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Phospholipase D in Tetrahymena: activity and significance
Comparative Biochemistry and Physiology Part B 124 (1999) 467 – 473 www.elsevier.com/locate/cbpb
Phospholipase D in Tetrahymena: activity and significance Morten Rasmussen *, Leif Rasmussen Department of Anatomy and Cytology, Institute of Medical Biology, Uni6ersity of Odense, DK-5230 Odense M, Denmark Received 16 April 1999; received in revised form 17 August 1999; accepted 19 August 1999
Abstract We detected phospholipase D in three species of ciliates: Tetrahymena: T. thermophila, T. pyriformis and T. setosa in nutrient medium supplemented with ethanol in in vivo systems, by the appearance of phosphatidylethanol. The calcium ionophore A23187 increased the synthesis of phosphatidylethanol, as compared with untreated controls. We suggest that Tetrahymena possess a calcium sensitive phospholipase D. Propranolol caused the cells in dense cultures to increase their average generation times or die, dependent on the drug concentration. This inhibition could be overcome by the addition of phospholipids or ethanol. Pure phosphatidylethanol had no effect on growth rates or generation times in cultures at high cell density, but postponed cell death in cultures at low cell density by a factor of 10. We suggest that an important role of phospholipase D in Tetrahymena is to supply the cell with diacylglycerol without which it can not enter the mode of proliferation from the lag phase of the culture. © 1999 Elsevier Science Inc. All rights reserved. Keywords: A23187; Ethanol; Phosphatidylethanol; Phospholipase D; Propranolol; Tetrahymena
1. Introduction Already the first cell culturists knew that cultures inoculated with too few cells have a high risk of dying before multiplication began. Raff [27] and Ishizaki et al. [17] recently studied this problem using lens epithelial cells at low cell concentrations. No matter which mixture of hormones they added to their cultures, their cells died. Here we address aspects in these important problems at the level of the implicated enzymes. Our model system consists of Tetrahymena cells inoculated into a nutritionally complete, lipid-free synthetic medium. Also these cells die at low cell density. Phosphatidylinositol specific phospholipase C (PIPLC) has long been implicated in intracellular cascades in connection with growth and multiplication via the products of diacylglycerol (DAG), IP3 and Ca. A PIfree mixture of phospholipids rescued our cells inoculated at low density. Therefore lipid-metabolising enzymes other than PI-PLC seemed to be involved in the control of cell survival/multiplication. Also ethanol * Corresponding author. Tel.: +45-6557-2344; fax: +45-65930480. E-mail address: [email protected] (M. Rasmussen)
was implicated in survival and we decided to investigate the possible role of phospholipase D in these processes. Phospholipase D (PLD) occurs in a wide variety of cells. It catalyses the breakdown of phospholipids, preferentially phosphatidylcholine (PC). If ethanol enters the reaction then phosphatidylethanol (Peth) — a dead end product — is formed. If water enters then phosphatidate (PA) is formed. PA may act as a second messenger. This has been inferred in studies of the secretion of histamine [11] and insulin [22], in the formation of progesterone [20] and in cell growth [31]. It may also be converted into another second messenger, diacylglycerol (DAG), in a reaction catalysed by phosphatidate phosphohydrolase (PPH), and addition of DAG rescued the cells at low density from dying [28]. These compounds and these enzymes are therefore closely connected to mechanisms in cell death/survival/ proliferation. Holz et al. [15,16] showed and Christensen et al. [7] confirmed that ethanol activated cell multiplication in T. setosa. Ethanol activates PLD in other cell types [23]. We therefore investigated the effects on cell proliferation of presumed activators and inhibitors of this enzyme.
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M. Rasmussen, L. Rasmussen / Comparati6e Biochemistry and Physiology, Part B 124 (1999) 467–473
2. Materials and methods
2.1. Cells Tetrahymena thermophila BIII-1868, T. pyriformis and T. setosa were used [16,25].
2.2. Nutrient medium The cells were grown in a synthetic nutrient medium, SSM, in pure cultures. The medium was adjusted to a pH-value of 7.5 and consists of 19 amino acids, four nucleosides, seven vitamins, salts and citrate [29]. The medium was sterilised by heating.
tracted samples were loaded onto the plates using thinly drawn-out glass pipettes. The sample volume would vary from pipette to pipette. To make sure that a similar volume of each sample was loaded onto the lanes, the same pipette was used for loading all samples and another for loading all standards. They were cleansed thoroughly in hexane between samples. The loaded plates were placed in a glass tank coated with filter paper. The solvent consisted of chloroform/acetone/methanol/acetic acid/water, 50:20:15:10:5. The developing distance was held between 11 and 13 cm. The lipids were detected by spraying the plates with phospho-molybdic acid reagent and heating at 120°C for 10–20 min. The plates were photocopied immediately after development.
2.3. Cultures 3.1. Lipids used Cultures were grown in plastic capped test tubes in volumes of 2 ml or in 125-ml capacity conical flasks in volumes of 10 ml or in conical flasks at a volume of 50 ml. T. thermophila was grown at 37°C, the other species at 26°C.
