ht. J. Biockm.
Vol. 23, No. S/6, pp. 605-608, Printed in Great Britain. All rightsreserved
0020-711X/91 $3.00 + 0.00
1991
Copyright 0 1991Pergamon Press plc
EFFECTS OF Ca2+ AND IONOPHORE A23187 ON PROTEIN SYNTHESIS IN INTACT RABBIT RETICULOCYTES Wlu LAMWONG, MARGARET A. BROSTROMand CHARLES0. BROSTROM Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, P&&away, NJ 08854, U.S.A. (Received I7 July 1990) Abstract-l. Amino acid incorporation in intact rabbit reticulocytes was unaffected by depletion of Ca*+ with EGTA. 2. The Cal+ ionophore A23187 strongly inhibited incorporation in reticulocytes incubated in 1mM Ca2+ but not in EGTA. Polysomal profiles and average ribosomal transit times of cells treated with Ca*+ ionophore at 1 mM Ca2+ were characteristic of translational elongation block. 3. The behavior of reticulocytes with respect to Ca2+ and A23187 contrasts with that of nucleated cells possessing endoplasmic reticulum in which protein synthesis is inhibited at translational initiation by either
Ca*+ depletion or by exposure to Ca2+ ionophore.
INTRODUCTION
eighth day blood was collected from the marginal ear vein
The endoplasmic reticulum is believed to function both in the processing of nascent peptides and as a major repository for intracellular sequestered CaZ+ sensitive to mobilization by inositol phosphates produced in response to a variety of hormones. Protein synthesis in most types of mammalian cells is rapidly inhibited by agents such as EGTA that withdraw internal Ca*+ into the extracellular fluid or by agents such as hormones or Ca*+ ionophores that mobilize internally sequestered Ca*+ (Brostrom et al., 1989a; Brostrom and Brostrom, 1990). These agents predominantly inhibit translational initiation rather than peptide chain elongation as determined by the disappearance of polysomes and unchanged average ribosomal transit times (Brostrom et al., 1989a). Maintenance by Ca*+ of high rates of translational initiation does not appear to depend on free cytosolic cation. In the present report the effects of Ca*+ on translation were studied in intact reticulocytes since these cells lack an endoplasmic reticulum. Ca*+ in conjunction with Ca*+ ionophore A23187 inhibited amino acid incorporation. The inhibition was expressed on peptide chain elongation rather than on translational initiation.
and reticulocytes prepared as described by Safer et al. (1979) after removing the top butTy coat of residual cells. The final washed preparation was resuspended in leucine-free Ham’s F-10 medium containing 1mM EGTA with or without I mM CaCl,.
[’ H&ucine incorporation Incorporations (0.5 ml) were conducted at 30°C for 90 min at 100 p M l&cine: Ca2+ ionophore was dissolved in dimethvlsulfoxide fMe,SO) for addition to the incubation: the concentration df M&S6 in the incubation (1%) was noi independently inhibitory. Following a 15-min equilibration period the incorporation was adjusted with [3H]~-leucine to a final specific radioactivity of 0.1 Ci/mmol. Incorporations were terminated with 1 ml of 10% trichloroacetic acid (TCA). The precipitated protein was prepared for analysis of radioactivity by scintillation counting as described previously (Brostrom et al., 1989b) except for the following modification to decolorize the samples. After the first collection of protein by centrifugation, redissolving with NaOH, and rep&pita&n with 5% TCA the samples were decolorized overnight at 4°C bv addition of 150 ul 30% H,O,. The protein was successively reprecipitated’and washed -3 more times to remove the peroxide.
purchased from ICN. hl othkr mat&ials were bf analytical grade.
Sucrose density gradient centrifugation Cells were collected by centrifugation and lysed with 0.5 ml lysis buffer (50 mM Tris, 25 mM KCl, 5 mM MgCI,, 1% Triton X-100, 1% sodium deoxycholate, 1 mg/ml heparin, lOpg/ml cycloheximide, pH 7.4). The lysate was clarified by centrifugation at 12,OOOg for IOmin. The post-mitochondrial supematant fraction (300 ~1) was subjected to sucrose density gradient centrifugation in an SW41 rotor as described previously (Brostrom et al., 1989b). Prior to unloading the gradients the top 0.5 ml containing the densely colored hemoglobin layer (approx. 0.5 ml) was removed by aspiration.
