found to be cyclic AMP dependent in fat cells [19]. PhosphoIipid biosynthesis was also examined by pulse Iabelling using [1-‘4C]acetate as a precursor. There was decreased incorporation of labelled acetate in total phospholipids of atropine grown cells as compared to control, further substantiating the observations presented in Table II. Inhibition in the incorporation of [Me- “HIcholine into phosphatidylcholine was earlier demonstrated in rat liver in the presence of cAMP analogues and phosphodiesterase inhibitors [ZO]. Turnover of total lipids and phospholipids was also examined by chase of [l- r4C]acetate labelled cells for 7 h. There was more leakage of radioactivity from total phosphoIipids in atropine grown cells as compared to control cells (Fig. 1) which suggests the high turnover rate of phospholipids in the presence of atropine. All these experiments indicate that atropine inhibits the lipid biosynthesis and enhances the turnover rate of lipids which accounts for the significant decrease in the
I
(30.92) (53.05) (68.91) (76.55) (82.07)
I
i
I
1
2
34
I
I
56
1
1
78
(h)
TIME
Fig. 1. Chase of radioactivity in total phosphohpids of control and atropine grown cells. The labelled cells (from pulse studies) were harvested, washed and resuspended in Kreb’s Ringer Buffer. The aliquots were taken at different time intervals ranging from O-7 h and processed for lipid extraction. 0, Control cells; A, atropine grown cells.
TABLE 11 P~~~~.sphol~pid ~o~zp~sitja~fof 84. gypseum grown in the pmence of atropine at 2.0 mM co~celztrat~~tz Ceils were harvested in their mid log phase. Lipids were extracted and phospholipids were quantitated. Individual phospholipids were separated as described in Materials and Methods. Values are mean k SD. of three independent batches. * P < 0.05; * * P < 0.01; NS, not significant. L,ipid fraction (mg/g dry wt.)
Control
Atropine
Total phospholipids Lysophosphatidylcholine Phosphatidylserine + phosphatidylinositol Phosphatidylcholine Phosphatidylethanolamine Unknown phospholipids
16.45 + 0.528 1.12+0.091
13.10 f 1.319 * 0.400~0.117 **
3.79 f 0.876 5.86iO.142 3.33&0.5X 2.59 f 0.749
3.78 4.98 1.91 1.70
kO.453 kO.448 kO.485 kO.216
NS * * Ns
188 TABLE
III
ACriiitie.s ofcslMP merabolizing emymes und ~~t~~cel~ui~r tevefs of CAMP uf A4 ~~pse~~ gmwtl in the presence of atropine ut 2 mM c~/fl~ertfruii~fz L_og phase control and atropine grown cells were harvested and washed with normal saline. For enzymes, homogenate was prepared in 40 mM Tris-I-ICI buffer (pH 7.5) and estimation were done as described in Materials and Methods. CAMP was first extracted and then measured by protein binding assay as described earlier. The values are mean+S.D. of three independent batches for enzymes and Four for CAMP Jevels. * P < 0.05. NS, not significant.
Cells
Adenylate cyclase (pmol/mg protein per min)
Phosphodiesterase fnmol/mg protein per min)
net amount of phosphoIipids in atropine grown cells (Table II). To understand the mechanism of inhibition of lipid biosynthesis in the presence of atropine, intracellular CAMP levels were measured (Table III). Supplementation of atropine caused 2.3-fold fall in CAMP levels which is in good agreement with the observations of Scott and Sotomon f21] in which a&opine decreased the cAMP levels of ~~e~~~u~~~#crassa. Further to find out the reason of reduced cAMP levels, activities of CAMP metaboljzing enzymes, i.e., adenylate cyclase and phosphodiesterase were measured (Table III>. The activity of adenylate cyclase decreased by 4’7% in atropine grown cells but no significant change was observed in the activity of phosphodiesterase. These results indicate that decreased phospholipid synthesis may be due to decreased CAMP levels in the presence of atropine. A correlation between membrane phospholipid composition