The following lipid standards were used for detecting lipids on TLC: 1,2-dipalmitoyl phosphatidylethanol, 99% pure, Avanti Polar Lipids, Inc., dipalmitoyl (C16:0) phosphatidylcholine, synthetic 99% pure; phosphatidylethanolamine, 99% pure.
2.4. Cell concentrations 3.2. Dissol6ing lipids Cell concentrations were in mass cultures determined on removed samples with the aid of an electronic cell counter. For each sample of 1 ml, cell density was determined three times, and the mean value was calculated. Control experiments have shown that this method is reliable down to 1000 cells per ml [8]. In 2 ml cultures with a few cells they were counted alive in a stereo microscope at low (25×) magnification.
2.5. Bligh and Dyer lipid extraction Cells were harvested, washed in 50 ml Tris/HCl buffer, resuspended in 5 ml water, and killed by sonication. To a separation flask were added: 5 ml chloroform, 10 ml methanol, and the cell suspension. After shaking, additional 5 ml chloroform were added along with 2.5 ml 0.5 M HCl. The sample was shaken again, and the chloroform layer was collected in a test tube, when it had separated from the methanol layer [3]. The collected chloroform layer was precipitated in the centrifuge and excess methanol and protein were removed and discarded. Blowing N2 into the test tube evaporated the remaining chloroform layer. The dried lipids were resuspended in 100 ml hexane and kept at − 20°C.
3. Thin layer chromatography Precoated TLC plates (DC-Alufolien Kieselgel 60W, Merck) were activated for 30 min at 140°C. The ex-
Lipids added to cultures were dissolved in distilled water and sonicated. The solutions were then sterilised. Lipids for TLC standards were dissolved in hexane, with the exception of Peth, which dissolved readily in distilled water.
3.3. Chemicals Chemicals were from Sigma Chemical Co.
4. Results
4.1. PLD The three Tetrahymena species were inoculated into separate cultures at 1000 cells per ml in four 50-ml portions of SSM supplemented with 0.3% ethanol. The cells were harvested the next day and their lipids were isolated and separated. Peth occurred in all three species (Fig. 1), indicating that PLD is active in them. We did not detect Peth in cells from ethanol-free media.
4.2. Propranolol Propranolol is a potent inhibitor of phosphatidyl phosphohydrolase in many cells and causes PA (and Peth in the presence of ethanol) to accumulate [21], because DAG synthesis is blocked. T. thermophila cells were inoculated into SSM supplemented with 0.3%
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ethanol and either 0, 30 or 60 mM propranolol. The highest concentration prevented cell division, 30 mM extended the doubling times from an average of 3–10 h. Ethanol and asolectin (a commercial mixture of phospholipids without phosphatidylinositol) reduced these long doubling times to 6 and 5 h, respectively (Fig. 2). A similar pattern of results was obtained with T. setosa (not shown).
4.3. PLD and Ca 2 + We inoculated T. thermophila at an initial cell density of 1000 cells per ml into four flasks of 50 ml of SSM without citrate and with the iron concentration reduced to 3 mM. We added 70 mM CaCl2 and 10 mM of the ionophore A 23187. Four similar cultures-but without the ionophore-were prepared as controls. The cells were
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harvested when the culture had reached a density of about 280.000 cells per ml and washed in a Tris/HClbuffer adjusted to pH 7.5. The lipids from each culture condition were extracted and separated by thin layer chromatography. In the presence of the ionophore, the synthesis of Peth was increased as compared to the controls (Fig. 3). These results suggest that T. thermophila cells possess a calcium sensitive PLD. It is not known whether there is more than one isotype. Similar results were obtained with T. pyriformis.
4.4. Peth To cultures of T. thermophila in SSM we added Peth at 3.3 and 10.5 mg per ml at either 15 or 1000 initial cells per ml (Fig. 4). The cells died at the low initial cell density without Peth in the course of a few hours. Peth
Fig. 1. Tetrahymena cells of the species T. thermophila, T. pyriformis and T. setosa were cultured in synthetic nutrient medium (SSM) to which 0.3% ethanol was added. The cells were harvested at late log phase, and the cellular lipids were extracted according to Bligh and Dyer [3]. Lanes 1: phosphatidylethanol standard; 2: T. thermophila, SSM; 3: T. thermophila, SSM+ ethanol; 4: phosphatidylethanol and phosphatidylcholine standards; 5: T. pyriformis, SSM; 6: T. pyriformis, SSM + ethanol; 7: T. setosa, SSM; 8: phosphatidylethanol and phosphatidylcholine standards; and 9: T. setosa, SSM + ethanol.