Preparation of intact rabbit reticulocytes Female (4-6 lb) New Zealand White rabbits were subcutaneously injected with 1% acetylphenylhydrazine dissolved in 40% ethanol (284pl/lb) for 5 consecutive days. On the
Ribosomai transit times Average ribosomal transit times were determined by the double isotope procedure of Ledford and Davis (1978) as described previously (Brostrom et al., 1989a).
MATERIALS AND METHODS
Materials A23 187 was purchased from Sigma. L-[4,5-’Hlleucine, 150 Ci/mmol. and L-IU-“Clleucine. 250 mCi/mmol. was
605
WAILAMWON0
606
et al.
IUSULTS Eflect of Ca2’ ionophore on amino acid ~cor~ration by intact rabbit retkulocytes The inhibitory effects on protein synthesis of Ca2+ depletion with EGTA or of Ca2+ mobilization with A23187 in various mammalian cell types normally occur within a few minutes of exposure and are reversible with increasing concentrations of extracellular Ca2+ (Brostrom et al., 1989a; Brostrom and Brostrom, 1990). The two agents in combination have been highly effective in rapidly suppressing amino acid incorporation in all cell lines tested. Amino acid incorporation in rabbit reticulocytes, however, was not subject to inhibition by EGTA and was only marginally affected by combined treatment with EGTA and A23187 (Table 1). A23187 strongly inhibited protein synthesis in reticulocytes incubated with Ca2’-containing medium.
Fig. 1.Effectof EGTA, Ca*+, and A23187on the pclysomal contents of intact rabbit reticulocytes. Reticulocytes were exposed for 30 rnin at 30°C to either 1 mM EGTA (A) and (C) or 1mM BGTA plus 2 mM CaCl, (B) and (D) with (C) and (D) or without (A) and (B) the addition of 0.5 PM A23187. The reticulocytes were lyzed and fractionated by sucrose density gradient centrifugation as described in Materials and Methods. 1989a), albeit at very different rates of initiation incorporation.
Eflects of CaZf and ionophore on thepolysome content of intact rabbit reticulocytes The nature of the inhibition by A23187 of translation in reticulocytes was examined by analyzing the effects of the agent on the size distribution of polysomes (Fig. 1). Polysomal size distributions were not changed by complexing Ca2+ in the incubation with EGTA [Fig. l(A)]. Polysome accumulation typical of elongation block occurred in response to 0.5 #hi A23187 for reticulocytes incubated with 1 mM free Ca2+ pig. l(D)]. Polysomes did not accumulate in response to A23187 when Ca” was complexed with EGTA [Fig. l(C)]. In contrast such mammalian cell types as GH, pituitary, HeLa, and CHO cells exhibit loss of polysomes when treated with similar concentrations of A23187 in Ca2+containing incubations (Brostrom et al., 1989a). Effect of Ca2+ and A23187 on reticulocyte average ribosomal transit times An analysis of average ribosomal transit times of reticulocytes treated with A23187 revealed that elongation times were extended by the ionophore (Table 2). With increasing con~nt~tions of A23187 in the presence of excess extracelhtlar Ca2+, the time needed to complete synthesis of an average protein rose progressively. In contrast nucleated cells, such as GH, cells, were previously found to have comparable average ribosomal transit times in samples treated with or without the ionophore (Brostrom et al.,
and
DISCUSSION
Protein synthesis in reticulocytes is largely involved with synthesizing the high concentrations of hemoglobin characteristic of mature red blood cells. Reticulocyte Iysates are a popular system for investigating the properties of translational initiation because the activity survives lysis of the cells and will support the synthesis of protein from exogenously added mRNA. This property is rem~kable since initiation in most mammalian cells is nearly eliminated by cell damage or disruption. In the present report protein synthesis in intact reticulocytes was observed to he unaffected by conditions that deplete intracellular Ca2+ stores including exposure to either EGTA or to combinations of EGTA and ionophore A23187. Comparable conditions have been consistently observed to inhibit translational initiation in a variety of nucleated cell types, including GHS pituitary, HeLa, CHO, and C6 glial tumor cells, that actively sequester Ca2+ in the endoplasmic reticulum, A considerable body of evidence suggests that the endoplasmic reticulum may control the rate of tr~slational initiation in such cells through an as yet undefined mechanism (Brostrom et al., 1989a; Brostrom and Brostrom, 1990). Clearly, initiation in reticulocytes Table 2. Average ribosomal transit times of intact rabbit reticulocvtes treated at various A23187 concentrations at I mM Ca2+ Ribosomal transit times (mm)
Table 1. Amino acid incorporation by intact rabbit reticulocytes treated at various A23187 concentrations in 1 mM ROTA with and without 2 mM CaCls L.eucine incorporation (pmol/mg protein) A23187 (nM)
-car+
0 - Me,SO 0+ Me,SG 30 50 100 300 1000
157 It 12 165i8 163k3 152 f 18 130 k 16 130*1 121 f 21
+Ca*+ 154f8 157 * 10 159 f 13 152*9 129k7 66 f I 29 + 4
Three replicates were conducted for each condition.