and total cAMP Ievels was also demonstrated by Gabrielides et al. f22f which supports our results. Cyclic AMP does not effect the metabolic pathways directly but through enzymes involved in those pathways. Therefore, activities of key enzymes of phospholipid biosynthetic pathway, i.e., glycerol kinase and glycerol-3-PO, acyltransferase were examined to find out the correlation between CAMP levels and phospholipid synthesis (Table IV). The activities of both of these enzymes decreased in the presence of atropine which suggests a link between CAMP levels and activities of these enzymes. These observations were sup-
Cyclic AMP -(nmol/g dry wt.1
ported by our earlier findings [6,23] in which increased CAMP levels enhanced these enzyme activities. Furthermore, dependence of glycerol-3-POA acyltransferase on CAMP was also shown by other workers in rat liver and human platelets [24,25] thus confirming the observations of this study. The primary effect of cAMP was studied by in vitro supplementing the cells with a&opine for I h and secondary effect was examined by growing the cehs in the presence of a&opine for 4 days. Similar effect&f, i.e., decreased synthesis of lipids were observed in both cases. It is in contrast to our earlier observations with aminophylline (an inhibitor of phosphodiesterasel wherein in vitro incubation inhibited the lipid synthesis while activation was seen in cells grown with aminophylline [6]. It is probably due to difference in the site of action of both the drugs, i.e., one effecting adenylatc cyclase (atropinef and other phosphodiesterase ~aminophylline~. It can be altuded that alterations induced in CAMP levels by changes in adenylate cyclasc were more stable (e.g., short and long term effects are simiIar> as compared to variations brought through inhibition of ph~~sphodiesterase, confirming that adenylate cyclase is the most important control point for regulating CAMP levels. Our results suggest that atropine, an inhibitor of adenylate cyclase control lipid biosynthesis by decreasing CAMP levels which in turn decrease the activities of key enzymes of phospholipid biosynthesis. It can be concluded from the results of this study alongwith our earlier report that there is a positive
TABLE IV ilcritiries c$’ key enzymes
uf~kosphQ~ip~ biu,TyF~t~es~s t@ h4. ,qypse~~ grown
itz the prmen~e qf afropine ut 2.0 n&f ~fl~cenr~f~t~u~j
Control and atropine grown cells were harvested in log phase and homogenate was prepared. The enzyme assays were done as described in Materials and Methods. The values are mean i S.D. of three independent batches. * P < 0.0.5; * * P < 0.01, Enzyme
Control (nmol/mg protein per h)
Glycerol kinase Glycerol-3-phosphateacyltransferase
10.41t1.311 17.70+ 0.894
Atropine (nmol/mg protein per h) 5.60 _t 0.855 * * t4._Wi* 1.2L *
189
correlation between intracellular CAMP levels and lipid biosynthesis in M. ~~s~u~. Acknowledgement
This work was supported by a grant from the Indian Council of Medical Research, New Delhi. References 1 Houslay, M.D. and Gordon, L.M. (1983) Curr. Top. Membr. Tramp. 18, 179-231. 2 Needham, L., Dodd, N.J.F. and Houslay, M.D. (1987) Biochim. Biophys. Acta 899, 44-50. 3 Pelech, XL., Pritchard, P.H. and Vance, D.E. (1982) Biochim. Biophys. Acta 713, 260-269. 4 Aeberhard, E.E., Scott, M.L., Barrett, C.T. and Kaplan, S.A. (1984) Biochim. Biophys. Acta 803, 29-38. 5 Vaidya, S. and Khuiler, G.K. (1988) Biochim. Biophys. Acta 960, 435-440. 6 Bindra, A. and Khuiler, G.K. (1991) Biochim. Biophys. Acta 10X1,61-64. 7 Folch, J., Lees, M. and Stanley, C.H.S. (1957) J. Biol. Chem. 226, 497-509. 8 Marinetti, G.V. (1962) J. Lipid Res. 3, l-11. 9 Khulier, G.K., Chopra, A., Bansal, V.S., Masih, R. (1981) Lipids 16, 20-22. IO Takeda, T., Kuno. T., Shuntoh, H. and Tanaka, C. (1989) J. Biochem. 105, 327-329.