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Fig. 2. Tetrahymena thermophila cells were inoculated at high initial cell density (1000 cells per ml) into synthetic nutrient medium to which propranolol (30 and 60 mM) was added. The untreated controls (squares) doubled about every 3 h. A total of 30 mM propranolol increased this mean generation time to an average of about 10 h. A total of 60 mM propranolol was lethal to the cells. Ethanol (0.3%) reduced the mean generation time to 6 h. A total of 50 mg asolectin per ml reduced the mean generation time to 5 h. Note that the ordinate is logarithmic with respect to cell densities. The experiments were repeated 36 times.
postponed cell death for more than 48 h. Thus Peth can postpone cell death at low cell densities, but not rescue them from dying. Peth added to cells at high initial density had no effect on cell multiplication rates (both are represented by diamonds in Fig. 4). The rescuing effect of ethanol on T. setosa at low cell density is thus likely to be a result of something different than generation of Peth. 5. Discussion Phospholipase D (PLD) may be an important but so far overlooked enzyme. Kovacs et al. [18,19] concluded that it was present in ciliate T. pyriformis. They added propanol to their cultures and saw that phosphatidyl propanol was formed. This type of reaction is restricted to phospholipase D activity by definition. Here we have confirmed the existence of this enzyme activity in ciliates and have shed light on its biological effects. Our three species of Tetrahymena must be inoculated above initial cell densities (‘critical initial densities’, CID) in our synthetic nutrient medium for the cultures ‘to take’ [6,8,10,28]. Below these densities all cells die within few hours [9,10]. Addition of a crude lipid extract from soybeans rescues the moribund cells in all three species of Tetrahymena, and thus lowers the CID [8]. Addition of ethanol (final concentrations 0.2 – 0.3%) has the same effect in T. setosa, but no effect was found in T. thermophila and T. pyriformis [7] prior to this report.
Ethanol has a dual role with respect to PLD. It stimulates the enzyme activity and it competes with water as the secondary substrate, giving rise to the dead end product Peth. Such stimulation has been reported from many sources [23], and we obtained indirect evidence for it (Fig. 2). Ethanol partially relieved the inhibition of 30 mM propranolol — a known inhibitor of phosphatidyl phosphohydrolase, PPH — on the PLD cascade. In spite of such stimulation, high concentrations of ethanol may drain the cellular content of PA and thus reduce the supply of DAG, and this could lead to poor growth [13]. Our results on T. setosa show, however, that moderate (0.2–0.3%) amounts of ethanol stimulate survival/proliferation [7] and that Peth also has positi6e effects on survival (Fig. 4) in contrast to conventional wisdom. It therefore seems likely that a higher net-yield of DAG can be gained at these moderate ethanol concentrations than in the absence of ethanol or at very high levels of ethanol. This is in line with our observation (Fig. 2) that the propranolol inhibition leading to reduced multiplication rates indeed can be counteracted by addition of ethanol in T. thermophila. This species has never before been shown to respond to ethanol. [We want to point out that we have under in vivo conditions used propranolol concentrations 100-fold lower than those shown to affect in vitro (!) b-adrenergic pathways [4,5,26,30].] Tetrahymena cells had longer average generation times in propranolol at 30 and at 60 mM they died (Fig. 2). The effects at 30 mM were partly restored by addi-
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tion phospholipids. When PPH is inhibited, PA accumulates [13,21]. We therefore propose that propranolol inhibits the activity of PPH in Tetrahymena in a concentration dependent way with respect to PA. We also propose that the extended generation times were caused primarily by inhibition of PPH and the subsequent reduction of DAG. This suggestion needs further investigation as no available source has commented on the kinetics of propranolol inhibition of PPH. It may be the lack of DAG rather than the higher levels of PA that is lethal to the cells. It is possible that PA stimulates PI turnover and Ca2 + accumulation in Tetrahymena, as shown in other cells [24]. Peth may here substitute for DAG in the activation of PKC, as shown in mammalian PKC-gamma [1,2]. Experiments with other cell types have shown that there are at least two isotypes of PLD [12,14]. One is Ca2 + dependent and has high
Fig. 3. Tetrahymena thermophila cells were cultured in synthetic nutrient medium to which ethanol and calcium ionophore A 23187 were added. The cells were harvested at late log phase and the cellular lipids were extracted. A 23187 increased phospholipase D activity as indicated by an increased level of phosphatidylethanol. Lanes 1: phosphatidylethanol and phosphatidylcholine standards; 2: T. thermophila, SSM; 3: T. thermophila, SSM + ethanol; 4: T. thermophila, SSM +ethanol+ A 23187; and 5: phosphatidylethanol and phosphatidylcholine standards.
