Changes in microtubular tau protein after morphine in a cultured human neuroblastoma cell line

ISSN 0306.3623/97 $t7.00 + .00 PII S0306-3623(97)00030-X All rights reserved

Gen. Pharmac. Vol. 29, No. 5, pp. 869-872, 1997 Copyright © 1997 Elsevier Science lnc. Printed in the USA. ELSEVIER

Changes in Microtubular Tau Protein after Morphine in a Cultured Human Neuroblastoma Cell Line G. M. Lew DEPARTMENTOF ANATOMY,COLLEGEOF HUMAN MEDICINE, MICHIGAN STATEUNIVERSITY,GILTNERHALL, EASTLANSING,MI 48824, USA

ABSTRACT. 1. Cultured human SH-SY5Y adrenergic neuroblastoma cells were used to examine the action of morphine sulfate on microtubular tau protein. 2. After 48 hr treatment morphine sulfate (200 IxM) reduced tau protein in the cytoplasmic (supernatant) fraction of undifferentiated cells, and in the cytoplasmic as well as membrane (pellet) fractions of differentiated cells. 3. A 71% increase ( P < 0.05) in total protein in the membrane ( pellet ) fraction of undifferentiated cells and a 188% increase (P<0.01) in that of differentiated cells accompanied the decrease in tau protein. 4. A 51% reduction (P<0.01) in the number of undifferentiated (but not differentiated) cells was seen after this drug (200 IxM). GEN PHAmaAC 29;5:869--872, 1997. © 1997 Elsevier Science Inc. KEY WORDS. Tau, morphine, SH-SY5Y cells

INTRODUCTION More research is necessary to explore the exact cellular and molecular mechanisms involved in morphine action. Previous studies have shown that morphine inhibits cell growth, DNA, RNA, and protein synthesis in epidermal carcinoma cells (Nassiri et al., 1991). Motphine has also been shown to modify the growth of astrocytes as well as the rate of DNA synthesis in cerebellar neuroblasts in vitro (Hauser et al., 1994). A decrease in intracellular protein synthesis and inhibition of proteolysis have also been reported after morphine treatment (Pan e t al., 1993 ). The results of Harlan and Song ( 1994 ) suggest that prenatal exposure to morphine disrupts neuronal migration and/or cell survival. The evidence also indicates that morphine and other commonly used opiates have profound effects on the proliferation of immature and mature immune cells (Loh et al., 1993). The results of Same et al. (1994) indicated that opiates exerted either inhibition or augmentation of transmitter release. Tau protein that promotes microtubule assembly and stabilizes microtubules (Drubin and Kirschner, 1986), thus affecting the transport of neurotransmitters, is a major component of paired helical filaments (PHFs), which are the main components of neurofibrillary tangles, that have been described in a variety of chronic neurological diseases such as Alzheimer disease, Down syndrome, and lead encephalopathy (Delacourte and Defossez, 1986; Kowall and Kosik, 1987; Wisneiwski et al., 1979). The adrenergic human neuroblastoma SH-SY5Y cell line has been found to be a useful model system for studying the action of opiates as they have been shown to express both the mu and delta opioid receptors (Kazmi and Mishra, 1986; Sadee et al., 1987). The purpose of this study was to examine the action of morphine sulfate on human neuroblastoma cell proliferation and on tau protein in these cells.

Received 18 March 1996; revised 9 December 1996; accepted 10 February 1997.