Experiment
Treatment -Ca2+ +Ca’+ +Ca2+ +Ca’+
+ + + c
1% Me,.‘30 1% Me,SO 0.8 pM A23187 I.OfiM A23187
1.1 0.7 2.8 5.4
1
Experiment 2 0.5 0.4 6.4 10.5
Reticutocytcs were equilibrated at 30°C for 30min in modiikd Ham’s medium wn~ni~ IOOpM lcucine and I mM EGTA with or without 2mM CaCI, and adjusted for 8min with Mess0 4 A23187. The sampks were then adjusted with [“C]Lleucine (2.5 &i/ml) for 60 min. PHjL-leucinc (25 @/ml) was added during the last 15 min of this period. Following addition of cycloheximide the cells were collected, lyzcd, and a post-mitochondriai fraction prepared for the subsoqucnt determination of transit times through analysis of isotope ratios in soluble vs soluble plus ribosomal bound peptides. Each value is calculated from values averaged from 6 replicate samplings for each post-mitochondrial and post-ribosomal supcmatant fraction.
Ca*+ and translation in reticulocytes
would not be subject to such regulation since these cells lack an endoplasmic reticulum. It is interesting to speculate that reticulocyte lysates retain high degrees of initiation because the process has been uncoupled from control by vesicular membranes prevalent in most other cell types. If so, the initiation system of reticulocytes may conceivably lack one or more key regulatory components. Exposure of intact reticulocytes to A23187 at 1 mM Ca2+ resulted in strong inhibition of amino acid incorporation. This inhibition was expressed at peptide chain elongation as evidenced by the accumulation of polysomes and in increased average ribosoma1 transit times. Reticulocyte lysates are well established to respond to Ca*+ with the phosphorylation and inhibition of elongation factor 2 (EF2) (Ryazanov, 1987; Ryazanov et al., 1988). This phosphorylation is calmodulin dependent (Ryazanov, 1987; Ryazanov et al., 1988; Naim et al., 1985; Naim and Palfrey, 1987) and is catalyzed by calmodulin dependent protein kinase III, an extensively characterized enzyme (Nairn et al., 1985; Naim and Palfrey, 1987). Phosphorylation of EF2 also occurs in various types of intact mammalian cells treated with agents that result in increased cytosolic free Ca’+ concentrations (Palfrey et ul., 1987; Mackie et al., 1989). Included here are fibroblasts treated with bradykinin, vasopressin, epidermal growth factor, or A23187 (Palfrey et al., 1987) and umbilical cord endothelial cells exposed to thrombin, histamine, or Ca2+ ionophore (Mackie et al., 1989). Inhibition of translation by this mechanism is normally of relatively short duration since increased free cytosolic Ca*+ concentrations are not sustained and dephosphorylation of EF2 occurs. Ca2+ ionophores, including A23187 and ionomycin, are hydrophobic molecules that have the ability to bind Ca*+ and facilitate diffusion of the cation across biological membranes. Relatively complex kinetics of Ca2+ movement prevail in cells that sequester the cation in internal vesicular structures such as the endoplasmic reticulum, since the ionophore facilitates passage of Ca*+ into the cytoplasm from these sites as well as by entry through the plasmalemma. While ionophores are conventionally thought to increase free cytosolic Ca2+, some cell types such as GHS pituitary cells respond with efflux of the cation with little change in cytosolic free concentration following a brief initial spike originating from internal release (Albert and Tashjian, 1984, 1986). The net direction of Ca2+ movement for a given cell type appears to be determined by whether ionophore fosters entry from the extracellular fluid at rates that overwhelm the plasmalemmal transport systems pumping the cation from the cell. Ca2+ is also pumped from cells in response to other substances that mobilize intracellular stores including hormones that elevate inositol trisphosphate (Berridge, 1987), small peptides, such as N-benzyloxycarbonyl-glycylphenylalanyl-amide, that mobilize Ca2+ independently of inositol phosphate generation (Lelkes and Pollard, 1987), and arachidonate (Wolf et al., 1986). For example, hepatocytes have been reported to extrude approx. 50% of their total intracellular Ca2+ in response to vasopressin or cr-adrenergic agonists (Berridge, 1987).