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affinity for PS. Another is Ca2 + insensitive and has high affinity for PC. Our results suggest that PS and PC help the cells to survive at low densities in a synergistic way. Our results also showed PLD activity at physiological concentrations of Ca2 + . This activity was increased in the presence of the Ca2 + ionophore A 23187. Tetrahymena may also have two istotypes of PLD. When added to cultures of high initial cell density, extracellular Peth had no effect on generation time at any of the tested concentrations. When added to cells inoculated below critical density, Peth postponed culture cell death to about 72 h (Fig. 4). Death can also be postponed by inhibition of protein synthesis [10] or by inoculating cells in a salts buffer. Both treatments are incompatible with multiplication in high-density cultures in contrast to Peth. Species of Tetrahymena have traditionally been divided into two groups according to their need for phospholipids: requirers and non-requirers. The former group included T. setosa, T. patula and others, the latter T. pyriformis, T. thermophila, T. 6orax, T. pigmentosa and others. Later it was shown that cells from both groups that die at low cell density are rescued by phospholipids [8,28], so under certain conditions they do require lipids. (By way of contrast, ‘requirers’ could be grown in the absence of phospholipids for more than 50 subcultivations, provided that all the initial cell densities were high, or in the case of T. setosa at lower concentrations if ethanol was added. So, at certain conditions, these cells do not need nutritional lipids.) We can specify the time period in culture growth, when the lipids are required. During the log phase, generation times were unaffected by phospholipids in T. thermophila, and it has been reported, that the cell commits itself to the paths leading to life or death within 4–30 min after inoculation in the absence of stimulating agents (unpubl. res.). Thus the requirement for phospholipids is most pronounced during the early lag phase in culture growth but disappears during the log phase. Our results indicate that PLD activity is implicated in the regulation of cell survival and that PLD may be a far more essential mechanism in intracellular signal transduction than formerly presumed, possibly being one of the key enzymes in regulation of cell growth and proliferation. We have understood the ethanol requirement of T. setosa by studying the lipid requirement of T. thermophila. We have understood cell death in T. thermophila by studying the ethanol requirement of T. setosa. These mechanisms would never come to light under rich nutritional conditions, but can be disclosed under controlled nutritional conditions in the laboratory. The obvious next problem is whether or not these issues can be transferred to other cell types.
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Fig. 4. Proliferation of Tetrahymena thermophila cells in the presence and absence of phosphatidylethanol. The cells were inoculated at two densities (10 or 1000 cells per ml) into synthetic medium, to which phosphatidylethanol (Peth) was added. At low initial cell density Peth (triangles) delayed cell death for 48 h compared to the controls (circles). At high initial cell density, the points for ‘with’ and ‘without’ Peth fall on top of each other (diamonds).
References [1] Asaoka Y, Kikkawa U, Sekiguchi K, Shearman MS, Kosaka Y, Nakano Y, et al. Activation of a brain-specific protein kinase C subspecies in the presence of phosphatidylethanol. FEBS Lett 1988;231(1):221– 4. [2] Asaoka Y. Distinct effects of phosphatidylethanol on three types of rat brain protein kinase C. Kobe J Med Sci 1989;35(4):229– 37. [3] Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959;37:911–7. [4] Blum JJ, Kirshner N, Utley J. The effect of reserpine on growth and catecholamine content of Tetrahymena. Mol Pharmacol 1966;2:606 – 23. [5] Blum JJ. An adrenergic control system in Tetrahymena. Proc Natl Acad Sci USA 1967;58:81–8. [6] Christensen ST, Leick V, Rasmussen L, Wheatley DN. Signalling in unicellular eukaryotes. Regulation of cell survival, proliferation, differentiation, mating, chemosensory behavior and programmed cell death. Int Rev Cytol 1998;177:181– 253. [7] Christensen ST, Schousboe P, Nielsen DS, Søndergaard G, Rasmussen L. Cell multiplication in Tetrahymena setosa and Tetrahymena thermophila in synthetic medium. Effects of ethanol, cholesterol and extracellular medium. Acta Protozool 1993;32:151 – 6. [8] Christensen ST, Schousboe P, Ghiladi M, Rasmussen L. Nutritional stress relieved by addition of hemin and phospholipids. J Comp Physiol B 1992;162:107–10. [9] Christensen ST, Rasmussen L. Evidence for growth factors which control cell multiplication in Tetrahymena thermophila. Acta Protozool 1992;31:215–9. [10] Christensen ST, Wheatley DN, Rasmussen MI, Rasmussen L. Mechanisms controlling death, survival and proliferation in a model unicellular eukaryotic cell system, Tetrahymena thermophila. Cell Death Diff 1995;2:301–8. [11] Gruchalla RS, Dinh TT, Kennerly DA. An indirect pathway of receptor-mediated 1,2-diacylglycerol formation in mast cells. I. IgE receptor-mediated activation of phospholipase D. J Immunol 1990;144:2334–42.