MATERIALS A N D METHODS Cell c u l t u r e , t r e a t m e n t , a n d h a r v e s t i n g Human adrenergic neuroblastoma SH-SY5Y cells, established by Biedler et o2. (1973), were obtained from the Cell Culture Facility at the University of California, San Francisco. The cells were grown in 100× 20-mm Falcon Culture dishes in a humidified incubator at 37°C in basal medium (1:1 mixture of Ham's F-12: Dulbecco's Modified Eagle Medium; Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 1% glutamine (Sigma). The medium, 10 ml/dish, was changed every other day. Upon reaching confluence in 5-6 days undifferentiated cells were split 1:3. One day after plating the medium was changed and undifferentiated cells were treated for 48 hr with morphine sulfate (200, 100, and 10 p~M) dissolved in water. Control plates were treated with the diluent. Dilutions were made immediately before use. SH-SY5Y cells differentiate and form processes morphologically similar to neurons upon exposure to retinoic acid (Sidell et al., 1983). SH-SY5Y cells were differentiated by culturing the ceils in medium supplemented with 10 I~M retinoic acid (Sigma) for 6 days before 48 hr treatment with morphine sulfate (200, 100, 10 txM). The retinoic acid medium was changed every other day. At the conclusion of the experiment the cells were washed briefly in phosphate-buffered saline (PBS), wanned to 37°C, pH 7.4, and harvested with the aid of a rubber policeman in 1 ml TE PBS (10 mM Tris, 1 mM EDTA in PBS). The cells were then centrifuged for 5 min at 3,000 g and resuspended in 1 ml TE PBS. The suspension was recentrifuged after a 10-ml aliquot was counted with the aid of a hemacytometer. The supematant was removed and each pellet was resuspended in 50 txl cell lysis buffer (0.01 M Tris, pH 8.3, 1 mM EDTA, 0.1 mM phenyl methyl sulfonyl fluoride, 10 I~g/ml leupeptin, 10 txg/ml pepstatin A, 10 I~g/ml aprotinin, 5 mM benzamidine) and frozen at -70°C. To separate the cytoplasmic (supematant) fraction from the membrane (pellet) fraction the samples were spun for 2 min after thawing at 8,800 g. After resuspension in 50 txI cell

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G.M. Lew TABLE 1. Decrease in undifferentiated SH-SY5Y neuroblastoma cells after morphine sulfate Number of cells in millions Morphine sulfate 200 i.tM 100 ~M 10 ~M

Experimental

Control

Difference (%)

P

2.9 -+ 0.2 7.5 -+ 0.6 4.0 --+ 0.15

6.0 -+ 0.03 6.6 +-- 0.03 6.3 -+ 0.12

51 13 36

<0.01 NS NS

P = probability based on Student-Fisher t-test. Each value represents the mean + SE of three individual cultures.

lysis buffer the pellet was sonicated at 30,000 Hz for 5 sec. In both supernatant and pellet fractions total protein was determined according to the method of Bradford (1976).

SDS.PAGE and Western blot analysis Using a modification of the method of Laemmli (1970) one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. Samples were prepared in a solution containing 10% glycerol, 18.4% SDS, 0.5 M Tris, pH 6.8, with a final concentration of 0.01 M dithiothreitol added as a reducing agent. After the samples were heated at 100°C for 3 min they were centrifuged to remove any precipitated material prior to loading gels. The supernatants were then electrophoresed in SDS polyacrylamide gels containing 12% acrylamide. Running buffer consisted of 0.025 M Tris-HCl, 0.192 M glycine, 0.1% SDS. Minigels were run at a constant voltage (90 V) until samples entered the separating gel (12% acrylamide, 0.8% bisacrylamide, Bio-Rad, Hercules, CA, USA), when the voltage was increased to 110 V. Proteins separated by SDS-PAGE were blotted onto nitrocellulose membrane (Bio-Rad; 0.45/~M) for 1 hr at 12 V in a horizontal blotting apparatus (Bio-Rad) with the use of a blot buffer (25 mM Tris, 192 mM glycine, and 20% methanol). The nitrocellulose was incubated overnight at 4°C in blocking buffer (5% milk powder, 0.05 Tween 20 in PBS), and then reincubated with fresh blocking buffer for 1 hr at room temperature with constant shaking. The blot was then washed five times with Tris/saline (0.01 M Tris-base, 0.14 M NaC|, pH 7.6), and incubated (2 hr, 23°C) with a 1:1,000 dilution of mouse monoclonal antibody to tau called Tau-1 (from Dr. L. I. Binder, University of Alabama, Birmingham, through Dr. Gene Mesco, Dept. of Biology, Moorehead State Univ., MN), or with a