1 I Salomon, Y., Londos, C. and Rodbell, M. (1974) Anal. Biochem. 58,541-548. 12 Aboud, M. and Burger, M. (1971) Biochem. Biophys. Res. Commun. 43, 174-182. 13 Kasinathan, C., Chopra, A. and Kh7uller, G.K. (1982) Lipids 17, 859-863. 14 Okuyama, H., Kankura, T. and Nojima, S. (1977) J. Biol. Chem. 252, 6682-6686. 15 Goldfine, H. (1966) J. Lipid Res. 7, 146-149. 16 Van den Bosch, H. and Vogeler. P.R. (1970) Biochim. Biophys. Acta 218, 233-248. 17 Tertov, V.V., Orekhov, A.N. and Smirnov, V.N. (1986) Atherosclerosis 62, 55-64. 18 Kennerly, D.A., Secosan, C.J., Parker, C.W. and Sullivan, T.J. 11979) J. lmmunol. 123, 1X9-1524. 19 Chiappe De Cingolani, G.E., Van den Bosch, H. and Van Deenen, L.L.M. (1972) Biochim. Biophys. Acta 260,387-392. 20 Pelech, S.L., Pritchard, P.H. and Vance, D.E. (1981) J. Biol. Chem. 256, 8283-8286. 21 Scott. W.A., Solomon, B. (1975) J. Bacterial. 122, 454-463. 22 Gabrielides, C., Zrike, J. and Scott, W.A. (1983) Arch. Microbial. 134, 10% 113. 23 Vaidya, S. and Khuller, G.K. (1989) Indian J. Biochem. Biophys. 26, 367-370. 24 Argilaga, C.S. Russell, R.L. and Heimberg, M. (1978) Arch. Biochem. and Biophys. 190, 367-372. 25 Imai, A., Hattori, H., Takahashi, M., Nakashima, S., Okano, Y., Hattori, T. and Nozawa, Y. (1984) Thrombosis Res. 35, 539-546.
Biochim~ca et Bju~~y~~caActa, 1124 (1992) 185-189 0 1992 Elsevier Science Publishers B.V. All rights resewed 0005-27~0/92/$05.00
BBALIP 53851
Correlation between intracellular CAMP levels and phospholipids of Microsporum gypseum Anu Bindra and G.K. Khuller department
ofBi~hemist~,
Postgraduate Institute
of Medical Education and Research, Cha~d~~arh Lindsay
(Received 14 August 1991)
Key words: Atropine; Adenylate cyclase; Intracellular cyclic-AMP; Phospholipid; (M. gypseum)
Atropine, a modulator of CAMP has been used to examine the relationship between phospholipids and intracellular levels of CAMP in Microsporum gypseum. A decreased phospholipid content was observed in atropine grown cells as a result of reduced levels of intracellular CAMP. This decline was caused by the inhibitory effect of atropine on adenylate cyclase. Lowered phospholipid content was supported by decreased [14Clacetate incorporation as well as reduced activities of key enzymes of phospholipid biosynthesis. In vitro supplementation of atropine in control cells also caused inhibition in lipid synthesis indicating similar effects of atropine and its meta~lites. These results in conjunction with our previous report, in which enhanced levels of CAMP resulted in increased phospholipid synthesis, suggest a direct correlation between phospholipid biosynthesis and intracellular levels of CAMP in M. gypseum.
Introduction
Cyclic adenosine monophosphate (CAMP) participates in many types of cellular processes in both prokaryotic and eukaryotic organisms. Usually cAMP levels change in response to the signals acting on the cell membrane, but no such signal has been identified in fungi. Therefore, CAMP function in fungi is determined only by its endogenous CAMP levels. These levels depend upon the balance between the activities of adenylate cyclase and phosphodiesterase. Out of these two enzymes, adenylate cyclase, which synthesizes cyclic AMP is thought to be a major control point of CAMP metabolism. It is a membrane bound enzyme and is known to be sensitive to its membrane lipid environment 111. Perturbations of adenylate cyclaselipid interactions can alter the activity of the enzyme [2]. But whether changes in this enzyme activity effect the lipid composition or not is not known. Various workers used CAMP analogues and phosphodiesterase inhibitors to modulate CAMP levels in mammalian cells [3,41. In our earlier communications [5,6], we reported that increased CAMP levels enhanced the lipid biosyn-
Correspondence: G.K. Khuller, Dept. Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, India.
thesis in Microsporum gypseum. However, no information is available whether a direct correlation between cAMP and lipid biosynthesis exists in this fungi or not. Therefore, a study has been initiated with atropine IC,,H,,NO,), an inhibitor of adenylate cyclase, which is known to decrease the CAMP levels. In this communication, we report that lower levels of intracellular CAMP results in decreased synthesis of lipids in M. gypseum.