[12] Guillemain I, Rossignol B. Receptor- and phorbol ester-mediated phospholipase D activation in rat parotid involves two different pathways. Am J Physiol 1994;266:692 – 9. [13] Gustavsson L. ESBRA 1994 award lecture. Phosphatidylethanol formation: specific effects of ethanol mediated via phospholipase D. Alcohol 1995;30(4):391– 406. [14] Halenda SP, Rehm AG. Evidence for the calcium-dependent activation of phospholipase D in thrombin-stimulated human erythroleukaemia cells. Biochem J 1990;267(2):479– 83. [15] Holz GG Jr. The nutrition of Tetrahymena: essential nutrients, feeding and digestion. In: Elliott AM, editor. Biology of Tetrahymena. Dowden: Hutchinson & Ross, Inc, 1973:89–98. [16] Holz GG, Erwin J, Wagner B, Rosenbaum N. The nutrition of Tetrahymena setifera HZ-1: sterol and alcohol requirements. J Protozool 1962;9(3):359 – 63. [17] Ishizaki Y, Voyvodic JT, Burne JF, Raff MC. Control of lens epithelial cell survival. J Cell Biol 1993;121:899 – 908. [18] Kovacs P, Csaba G, Ito Y, Nozawa Y. Effect of insulin on the phospholipase D activity of untreated and insulin-pretreated (hormonally imprinted) Tetrahymena. Biochem Biophys Res Commun 1996;222(2):359– 61. [19] Kovacs P, Csaba G, Nakashima S, Nozawa Y. Phospholipase D activity in the Tetrahymena pyriformis GL. Cell Biochem Funct 1997;15(1):53 – 60. [20] Liscovitch ML, Amsterdam A. Gonadotropin-releasing hormone activates phospholipase D in ovarian granulosa cells. Possible role in signal transduction. J Biol Chem 1989;264:11762–7. [21] Meier KE, Gause KC, Wisehart-Johnson AE, Gore AC, Finley EL, Jones LG, et al. Effects of propranolol on phosphatidate phosphohydrolase and mitogen-activated protein kinase activities in A7r5 vascular smooth muscle cells. Cell Signal 1998;10(6):415– 26. [22] Metz SA, Dunlop M. Stimulation of insulin release by phospholipase D. A potential role for endogenous phosphatidic acid in pancreatic islet function. Biochem J 1990;270:427 – 35. [23] Miller RR, Yates JW, Geer BW. Dietary ethanol stimulates the activity of phosphatidylcholine-specific phospholipase D and the formation of phosphatidylethanol in Drosophila melanogaster larvae. Insect Biochem Mol Biol 1993;23(6):749– 55.
M. Rasmussen, L. Rasmussen / Comparati6e Biochemistry and Physiology, Part B 124 (1999) 467–473 [24] Moolenaar WH, Kruijer W, Tilly BC, Verlaan I, Biermann AJ, deLaat SW. Growth factor-like action of phosphatidic acid. Nature 1986;323:171–3. [25] Nanney DL, McCoy JW. Characterization of the species of the Tetrehymena pyriformis complex. Trans Am Microsc Soc 1976;95:664 – 82. [26] Ness J, Morse DJ. Regulation of galactokinase gene expression in Tetrahymena thermophila. I. Intracellular catecholamine control of galactokinase expression. J Biol Chem 1985;260(18):10001– 12. [27] Raff MC. Social controls on cell survival and cell death. Nature 1992;356:397 – 400.
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[28] Schousboe P, Rasmussen L. Survival of Tetrahymena thermophila at low cell densities. Effects of lipids and long-chain alcohols. J Eukaryot Microbiol 1994;41:195 – 9. [29] Szablewzki L, Andersen PH, Florin-Christensen J, Florin-Christensen M, Rasmussen L. Tetrahymena thermophila: growth in synthetic medium in the presence and in the absence of glucose. J Protozool 1991;38:62 – 5. [30] Umeki S, Nozawa Y. Effect of local anesthetics on stearoyl-CoA desaturase of Tetrahymena microsomes. Biol Chem Hoppe-Seyler 1986;367:61 – 5. [31] Yu CL, Tsai MH, Stacey DW. Cellular ras activity and phospholipid metabolism. Cell 1988;52:63 – 71.
Phospholipase D in Tetrahymena: activity and significance Morten Rasmussen *, Leif Rasmussen Department of Anatomy and Cytology, Institute of Medical Biology, Uni6ersity of Odense, DK-5230 Odense M, Denmark Received 16 April 1999; received in revised form 17 August 1999; accepted 19 August 1999
Abstract We detected phospholipase D in three species of ciliates: Tetrahymena: T. thermophila, T. pyriformis and T. setosa in nutrient medium supplemented with ethanol in in vivo systems, by the appearance of phosphatidylethanol. The calcium ionophore A23187 increased the synthesis of phosphatidylethanol, as compared with untreated controls. We suggest that Tetrahymena possess a calcium sensitive phospholipase D. Propranolol caused the cells in dense cultures to increase their average generation times or die, dependent on the drug concentration. This inhibition could be overcome by the addition of phospholipids or ethanol. Pure phosphatidylethanol had no effect on growth rates or generation times in cultures at high cell density, but postponed cell death in cultures at low cell density by a factor of 10. We suggest that an important role of phospholipase D in Tetrahymena is to supply the cell with diacylglycerol without which it can not enter the mode of proliferation from the lag phase of the culture. © 1999 Elsevier Science Inc. All rights reserved. Keywords: A23187; Ethanol; Phosphatidylethanol; Phospholipase D; Propranolol; Tetrahymena