1:200 dilution of a mouse monoclonal antibody to tau from Boehringer-Mannheim Co. (Indianapolis, IN, USA). The blot was washed five times with Tris/saline, pH 7.6 after the incubation, and was then placed on the shaker for 5 min in fresh Tris/saline. The blot was then incubated for 30 min with an alkaline-phosphataseconjugated second antibody (Sigma) diluted 1:1,000, and then washed for 1 hr. It was then developed with the use of substrates, nitroblue tetrazolium (50 mg/ml in 70% dimethyl formamide and 5bromo-4-chloro-3-indolyl phosphate) (50 mg/ml in 100% dimethyl formamide) (Sigma) in Tris/saline, pH 8.9. RESULTS Treatment of undifferentiated SH-SY5Y human neuroblastoma cells with morphine sulfate (200 IxM) caused a 51% reduction (P<0.01) in cell number (Table 1) and a 71% increase (P<0.05) in total protein in the membrane (pellet) faction (Table 2). Tau protein (phosphorylated and nonphosphorylated forms), which appeared in these cells chiefly as an electrophoretically separated 50-kDa band, was reduced in the cytoplasmic (supernatant) fraction (Figs. 1 and 2) by morphine sulfate (200 ~M). In differentiated SH-SY5Y neuroblastoma cells tau protein ap-

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TABLE 2. Total protein in undifferentiated SH-SY5Y neuroblastoma cells after morphine sulfate, as determined by the method of Bradford (1976), is expressed as mean - SE of three individual cultures Total protein (Ixg/million cells) Morphine sulfate

Cytoplasmic fraction Experimental Control Difference (%) P Membrane fraction Experimental Control Difference (%) P

200 IxM

100 ~M

10 ~M

87.2 -+ 11 58.3 + 10 50 NS

42.5 + 16 57.5 + 8 26 NS

67.0 + 9 50.5 + 10 33 NS

177.5 + 22 103.8 + 7.5 71 < 0.05

80.5 + 9 73.5 + 3 10 NS

108 + 8 73.5 + 13 47 NS

P = probability based on Student-Fisher t-test.

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2

FIGURE 1. Western analysis of cytoplasmic (supernatant) fraction of undifferentiated SH-SY5Y human neuroblastoma cells after 48 hr treatment with morphine sulfate (200 IxM). Samples were prepared in Laemmli sample buffer; 40 p.g of protein was loaded onto each lane and separated by SDS-PAGE. Western blots were performed using a mouse monoclonal antibody to tau protein (Boehringer-Mannheim) that recognizes phosphorylated and nonphosphorylated forms of tau. Lane 1,200 IxM morphine sulfate; lane 2, control.

Tau after Morphine in SH-SY5Y Cells

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50 kD

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35kD

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FIGURE 2. Western analysis of cytoplasmic (supernatant) fraction of undifferentiated SH-SY5Y human neuroblastoma cells after 48 hr treatment with morphine sulfate (200 p.M). Samples were prepared in Laemmli sample buffer; 40 p-g of protein was loaded onto each lane and separated by SDS-PAGE. Western blots were performed using a mouse monoc|onal antibody to tau protein, tau-1, which recognizes nonphosphorylated tau. Lane 1,200 p-M; lane 2, control.

peared as three major electrophoretically separated 35-, 42-, and 50kDa bands with some differences between cytoplasmic and membrane fractions (Figs. 3 and 4). Treatment with morphine sulfate resulted in no significant changes in cell numbers but in a significant 188% increase (P<0.01) in the total protein of the membrane (pellet) fractions of these differentiated cells (Table 3). Tau protein was markedly reduced in the cytoplasmic (supemarant) as well as the membrane (pellet) fractions of these differentiated cells showing decreases in phosphorylated and nonphosphorylated forms of tau protein (Figs. 3 and 4). DISCUSSION The reduction in the number of undifferentiated cells observed after morphine sulfate treatment is generally consistent with the decrease in cell number in the caudate-putamen region of the embryonic rodent brain (Harlan and Song, 1994) after prenatal morphine treatment, and with the reported inhibitory effect of opiates on neuronal development in vivo and in vitro (Hammer et al., 1989; Zagon and McLaughlin, 1984). It is also consistent with the reported 70% decrease in bone marrow cell proliferation after 72-hr morphine treatment (Loh et al., 1993) and with the reported inhibition of cell growth in human epidermal carcinoma cells after morphine treatment (Nassiri et al., 1991). The decrease in tau protein is consistent with the inhibition in protein synthesis described by the latter in epidermal carcinoma cells and by Pan eta/. (1993) in cultured mesangial cells. The increases in total protein in the membrane (pellet) fractions of undifferentiated and differentiated cells may be explained by a decrease in degradation of cellular protein as described by Pan et al. (1993) as accompanying the decrease in intracellular protein synthesis. More studies are needed to determine the exact cellular and molecular mechanisms responsible for the role of morphine and other