Materials
and Methods
Radiolabelled {‘4C]acetate (spec. act. 56.2 mCi/ mmol), (‘4Clglycerol-3-phosphate (spec. act. 4.75 mCi/mmol), E3H]ATP (13.5 Ci/mmoI) were obtained from BARC, Bombay. 13HlGlycerol (spec. act. 2.5 Ci/mmol), [ 3Hladenosine 3’-5’-monophosphate were purchased from Amersham (U.K.). Atropine, acetyl coenzyme A and dibutyryl CAMP were obtained from Sigma (St. Louis, U.S.A.). Other chemicals used were of analytical grade. Growth of the culture M. gypseum NCPF 412 obtained from the Mycologi-
cal Reference Laboratory (School of Hygiene and Tropical Medicine, London) was grown as shake culture in Sabouraud’s broth (4% glucose, 1% peptone,
186
pH 5.4-5.6) at 27°C. Celis were harvested in the mid log phase (4 days) and processed for different studies. Pulse and chase studies
[‘4ClAcetate was used as radiolabelled precursor for these studies. To study in vitro uptake of [14C]acetate into lipids, log phase cells were harvested, washed and resuspended in Kreb’s Ringer Buffer (pH 5.4). The cell suspensions were incubated with different concentration of atropine alongwith labelled acetate for 1 h at 37°C with shaking. After 1 h, 10% TCA was added to stop the reaction. Cells were harvested, washed with normal saline, dried and processed for lipid extraction. For in vivo studies, cells were grown in the presence of atropine at its optimum cont. Cells were harvested and [ ‘“Clacetate pulse was given for different time intervals ranging from 10 to 60 min. After appropriate incubation, cells were filtered and lipids were extracted. To study the turnover of phospholipids, the labelled cells were harvested, washed and resuspended in fresh Kreb’s Ringer Buffer. The aliquots were taken at different time intervals ranging from 0 to 7 h. Phospholipids were extracted from dried cells and radioactivity incorporated was measured. ~u~nt~tatio~ of lipids
Lipids were extracted by the method of Folch et al. [7]. Phospholipids were quantitated by the method of Marinetti [S]. Individual phospholipids were separated by single dimensional thin-layer chromatography in chloroform/methanol/7 M ammonia (65 : 25 : 4, v/v) and identified as detailed earIier [9]. Total phospholipids from pulse and chase studies were seperated by thin-layer chromatography in acetone. The respective spots were scraped off the plate and transferred into vials containing toluene-based scintillation fluid for radioactivity determination. Determination of intracellular CAMP leuels
Cells were harvested in their mid log phase. After light homogenization in the cold, the cells were boiled in 2% perchloric acid for 15 min. The homogenate was cooled and centrifuged at 10000 X g for 5 min at 0-4°C. The supernatant was collected and its pH was adjusted to 7.4-7.6 with NaOH. This extract was then passed through Dowex 1 X 8 ~2~-400 mesh, Cl - form) equilibrated with water. The column was washed with 10 ml of water to remove any unabsorbed material. cAMP was selectively eluted with 0.05 M HCl and was measured by the method of Takeda et al. [lo]. Enzyme assays
The activity of adenylate cyclase was measured by the method of Salomon et al. 1111with slight modification. The labelled CAMP was purified by ZnSO,Na,CO, precipitation instead of ZnSO,-BatOH), pre-
cipitation as described in the method. Phosphodiesterase was assayed by the method of Aboud and Burger [ 121. The activity of glycerol kinase was determined by the method of Kasinathan et al. [131. For the activity of acyltransferase, the assay system used was of Okuyama et al. [14] and filter disc method of Goldfine [ 151 as modified by Van den Bosch and Vogclos [ 161 was employed. The homogenate for adenylate cyclase, phosphodiesterase and acyltransferase was prepared by harvesting and sonicating the cells at 4°C at 40 mM Tris-HCl buffer (pH 7.51. For glycerol kinase, homogenate was prepared in 10 mM Tris-HCl buffer (pH 8.0) with 250 mM sucrose. The homogenate was centrifuged at 5000 x g for 20 min to remove cell debris and the supernatant was used for various enzyme assays. Results and Discussion