1. Introduction Already the first cell culturists knew that cultures inoculated with too few cells have a high risk of dying before multiplication began. Raff [27] and Ishizaki et al. [17] recently studied this problem using lens epithelial cells at low cell concentrations. No matter which mixture of hormones they added to their cultures, their cells died. Here we address aspects in these important problems at the level of the implicated enzymes. Our model system consists of Tetrahymena cells inoculated into a nutritionally complete, lipid-free synthetic medium. Also these cells die at low cell density. Phosphatidylinositol specific phospholipase C (PIPLC) has long been implicated in intracellular cascades in connection with growth and multiplication via the products of diacylglycerol (DAG), IP3 and Ca. A PIfree mixture of phospholipids rescued our cells inoculated at low density. Therefore lipid-metabolising enzymes other than PI-PLC seemed to be involved in the control of cell survival/multiplication. Also ethanol * Corresponding author. Tel.: +45-6557-2344; fax: +45-65930480. E-mail address: [email protected] (M. Rasmussen)
was implicated in survival and we decided to investigate the possible role of phospholipase D in these processes. Phospholipase D (PLD) occurs in a wide variety of cells. It catalyses the breakdown of phospholipids, preferentially phosphatidylcholine (PC). If ethanol enters the reaction then phosphatidylethanol (Peth) — a dead end product — is formed. If water enters then phosphatidate (PA) is formed. PA may act as a second messenger. This has been inferred in studies of the secretion of histamine [11] and insulin [22], in the formation of progesterone [20] and in cell growth [31]. It may also be converted into another second messenger, diacylglycerol (DAG), in a reaction catalysed by phosphatidate phosphohydrolase (PPH), and addition of DAG rescued the cells at low density from dying [28]. These compounds and these enzymes are therefore closely connected to mechanisms in cell death/survival/ proliferation. Holz et al. [15,16] showed and Christensen et al. [7] confirmed that ethanol activated cell multiplication in T. setosa. Ethanol activates PLD in other cell types [23]. We therefore investigated the effects on cell proliferation of presumed activators and inhibitors of this enzyme.
0305-0491/99/$ - see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 3 0 5 - 0 4 9 1 ( 9 9 ) 0 0 1 4 4 - 3
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M. Rasmussen, L. Rasmussen / Comparati6e Biochemistry and Physiology, Part B 124 (1999) 467–473
2. Materials and methods
2.1. Cells Tetrahymena thermophila BIII-1868, T. pyriformis and T. setosa were used [16,25].
2.2. Nutrient medium The cells were grown in a synthetic nutrient medium, SSM, in pure cultures. The medium was adjusted to a pH-value of 7.5 and consists of 19 amino acids, four nucleosides, seven vitamins, salts and citrate [29]. The medium was sterilised by heating.
tracted samples were loaded onto the plates using thinly drawn-out glass pipettes. The sample volume would vary from pipette to pipette. To make sure that a similar volume of each sample was loaded onto the lanes, the same pipette was used for loading all samples and another for loading all standards. They were cleansed thoroughly in hexane between samples. The loaded plates were placed in a glass tank coated with filter paper. The solvent consisted of chloroform/acetone/methanol/acetic acid/water, 50:20:15:10:5. The developing distance was held between 11 and 13 cm. The lipids were detected by spraying the plates with phospho-molybdic acid reagent and heating at 120°C for 10–20 min. The plates were photocopied immediately after development.
2.3. Cultures 3.1. Lipids used Cultures were grown in plastic capped test tubes in volumes of 2 ml or in 125-ml capacity conical flasks in volumes of 10 ml or in conical flasks at a volume of 50 ml. T. thermophila was grown at 37°C, the other species at 26°C.
The following lipid standards were used for detecting lipids on TLC: 1,2-dipalmitoyl phosphatidylethanol, 99% pure, Avanti Polar Lipids, Inc., dipalmitoyl (C16:0) phosphatidylcholine, synthetic 99% pure; phosphatidylethanolamine, 99% pure.
2.4. Cell concentrations 3.2. Dissol6ing lipids Cell concentrations were in mass cultures determined on removed samples with the aid of an electronic cell counter. For each sample of 1 ml, cell density was determined three times, and the mean value was calculated. Control experiments have shown that this method is reliable down to 1000 cells per ml [8]. In 2 ml cultures with a few cells they were counted alive in a stereo microscope at low (25×) magnification.
2.5. Bligh and Dyer lipid extraction Cells were harvested, washed in 50 ml Tris/HCl buffer, resuspended in 5 ml water, and killed by sonication. To a separation flask were added: 5 ml chloroform, 10 ml methanol, and the cell suspension. After shaking, additional 5 ml chloroform were added along with 2.5 ml 0.5 M HCl. The sample was shaken again, and the chloroform layer was collected in a test tube, when it had separated from the methanol layer [3]. The collected chloroform layer was precipitated in the centrifuge and excess methanol and protein were removed and discarded. Blowing N2 into the test tube evaporated the remaining chloroform layer. The dried lipids were resuspended in 100 ml hexane and kept at − 20°C.
3. Thin layer chromatography Precoated TLC plates (DC-Alufolien Kieselgel 60W, Merck) were activated for 30 min at 140°C. The ex-
Lipids added to cultures were dissolved in distilled water and sonicated. The solutions were then sterilised. Lipids for TLC standards were dissolved in hexane, with the exception of Peth, which dissolved readily in distilled water.