607
The lack of a prominent system within reticulocytes for the storage and release of Ca*+ in response to external stimuli simplifies Ca2+ homeostasis in these cells as monitored by effects on protein synthesis. Protein synthesis in intact reticulocytes was not affected by N-benzyloxycarbonyl-glycyl-phenylalanyl-amide (data not shown). Reticulocytes incubated with A23187 at 1 mM free Ca2+ as opposed to 1 mM EGTA exhibited a sustained inhibition of peptide chain elongation. A protracted elevation of intracellular Ca*+ would be predicted to maintain the phosphorylation and inhibition of EF2.
SUhlMARY
Rates of amino acid incorporation in intact rabbit reticulocytes were unaffected by depletion of Ca2+ with EGTA. The Ca2+ ionophore A23187 strongly inhibited incorporation in reticulocytes incubated in 1 mM Ca2+ but not in EGTA. Polysomal profiles and the extension of average ribosomal transit times of cells treated with Ca*+ ionophore at 1 mM Ca2+ were characteristic of translational elongation block. The behavior of reticulocytes with respect to Ca*+ and A23187 is at variance with that of nucleated cells possessing endoplasmic reticulum in which protein synthesis is inhibited at translational initiation by either Ca2+ depletion or by exposure to Ca*+ ionophore. The role of the endoplasmic reticulum in imposing a Ca2+ requirement on protein synthesis is discussed. Acknowledgemenl-This work was supported by National Institute of Health Grant DK 35393.
RRFERRNCES
Albert P. R. and Tashjian A. H. Jr (1984) Relationship of thyrotropin-releasing hormone-induced spike and plateau phases in cytosolic free Ca*+ concentrations to hormone secretion. Selective blockage using ionomycin and nifedipine. J. biol. Chem. 259, 15,350-l 5,363. Albert P. R. and Tashjian A. H. Jr (1986) Ionomycin acts as an ionophore to release TRH-regulated calcium stores from GH,?, cells. Am. J. PhysioL-251, C8874891. Berridne M. J. (1987) Inositol trisnhosohate and diacvlglycerol: two ‘interacting second me&igers. A. R& Eiochem. 56, 159-193. Brostrom C. 0. and Brostrom M. A. (1990) Calciumdependent regulation of protein synthesis in intact mammalian cells. A. Rev. Ph$ol. 52,- 577-590. Brostrom C. 0.. Chin K. V.. Wonn W. L.. Cade C. and Brostrom M. ‘A. (1989a) Inhibit& of translational initiation in eukaryotic cells by calcium ionophore. J. biol. Chem. 264, 16461649. Brostrom M. A., Lin X., Cade C., Gmitter D. and Brostrom C. 0. (1989b) Loss of a calcium requirement for protein synthesis in pituitary cells following thermal or chemical stress. J. biol. Chem. 264, 1638-1643. Ledford B. E. and Davis D. F. (1978) Analysis of translational parameters in cultured cells. Biochim. biophys. Acra 519, 201-212. Lelkes P. I. and Pollard H. B. (1987) Oligopeptide inhibitors of metalloendoprotease activity inhibit catecholamine secretion from bovine adrenal chromaIIin cells by modulating intracellular calcium homeostasis. J. biol. Gem. 262, 15,49615,505.
608
WN LMI WONG et al.