The roIe of CAMP in phospholipid biosynthesis of JW. gypseum was studied by using atropine, a known effector of CAMP levels. The studies were done by both in vitro and in vivo supplementing the atropine in order to study the primary and secondary effects on CAMP on lipids. During in vitro studies, [ ‘“Cjacetate incorporation into lipids was measured in the presence of different concentrations of atropine ranging from 1.0 to 8.0 mM supplied in the incubation mixture for optimum time (1 h). There was gradual decrease in the incorporation into total lipids and total phospholipids with increasing concentration of atropine (Table I>. At 2.0 mM, 50% decrease was noticed in the incorporation into total phospholipids. Hence, this concentration was used for further studies. This decrease in incorporation into phospholipids was supported by observations of Tertov et al. [17] and Kennerly et al. [18] in which methylxanthine compounds (known modulators of CAMP levels) inhibited the incorporation of [ ‘HIacetate and .12PO, into phospholipids of aortic cells and unstimulated mast cells, respectively. After determining the optimum concentration, atropine (2.0 mM) was supplied in the growth medium for in vivo studies. Cells grown in the presence of atropine exhibited decreased content of phospholipids as compared to non”supplemented cells (Table 111. Among individual phospholipids, maximum decrease was observed in lysophosphatidylcholine fraction (P < 0.01). A significant change was atso seen in PC and PE fractions (P < 0.05) while PS, PI and unknown fractions did not exhibit any change. In order to find out the reason for the maximum decrease in LPC levels, activity of phospholipase A (enzyme that converts PC into LPC) was examined. A 46% decrease (from 32.91 to 17.53 nmol/mg protein per h) in the activity of phospholipase A was seen in atropine grown cells thus
187 TABLE 1 Effect
of different concentrations of atropine on /“C]acetate incorporation into total lipids and total phospholipids of control cells
L.og phase ceils were harvested, washed and resuspended in Kreb’s Ringer buffer (pH 5.4). Cells were incubated with different concentrations of atropine (1.0-8.0 mM) and labelled with [l-‘4C]acetate for 1 h at 37°C with shaking. Cells were filtered and lipids were extracted. Phospholipids were separated from total lipids by TLC and radioactivity incorporated was determined. Values are mean + SD. of three independent batches. * P < 0.05; * * P < 0.01. Number in parenthesis depicts % age inhibition as compared to control. Atropine tmM) Control
1.o 2.0 4.0 6.0 X.0
Acetate incorporation (CPM/mg dry wt.) (total lipids)
(total phosphoiipids)
453Oi210 3257+206 * (28.10) 2902i326 * (35.93) 1832i_ 173 * * (59.55) 1043+ 129 * * (76.97) S92& 141 * * (86.93)
1950+ 170 1349k 130 * 9175 88 * 607% 75 ** 458 + 5X * * 350:_ 42 **
confirming the decreased content of LPC in these cells. Although no information is available regarding the dependence of phospholipase on CAMP in microorganisms but phospholipase A was found to be cyclic AMP dependent in fat cells [19]. PhosphoIipid biosynthesis was also examined by pulse Iabelling using [1-‘4C]acetate as a precursor. There was decreased incorporation of labelled acetate in total phospholipids of atropine grown cells as compared to control, further substantiating the observations presented in Table II. Inhibition in the incorporation of [Me- “HIcholine into phosphatidylcholine was earlier demonstrated in rat liver in the presence of cAMP analogues and phosphodiesterase inhibitors [ZO]. Turnover of total lipids and phospholipids was also examined by chase of [l- r4C]acetate labelled cells for 7 h. There was more leakage of radioactivity from total phosphoIipids in atropine grown cells as compared to control cells (Fig. 1) which suggests the high turnover rate of phospholipids in the presence of atropine. All these experiments indicate that atropine inhibits the lipid biosynthesis and enhances the turnover rate of lipids which accounts for the significant decrease in the
I
(30.92) (53.05) (68.91) (76.55) (82.07)
I
i
I
1
2
34
I
I
56
1
1
78
(h)
TIME
Fig. 1. Chase of radioactivity in total phosphohpids of control and atropine grown cells. The labelled cells (from pulse studies) were harvested, washed and resuspended in Kreb’s Ringer Buffer. The aliquots were taken at different time intervals ranging from O-7 h and processed for lipid extraction. 0, Control cells; A, atropine grown cells.
TABLE 11 P~~~~.sphol~pid ~o~zp~sitja~fof 84. gypseum grown in the pmence of atropine at 2.0 mM co~celztrat~~tz Ceils were harvested in their mid log phase. Lipids were extracted and phospholipids were quantitated. Individual phospholipids were separated as described in Materials and Methods. Values are mean k SD. of three independent batches. * P < 0.05; * * P < 0.01; NS, not significant. L,ipid fraction (mg/g dry wt.)