3.3. Chemicals Chemicals were from Sigma Chemical Co.
4. Results
4.1. PLD The three Tetrahymena species were inoculated into separate cultures at 1000 cells per ml in four 50-ml portions of SSM supplemented with 0.3% ethanol. The cells were harvested the next day and their lipids were isolated and separated. Peth occurred in all three species (Fig. 1), indicating that PLD is active in them. We did not detect Peth in cells from ethanol-free media.
4.2. Propranolol Propranolol is a potent inhibitor of phosphatidyl phosphohydrolase in many cells and causes PA (and Peth in the presence of ethanol) to accumulate [21], because DAG synthesis is blocked. T. thermophila cells were inoculated into SSM supplemented with 0.3%
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ethanol and either 0, 30 or 60 mM propranolol. The highest concentration prevented cell division, 30 mM extended the doubling times from an average of 3–10 h. Ethanol and asolectin (a commercial mixture of phospholipids without phosphatidylinositol) reduced these long doubling times to 6 and 5 h, respectively (Fig. 2). A similar pattern of results was obtained with T. setosa (not shown).
4.3. PLD and Ca 2 + We inoculated T. thermophila at an initial cell density of 1000 cells per ml into four flasks of 50 ml of SSM without citrate and with the iron concentration reduced to 3 mM. We added 70 mM CaCl2 and 10 mM of the ionophore A 23187. Four similar cultures-but without the ionophore-were prepared as controls. The cells were
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harvested when the culture had reached a density of about 280.000 cells per ml and washed in a Tris/HClbuffer adjusted to pH 7.5. The lipids from each culture condition were extracted and separated by thin layer chromatography. In the presence of the ionophore, the synthesis of Peth was increased as compared to the controls (Fig. 3). These results suggest that T. thermophila cells possess a calcium sensitive PLD. It is not known whether there is more than one isotype. Similar results were obtained with T. pyriformis.
4.4. Peth To cultures of T. thermophila in SSM we added Peth at 3.3 and 10.5 mg per ml at either 15 or 1000 initial cells per ml (Fig. 4). The cells died at the low initial cell density without Peth in the course of a few hours. Peth
Fig. 1. Tetrahymena cells of the species T. thermophila, T. pyriformis and T. setosa were cultured in synthetic nutrient medium (SSM) to which 0.3% ethanol was added. The cells were harvested at late log phase, and the cellular lipids were extracted according to Bligh and Dyer [3]. Lanes 1: phosphatidylethanol standard; 2: T. thermophila, SSM; 3: T. thermophila, SSM+ ethanol; 4: phosphatidylethanol and phosphatidylcholine standards; 5: T. pyriformis, SSM; 6: T. pyriformis, SSM + ethanol; 7: T. setosa, SSM; 8: phosphatidylethanol and phosphatidylcholine standards; and 9: T. setosa, SSM + ethanol.
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Fig. 2. Tetrahymena thermophila cells were inoculated at high initial cell density (1000 cells per ml) into synthetic nutrient medium to which propranolol (30 and 60 mM) was added. The untreated controls (squares) doubled about every 3 h. A total of 30 mM propranolol increased this mean generation time to an average of about 10 h. A total of 60 mM propranolol was lethal to the cells. Ethanol (0.3%) reduced the mean generation time to 6 h. A total of 50 mg asolectin per ml reduced the mean generation time to 5 h. Note that the ordinate is logarithmic with respect to cell densities. The experiments were repeated 36 times.
postponed cell death for more than 48 h. Thus Peth can postpone cell death at low cell densities, but not rescue them from dying. Peth added to cells at high initial density had no effect on cell multiplication rates (both are represented by diamonds in Fig. 4). The rescuing effect of ethanol on T. setosa at low cell density is thus likely to be a result of something different than generation of Peth. 5. Discussion Phospholipase D (PLD) may be an important but so far overlooked enzyme. Kovacs et al. [18,19] concluded that it was present in ciliate T. pyriformis. They added propanol to their cultures and saw that phosphatidyl propanol was formed. This type of reaction is restricted to phospholipase D activity by definition. Here we have confirmed the existence of this enzyme activity in ciliates and have shed light on its biological effects. Our three species of Tetrahymena must be inoculated above initial cell densities (‘critical initial densities’, CID) in our synthetic nutrient medium for the cultures ‘to take’ [6,8,10,28]. Below these densities all cells die within few hours [9,10]. Addition of a crude lipid extract from soybeans rescues the moribund cells in all three species of Tetrahymena, and thus lowers the CID [8]. Addition of ethanol (final concentrations 0.2 – 0.3%) has the same effect in T. setosa, but no effect was found in T. thermophila and T. pyriformis [7] prior to this report.