Mackie K. P., Naim A. C., Hampel G., Lam G. and JaiTe E. A. (1989) Thrombin and histamine stimulate the phosphdrylation of elongation factor 2 in human umbilical vein endothelial cells. J. biol. Chem. 264, 1748-1753. Naim A. C. and Palfrey H. C. (1987) Identification of the major Mr 100,000 substrate for calmodulin-dependent protein kinase III in mammalian cells as elongation factor-2. J. biol. Chem. 262, 17,299-17,303. Naim A. C., Bhagat B. and Palfrey H. C. (1985) Identification of calmodulin-dependent protein kinase III and its maior Mr 100,000 substrate in mammalian tissues. Proc. t&n. Acad. Sk. U.S.A. 82, 793F7943. Palfrey H. C., Nairn A. C., Muldoon L. L. and Villereal M. L. (1987) Ranid activation of calmodulin-deuendent protein‘ kin&e III in mitogen-stimulated human fibroblasts. Correlation with intracellular Ca2+ transients. J. biol. Chem. 262, 9785-9792.
Ryszanov A. G. (1987) Ca2+/cahnodulindependent phosnhorvlation of elongation factor 2. FEBS L&r. 214, j31-334.
Ryaranov A. G., Natapov P. G., Shestakova E. A., Severin F. F. and Spirin A. S. (1988) Phosphorylation of the elongation factor 2: the f&h Ca2+/cahnodulindependent system of protein phosphorylation. Biochimie 70, 619626. Safer B., Jagus R. and Kemper W. (1979) Analysis of initiation factor function in highly fractionated and unfractionated reticulocvte Meth. Enzvm. 60, __ lvsate svstems. _ 61-65. Wolf B. A., Turk J., Sherman W. R. and McDaniel M. L. (1986) Intracellular calcium mobilization bv arachidonic acid. Comparison with myo-inositol 1,4,E&isphosphate in isolated pancreatic islets. J. biof. Chem. 261, 3501-3511.
Vol. 23, No. S/6, pp. 605-608, Printed in Great Britain. All rightsreserved
0020-711X/91 $3.00 + 0.00
1991
Copyright 0 1991Pergamon Press plc
EFFECTS OF Ca2+ AND IONOPHORE A23187 ON PROTEIN SYNTHESIS IN INTACT RABBIT RETICULOCYTES Wlu LAMWONG, MARGARET A. BROSTROMand CHARLES0. BROSTROM Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, P&&away, NJ 08854, U.S.A. (Received I7 July 1990) Abstract-l. Amino acid incorporation in intact rabbit reticulocytes was unaffected by depletion of Ca*+ with EGTA. 2. The Cal+ ionophore A23187 strongly inhibited incorporation in reticulocytes incubated in 1mM Ca2+ but not in EGTA. Polysomal profiles and average ribosomal transit times of cells treated with Ca*+ ionophore at 1 mM Ca2+ were characteristic of translational elongation block. 3. The behavior of reticulocytes with respect to Ca2+ and A23187 contrasts with that of nucleated cells possessing endoplasmic reticulum in which protein synthesis is inhibited at translational initiation by either
Ca*+ depletion or by exposure to Ca2+ ionophore.
INTRODUCTION
eighth day blood was collected from the marginal ear vein
The endoplasmic reticulum is believed to function both in the processing of nascent peptides and as a major repository for intracellular sequestered CaZ+ sensitive to mobilization by inositol phosphates produced in response to a variety of hormones. Protein synthesis in most types of mammalian cells is rapidly inhibited by agents such as EGTA that withdraw internal Ca*+ into the extracellular fluid or by agents such as hormones or Ca*+ ionophores that mobilize internally sequestered Ca*+ (Brostrom et al., 1989a; Brostrom and Brostrom, 1990). These agents predominantly inhibit translational initiation rather than peptide chain elongation as determined by the disappearance of polysomes and unchanged average ribosomal transit times (Brostrom et al., 1989a). Maintenance by Ca*+ of high rates of translational initiation does not appear to depend on free cytosolic cation. In the present report the effects of Ca*+ on translation were studied in intact reticulocytes since these cells lack an endoplasmic reticulum. Ca*+ in conjunction with Ca*+ ionophore A23187 inhibited amino acid incorporation. The inhibition was expressed on peptide chain elongation rather than on translational initiation.
and reticulocytes prepared as described by Safer et al. (1979) after removing the top butTy coat of residual cells. The final washed preparation was resuspended in leucine-free Ham’s F-10 medium containing 1mM EGTA with or without I mM CaCl,.