I

2

3

4

FIGURE 3. Western analysis of cytoplasmic (supernatant) and membrane (pellet) fractions of differentiated SH-SY5Y human neuroblastoma cells after 48 hr treatment with morphine sulfate (200 p-M). Samples were prepared in Laemmli sample buffer, 40 p-g of protein was loaded onto each lane and separated by SDSPAGE. Western blots were performed by using a mouse monoclonal antibody to tau protein (Boehringer-Mannheim), which recognized phosphorylated and nonphosphorylated forms of tau. Cytoplasmic fractions: Lane 1,200 p-M morphine sulfate; lane 2, control. Membrane fractions: Lane 3 , 2 0 0 p-M morphine sulfate; lane 4, control.

drugs of abuse in altering the expression of specific genes in different neuronal types, thereby producing the physical and psychologic actions of drug addiction (Nestler, 1993). However, it is known that chronic exposure to morphine induces mu opiate receptor down-regulation (Zadina et al., 1994). In addition, in the locus coeruleus up-regulation of the cyclic AMP pathway has been shown to contribute to opiate tolerance, dependence and withdrawal. In the mesolimbic dopamine system, which is implicated in the psychological aspects of drug addiction, a similar up-regulation of the cyclic AMP pathway has been seen in response to chronic opiate treatment (Nestler, 1993). Attempts are being made to identify the mechanisms by which opiates regulate the expression of intracellular messenger proteins in the locus coeruleus, which is a relatively homogeneous brain region, and that has been characterized electrophysiologically and anatomically (Nestler et al., 1994). The decrease in tau protein observed after morphine sulfate in this experiment probably resulted, via genomic action of this drug on tau, in a reduction in microtubule assembly leading to a decrease in the transport of neurotransmitters. However, more studies are needed with the use of these undifferentiated and differentiated SH-SY5Y neuroblastoma cells to examine the precise cellular and molecular mechanisms by which morphine exerts its effects on tau protein and other microtubule-associated proteins. SUMMARY Human SH-SY5Y adrenergic neuroblastoma cells were used to examine the effect of morphine sulfate on cell number, total protein, and tau protein, which promotes microtubule assembly. The cells

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G . M . Lew crotubular tau protein in the cytoplasmic (supematant) fraction. The drug (200 IxM) also caused a 71% increase in total protein in the membrane (pellet) fraction of these undifferentiated cells and a 188% increase in. total protein in this same fraction of differentiated cells. In addition, morphine sulfate (200/~M) markedly reduced tau protein in cytoplasmic (supernatant) and membrane (pellet) fractions of differentiated cells.

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References

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3

4

F I G U R E 4. Western analysis of cytoplasmic (supernatant) and membrane (pellet) fractions of differentiated SH-SY5Y human neuroblastoma cells after 48 hr treatment with morphine sulfate (200 IzM). Samples were prepared in Laemmli sample buffer; 40 l~g protein were loaded onto each lane and separated by SDSP A G E . Western blots were performed by using a mouse monoclonat antibody to tau protein, tau-1, which recognizes nonphosphorylated tau. Cytoplasmic fractions: Lane 1 , 2 0 0 ~ M morphine sulfate; lane 2, control. Membrane fractions: Lane 3, 200 I~M morphine sulfate; lane 4, control.

were treated for 48 hr with morphine sulfate (200, 100, and 10 p-M), and were then harvested and counted with the aid of a hemacytometer. Total protein was determined according to the method of Bradford. Separation of proteins by SDS-PAGE was followed by Western blots. Morphine sulfate (200 p.M) caused a 51% reduction in the number of undifferentiated cells as well as a decrease in mi-

T A B L E 3 . Total protein in differentiated SH-SY5Y neuroblastoma cells after morphine sulfate, as determined by the method of Bradford (1976), is expressed as mean -+ SE of three individual cultures Total protein (i~g/million cells) Morphine sulfate

Cytoplasmic fraction Experimental Control Difference (%) P Membrane fraction Experimental Control Difference (%) P

200 IzM

100 I~M

10 IzM

44.0 + 5 47.4 + 7 7 NS

65.7 + 19 55.6 -+ 6 18 NS

106 +- 31 133 +- 35 20 NS

130.5 -+ 25 45.3 -+ 3 188 < 0.01

59.8 -+ 3 5 54.4 -+ 7 10 NS

85.1 + 22 106 -+ 29 20 NS

P = probability based on Student-Fisher t-test.

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