Ethanol has a dual role with respect to PLD. It stimulates the enzyme activity and it competes with water as the secondary substrate, giving rise to the dead end product Peth. Such stimulation has been reported from many sources [23], and we obtained indirect evidence for it (Fig. 2). Ethanol partially relieved the inhibition of 30 mM propranolol — a known inhibitor of phosphatidyl phosphohydrolase, PPH — on the PLD cascade. In spite of such stimulation, high concentrations of ethanol may drain the cellular content of PA and thus reduce the supply of DAG, and this could lead to poor growth [13]. Our results on T. setosa show, however, that moderate (0.2–0.3%) amounts of ethanol stimulate survival/proliferation [7] and that Peth also has positi6e effects on survival (Fig. 4) in contrast to conventional wisdom. It therefore seems likely that a higher net-yield of DAG can be gained at these moderate ethanol concentrations than in the absence of ethanol or at very high levels of ethanol. This is in line with our observation (Fig. 2) that the propranolol inhibition leading to reduced multiplication rates indeed can be counteracted by addition of ethanol in T. thermophila. This species has never before been shown to respond to ethanol. [We want to point out that we have under in vivo conditions used propranolol concentrations 100-fold lower than those shown to affect in vitro (!) b-adrenergic pathways [4,5,26,30].] Tetrahymena cells had longer average generation times in propranolol at 30 and at 60 mM they died (Fig. 2). The effects at 30 mM were partly restored by addi-
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tion phospholipids. When PPH is inhibited, PA accumulates [13,21]. We therefore propose that propranolol inhibits the activity of PPH in Tetrahymena in a concentration dependent way with respect to PA. We also propose that the extended generation times were caused primarily by inhibition of PPH and the subsequent reduction of DAG. This suggestion needs further investigation as no available source has commented on the kinetics of propranolol inhibition of PPH. It may be the lack of DAG rather than the higher levels of PA that is lethal to the cells. It is possible that PA stimulates PI turnover and Ca2 + accumulation in Tetrahymena, as shown in other cells [24]. Peth may here substitute for DAG in the activation of PKC, as shown in mammalian PKC-gamma [1,2]. Experiments with other cell types have shown that there are at least two isotypes of PLD [12,14]. One is Ca2 + dependent and has high
Fig. 3. Tetrahymena thermophila cells were cultured in synthetic nutrient medium to which ethanol and calcium ionophore A 23187 were added. The cells were harvested at late log phase and the cellular lipids were extracted. A 23187 increased phospholipase D activity as indicated by an increased level of phosphatidylethanol. Lanes 1: phosphatidylethanol and phosphatidylcholine standards; 2: T. thermophila, SSM; 3: T. thermophila, SSM + ethanol; 4: T. thermophila, SSM +ethanol+ A 23187; and 5: phosphatidylethanol and phosphatidylcholine standards.
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affinity for PS. Another is Ca2 + insensitive and has high affinity for PC. Our results suggest that PS and PC help the cells to survive at low densities in a synergistic way. Our results also showed PLD activity at physiological concentrations of Ca2 + . This activity was increased in the presence of the Ca2 + ionophore A 23187. Tetrahymena may also have two istotypes of PLD. When added to cultures of high initial cell density, extracellular Peth had no effect on generation time at any of the tested concentrations. When added to cells inoculated below critical density, Peth postponed culture cell death to about 72 h (Fig. 4). Death can also be postponed by inhibition of protein synthesis [10] or by inoculating cells in a salts buffer. Both treatments are incompatible with multiplication in high-density cultures in contrast to Peth. Species of Tetrahymena have traditionally been divided into two groups according to their need for phospholipids: requirers and non-requirers. The former group included T. setosa, T. patula and others, the latter T. pyriformis, T. thermophila, T. 6orax, T. pigmentosa and others. Later it was shown that cells from both groups that die at low cell density are rescued by phospholipids [8,28], so under certain conditions they do require lipids. (By way of contrast, ‘requirers’ could be grown in the absence of phospholipids for more than 50 subcultivations, provided that all the initial cell densities were high, or in the case of T. setosa at lower concentrations if ethanol was added. So, at certain conditions, these cells do not need nutritional lipids.) We can specify the time period in culture growth, when the lipids are required. During the log phase, generation times were unaffected by phospholipids in T. thermophila, and it has been reported, that the cell commits itself to the paths leading to life or death within 4–30 min after inoculation in the absence of stimulating agents (unpubl. res.). Thus the requirement for phospholipids is most pronounced during the early lag phase in culture growth but disappears during the log phase. Our results indicate that PLD activity is implicated in the regulation of cell survival and that PLD may be a far more essential mechanism in intracellular signal transduction than formerly presumed, possibly being one of the key enzymes in regulation of cell growth and proliferation. We have understood the ethanol requirement of T. setosa by studying the lipid requirement of T. thermophila. We have understood cell death in T. thermophila by studying the ethanol requirement of T. setosa. These mechanisms would never come to light under rich nutritional conditions, but can be disclosed under controlled nutritional conditions in the laboratory. The obvious next problem is whether or not these issues can be transferred to other cell types.
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Fig. 4. Proliferation of Tetrahymena thermophila cells in the presence and absence of phosphatidylethanol. The cells were inoculated at two densities (10 or 1000 cells per ml) into synthetic medium, to which phosphatidylethanol (Peth) was added. At low initial cell density Peth (triangles) delayed cell death for 48 h compared to the controls (circles). At high initial cell density, the points for ‘with’ and ‘without’ Peth fall on top of each other (diamonds).
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