[’ H&ucine incorporation Incorporations (0.5 ml) were conducted at 30°C for 90 min at 100 p M l&cine: Ca2+ ionophore was dissolved in dimethvlsulfoxide fMe,SO) for addition to the incubation: the concentration df M&S6 in the incubation (1%) was noi independently inhibitory. Following a 15-min equilibration period the incorporation was adjusted with [3H]~-leucine to a final specific radioactivity of 0.1 Ci/mmol. Incorporations were terminated with 1 ml of 10% trichloroacetic acid (TCA). The precipitated protein was prepared for analysis of radioactivity by scintillation counting as described previously (Brostrom et al., 1989b) except for the following modification to decolorize the samples. After the first collection of protein by centrifugation, redissolving with NaOH, and rep&pita&n with 5% TCA the samples were decolorized overnight at 4°C bv addition of 150 ul 30% H,O,. The protein was successively reprecipitated’and washed -3 more times to remove the peroxide.
purchased from ICN. hl othkr mat&ials were bf analytical grade.
Sucrose density gradient centrifugation Cells were collected by centrifugation and lysed with 0.5 ml lysis buffer (50 mM Tris, 25 mM KCl, 5 mM MgCI,, 1% Triton X-100, 1% sodium deoxycholate, 1 mg/ml heparin, lOpg/ml cycloheximide, pH 7.4). The lysate was clarified by centrifugation at 12,OOOg for IOmin. The post-mitochondrial supematant fraction (300 ~1) was subjected to sucrose density gradient centrifugation in an SW41 rotor as described previously (Brostrom et al., 1989b). Prior to unloading the gradients the top 0.5 ml containing the densely colored hemoglobin layer (approx. 0.5 ml) was removed by aspiration.
Preparation of intact rabbit reticulocytes Female (4-6 lb) New Zealand White rabbits were subcutaneously injected with 1% acetylphenylhydrazine dissolved in 40% ethanol (284pl/lb) for 5 consecutive days. On the
Ribosomai transit times Average ribosomal transit times were determined by the double isotope procedure of Ledford and Davis (1978) as described previously (Brostrom et al., 1989a).
MATERIALS AND METHODS
Materials A23 187 was purchased from Sigma. L-[4,5-’Hlleucine, 150 Ci/mmol. and L-IU-“Clleucine. 250 mCi/mmol. was
605
WAILAMWON0
606
et al.
IUSULTS Eflect of Ca2’ ionophore on amino acid ~cor~ration by intact rabbit retkulocytes The inhibitory effects on protein synthesis of Ca2+ depletion with EGTA or of Ca2+ mobilization with A23187 in various mammalian cell types normally occur within a few minutes of exposure and are reversible with increasing concentrations of extracellular Ca2+ (Brostrom et al., 1989a; Brostrom and Brostrom, 1990). The two agents in combination have been highly effective in rapidly suppressing amino acid incorporation in all cell lines tested. Amino acid incorporation in rabbit reticulocytes, however, was not subject to inhibition by EGTA and was only marginally affected by combined treatment with EGTA and A23187 (Table 1). A23187 strongly inhibited protein synthesis in reticulocytes incubated with Ca2’-containing medium.
Fig. 1.Effectof EGTA, Ca*+, and A23187on the pclysomal contents of intact rabbit reticulocytes. Reticulocytes were exposed for 30 rnin at 30°C to either 1 mM EGTA (A) and (C) or 1mM BGTA plus 2 mM CaCl, (B) and (D) with (C) and (D) or without (A) and (B) the addition of 0.5 PM A23187. The reticulocytes were lyzed and fractionated by sucrose density gradient centrifugation as described in Materials and Methods. 1989a), albeit at very different rates of initiation incorporation.