Control
Atropine
Total phospholipids Lysophosphatidylcholine Phosphatidylserine + phosphatidylinositol Phosphatidylcholine Phosphatidylethanolamine Unknown phospholipids
16.45 + 0.528 1.12+0.091
13.10 f 1.319 * 0.400~0.117 **
3.79 f 0.876 5.86iO.142 3.33&0.5X 2.59 f 0.749
3.78 4.98 1.91 1.70
kO.453 kO.448 kO.485 kO.216
NS * * Ns
188 TABLE
III
ACriiitie.s ofcslMP merabolizing emymes und ~~t~~cel~ui~r tevefs of CAMP uf A4 ~~pse~~ gmwtl in the presence of atropine ut 2 mM c~/fl~ertfruii~fz L_og phase control and atropine grown cells were harvested and washed with normal saline. For enzymes, homogenate was prepared in 40 mM Tris-I-ICI buffer (pH 7.5) and estimation were done as described in Materials and Methods. CAMP was first extracted and then measured by protein binding assay as described earlier. The values are mean+S.D. of three independent batches for enzymes and Four for CAMP Jevels. * P < 0.05. NS, not significant.
Cells
Adenylate cyclase (pmol/mg protein per min)
Phosphodiesterase fnmol/mg protein per min)
net amount of phosphoIipids in atropine grown cells (Table II). To understand the mechanism of inhibition of lipid biosynthesis in the presence of atropine, intracellular CAMP levels were measured (Table III). Supplementation of atropine caused 2.3-fold fall in CAMP levels which is in good agreement with the observations of Scott and Sotomon f21] in which a&opine decreased the cAMP levels of ~~e~~~u~~~#crassa. Further to find out the reason of reduced cAMP levels, activities of CAMP metaboljzing enzymes, i.e., adenylate cyclase and phosphodiesterase were measured (Table III>. The activity of adenylate cyclase decreased by 4’7% in atropine grown cells but no significant change was observed in the activity of phosphodiesterase. These results indicate that decreased phospholipid synthesis may be due to decreased CAMP levels in the presence of atropine. A correlation between membrane phospholipid composition and total cAMP Ievels was also demonstrated by Gabrielides et al. f22f which supports our results. Cyclic AMP does not effect the metabolic pathways directly but through enzymes involved in those pathways. Therefore, activities of key enzymes of phospholipid biosynthetic pathway, i.e., glycerol kinase and glycerol-3-PO, acyltransferase were examined to find out the correlation between CAMP levels and phospholipid synthesis (Table IV). The activities of both of these enzymes decreased in the presence of atropine which suggests a link between CAMP levels and activities of these enzymes. These observations were sup-
Cyclic AMP -(nmol/g dry wt.1
ported by our earlier findings [6,23] in which increased CAMP levels enhanced these enzyme activities. Furthermore, dependence of glycerol-3-POA acyltransferase on CAMP was also shown by other workers in rat liver and human platelets [24,25] thus confirming the observations of this study. The primary effect of cAMP was studied by in vitro supplementing the cells with a&opine for I h and secondary effect was examined by growing the cehs in the presence of a&opine for 4 days. Similar effect&f, i.e., decreased synthesis of lipids were observed in both cases. It is in contrast to our earlier observations with aminophylline (an inhibitor of phosphodiesterasel wherein in vitro incubation inhibited the lipid synthesis while activation was seen in cells grown with aminophylline [6]. It is probably due to difference in the site of action of both the drugs, i.e., one effecting adenylatc cyclase (atropinef and other phosphodiesterase ~aminophylline~. It can be altuded that alterations induced in CAMP levels by changes in adenylate cyclasc were more stable (e.g., short and long term effects are simiIar> as compared to variations brought through inhibition of ph~~sphodiesterase, confirming that adenylate cyclase is the most important control point for regulating CAMP levels. Our results suggest that atropine, an inhibitor of adenylate cyclase control lipid biosynthesis by decreasing CAMP levels which in turn decrease the activities of key enzymes of phospholipid biosynthesis. It can be concluded from the results of this study alongwith our earlier report that there is a positive
TABLE IV ilcritiries c$’ key enzymes
uf~kosphQ~ip~ biu,TyF~t~es~s t@ h4. ,qypse~~ grown
itz the prmen~e qf afropine ut 2.0 n&f ~fl~cenr~f~t~u~j
Control and atropine grown cells were harvested in log phase and homogenate was prepared. The enzyme assays were done as described in Materials and Methods. The values are mean i S.D. of three independent batches. * P < 0.0.5; * * P < 0.01, Enzyme
Control (nmol/mg protein per h)
Glycerol kinase Glycerol-3-phosphateacyltransferase
10.41t1.311 17.70+ 0.894
Atropine (nmol/mg protein per h) 5.60 _t 0.855 * * t4._Wi* 1.2L *
189
correlation between intracellular CAMP levels and lipid biosynthesis in M. ~~s~u~. Acknowledgement
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