Eflects of CaZf and ionophore on thepolysome content of intact rabbit reticulocytes The nature of the inhibition by A23187 of translation in reticulocytes was examined by analyzing the effects of the agent on the size distribution of polysomes (Fig. 1). Polysomal size distributions were not changed by complexing Ca2+ in the incubation with EGTA [Fig. l(A)]. Polysome accumulation typical of elongation block occurred in response to 0.5 #hi A23187 for reticulocytes incubated with 1 mM free Ca2+ pig. l(D)]. Polysomes did not accumulate in response to A23187 when Ca” was complexed with EGTA [Fig. l(C)]. In contrast such mammalian cell types as GH, pituitary, HeLa, and CHO cells exhibit loss of polysomes when treated with similar concentrations of A23187 in Ca2+containing incubations (Brostrom et al., 1989a). Effect of Ca2+ and A23187 on reticulocyte average ribosomal transit times An analysis of average ribosomal transit times of reticulocytes treated with A23187 revealed that elongation times were extended by the ionophore (Table 2). With increasing con~nt~tions of A23187 in the presence of excess extracelhtlar Ca2+, the time needed to complete synthesis of an average protein rose progressively. In contrast nucleated cells, such as GH, cells, were previously found to have comparable average ribosomal transit times in samples treated with or without the ionophore (Brostrom et al.,
and
DISCUSSION
Protein synthesis in reticulocytes is largely involved with synthesizing the high concentrations of hemoglobin characteristic of mature red blood cells. Reticulocyte Iysates are a popular system for investigating the properties of translational initiation because the activity survives lysis of the cells and will support the synthesis of protein from exogenously added mRNA. This property is rem~kable since initiation in most mammalian cells is nearly eliminated by cell damage or disruption. In the present report protein synthesis in intact reticulocytes was observed to he unaffected by conditions that deplete intracellular Ca2+ stores including exposure to either EGTA or to combinations of EGTA and ionophore A23187. Comparable conditions have been consistently observed to inhibit translational initiation in a variety of nucleated cell types, including GHS pituitary, HeLa, CHO, and C6 glial tumor cells, that actively sequester Ca2+ in the endoplasmic reticulum, A considerable body of evidence suggests that the endoplasmic reticulum may control the rate of tr~slational initiation in such cells through an as yet undefined mechanism (Brostrom et al., 1989a; Brostrom and Brostrom, 1990). Clearly, initiation in reticulocytes Table 2. Average ribosomal transit times of intact rabbit reticulocvtes treated at various A23187 concentrations at I mM Ca2+ Ribosomal transit times (mm)
Table 1. Amino acid incorporation by intact rabbit reticulocytes treated at various A23187 concentrations in 1 mM ROTA with and without 2 mM CaCls L.eucine incorporation (pmol/mg protein) A23187 (nM)
-car+
0 - Me,SO 0+ Me,SG 30 50 100 300 1000
157 It 12 165i8 163k3 152 f 18 130 k 16 130*1 121 f 21
+Ca*+ 154f8 157 * 10 159 f 13 152*9 129k7 66 f I 29 + 4
Three replicates were conducted for each condition.
Experiment
Treatment -Ca2+ +Ca’+ +Ca2+ +Ca’+
+ + + c
1% Me,.‘30 1% Me,SO 0.8 pM A23187 I.OfiM A23187
1.1 0.7 2.8 5.4
1
Experiment 2 0.5 0.4 6.4 10.5
Reticutocytcs were equilibrated at 30°C for 30min in modiikd Ham’s medium wn~ni~ IOOpM lcucine and I mM EGTA with or without 2mM CaCI, and adjusted for 8min with Mess0 4 A23187. The sampks were then adjusted with [“C]Lleucine (2.5 &i/ml) for 60 min. PHjL-leucinc (25 @/ml) was added during the last 15 min of this period. Following addition of cycloheximide the cells were collected, lyzcd, and a post-mitochondriai fraction prepared for the subsoqucnt determination of transit times through analysis of isotope ratios in soluble vs soluble plus ribosomal bound peptides. Each value is calculated from values averaged from 6 replicate samplings for each post-mitochondrial and post-ribosomal supcmatant fraction.
Ca*+ and translation in reticulocytes
would not be subject to such regulation since these cells lack an endoplasmic reticulum. It is interesting to speculate that reticulocyte lysates retain high degrees of initiation because the process has been uncoupled from control by vesicular membranes prevalent in most other cell types. If so, the initiation system of reticulocytes may conceivably lack one or more key regulatory components. Exposure of intact reticulocytes to A23187 at 1 mM Ca2+ resulted in strong inhibition of amino acid incorporation. This inhibition was expressed at peptide chain elongation as evidenced by the accumulation of polysomes and in increased average ribosoma1 transit times. Reticulocyte lysates are well established to respond to Ca*+ with the phosphorylation and inhibition of elongation factor 2 (EF2) (Ryazanov, 1987; Ryazanov et al., 1988). This phosphorylation is calmodulin dependent (Ryazanov, 1987; Ryazanov et al., 1988; Naim et al., 1985; Naim and Palfrey, 1987) and is catalyzed by calmodulin dependent protein kinase III, an extensively characterized enzyme (Nairn et al., 1985; Naim and Palfrey, 1987). Phosphorylation of EF2 also occurs in various types of intact mammalian cells treated with agents that result in increased cytosolic free Ca’+ concentrations (Palfrey et ul., 1987; Mackie et al., 1989). Included here are fibroblasts treated with bradykinin, vasopressin, epidermal growth factor, or A23187 (Palfrey et al., 1987) and umbilical cord endothelial cells exposed to thrombin, histamine, or Ca2+ ionophore (Mackie et al., 1989). Inhibition of translation by this mechanism is normally of relatively short duration since increased free cytosolic Ca*+ concentrations are not sustained and dephosphorylation of EF2 occurs. Ca2+ ionophores, including A23187 and ionomycin, are hydrophobic molecules that have the ability to bind Ca*+ and facilitate diffusion of the cation across biological membranes. Relatively complex kinetics of Ca2+ movement prevail in cells that sequester the cation in internal vesicular structures such as the endoplasmic reticulum, since the ionophore facilitates passage of Ca*+ into the cytoplasm from these sites as well as by entry through the plasmalemma. While ionophores are conventionally thought to increase free cytosolic Ca2+, some cell types such as GHS pituitary cells respond with efflux of the cation with little change in cytosolic free concentration following a brief initial spike originating from internal release (Albert and Tashjian, 1984, 1986). The net direction of Ca2+ movement for a given cell type appears to be determined by whether ionophore fosters entry from the extracellular fluid at rates that overwhelm the plasmalemmal transport systems pumping the cation from the cell. Ca2+ is also pumped from cells in response to other substances that mobilize intracellular stores including hormones that elevate inositol trisphosphate (Berridge, 1987), small peptides, such as N-benzyloxycarbonyl-glycylphenylalanyl-amide, that mobilize Ca2+ independently of inositol phosphate generation (Lelkes and Pollard, 1987), and arachidonate (Wolf et al., 1986). For example, hepatocytes have been reported to extrude approx. 50% of their total intracellular Ca2+ in response to vasopressin or cr-adrenergic agonists (Berridge, 1987).
607
The lack of a prominent system within reticulocytes for the storage and release of Ca*+ in response to external stimuli simplifies Ca2+ homeostasis in these cells as monitored by effects on protein synthesis. Protein synthesis in intact reticulocytes was not affected by N-benzyloxycarbonyl-glycyl-phenylalanyl-amide (data not shown). Reticulocytes incubated with A23187 at 1 mM free Ca2+ as opposed to 1 mM EGTA exhibited a sustained inhibition of peptide chain elongation. A protracted elevation of intracellular Ca*+ would be predicted to maintain the phosphorylation and inhibition of EF2.
SUhlMARY
Rates of amino acid incorporation in intact rabbit reticulocytes were unaffected by depletion of Ca2+ with EGTA. The Ca2+ ionophore A23187 strongly inhibited incorporation in reticulocytes incubated in 1 mM Ca2+ but not in EGTA. Polysomal profiles and the extension of average ribosomal transit times of cells treated with Ca*+ ionophore at 1 mM Ca2+ were characteristic of translational elongation block. The behavior of reticulocytes with respect to Ca*+ and A23187 is at variance with that of nucleated cells possessing endoplasmic reticulum in which protein synthesis is inhibited at translational initiation by either Ca2+ depletion or by exposure to Ca*+ ionophore. The role of the endoplasmic reticulum in imposing a Ca2+ requirement on protein synthesis is discussed. Acknowledgemenl-This work was supported by National Institute of Health Grant DK 35393.
RRFERRNCES
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