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1 , , 51 (6), Acta Physiologica Sinica SK2N2SH (, 2004) SK2N2SH, SK2N2SH : SK2N2SH Na + Cl -,, mol/ L, Ca 2 + SK2N2SH : ; SK2N2SH ; ; ; : Q46,, [1 ],,, [2, ],, SK2N2SH, SK2N2SH,, SK2N2SH SK2N2SH, (No190100, ) :, Tel : E2mail :

2 (CORT) ; (P) ; ( E 2 ) ; (DEX) (Sigma ), : 10-6 mol/ L 10-7 mol/ L (DOC) (Sigma ), 10-2 mol/ L, 10-6 mol/ L 10-7 mol/ L 11 H2, 548 GBq/ mmol, 7 MBq/ ml, 9716 % (Amersham ) 114 HEPES, Merck, Canberra Company (B2BSA) ( GLY) Sigma 115 RPM1640 ( 10 % ), 5 % CO 2 95 % 7 CO 2,, 0125 %,, 1000 / min 5 min, RPM1640, 24, 4 d, [ 116 Zafra ], 24, 1 ml Krebs2HEPES 2 Krebs2HEPES 015 ml, : (1) : 1 mol/ L, 7 kbq (2) : 1 mol/ L, 7 kbq () : 1 mol/ L, 7 kbq,, 10 6, 10-6,, Na + Cl -, Krebs2HEPES, Ca 2 +, Krebs2HEPES Ca 2 +, EGTA ( 2 mmol/ L), CO 2 15 min( 5, 10, 15, 20 min),, Krebs2HEPES, 015 mol/ L NaOH 015 ml 5 6 h, 01 ml 2 ml, cpm dpm, 011 ml SK2N2SH, 10-5 mol/ L 015 ml 50 min, , 5, 4, x s x, t SK2N2SH 10-6 mol/ L Na + Cl -, SK2N2SH, Krebs2HEPES NaCl Na 2 SO 4, Na + Cl -, Na + Cl - 4, 5, x s x, Na +, Cl pmol/ (g1min), pmol/ (g1 min), pmol/

3 6 : SK2N2SH 605 (g1min) : Na + Cl -, SK2N2SH ( P 0101), SK2N2SH Na + Cl SK2N2SH mol/ L, mol/ L, 10-6 mol/ L ( 1) 1 SK2N2SH Fig11 Rapid effects of CORT of different concentration on GL Y uptake in SK2N2SH cells Numbers in abscissa represent concen2 tration in mol/ L. P < 0105 vs control ; P < 0101, vs control ; P < 01001, vs control. Con represents control SK2N2SH 15 min, 15 min ( 2) 2 SK2N2SH Fig12 Time course of GL Y uptake in SK2N2 SH cells in the presence of CORT Values are expressed as percentage s x of changes with respect to control uptake and represent the aver2 age of five experiments in quadruplicate. The results of control at 5, 10, 15, 20 min are respectively are , , , pmol/ (g1min). The concentration of CORT was at 10-7 mol/ L , V max, V max Km, 1 CMP SK2N2SH V max Km Table 1 changes of V max and Km of GLY uptake in SK2N2SH cells in the presence of CORT or CMP Item Control CORT (10-7 ) CMP (10-4 ) V max [ nmol/ (g1min) ] Km ( M) Numbers in brackets represent concentration in mol/ L1 CPM is the inhibitor of glycine transporter. 0101, P < 01001, vs control. P <

4 Ca 2 + SK2N2SH 2 Ca 2 +, SK2N2SH 2 Ca 2 + SK2 SK2N2SH N2SH Table 2 Influence of Ca 2 + in the external me2 dium on the rapid stimulating effects of CORT on GLY uptake in SK2N2SH cells Treatment Uptake [ nmol/ (g1min) ] Control 2Ca 2 + CORT(10-7 ) + Ca 2 + CORT (10-7 ) - Ca # Numbers in brackets represent concentration in mol/ L. P < 0105, CORT + Ca 2 +. P > 0105 vs control ; # P < 0105 vs Table Influence of cycloheximide on the rapid stimulating effects of CORT on the GLY uptake in SK2N2SH cells Treatment Uptake [pmol/ (g1min) ] Control CORT (10-7 ) Cycloheximide (10-5 ) CORT(10-7 ) + cycloheximide(10-5 ) Numbers in brackets represent concentration in mol/ L. P < 0101 vs control. 21 SK2N2SH 2111 B2BSA 10-7 mol/ L :, pmol/ (g1min) ; B2BSA, pmol/ (g1min) ;, pmol/ (g1min) ; B2BSA ( P < 0101), ( P > 0105) 2112 SK2N2SH, 211 SK2N2SH,, ( ) SK2N2SH Fig1 Rapid effects of various steroids on GLY uptake in SK2N2SH cells Values are expressed as x s x and repre2 sent the average of five experiments in tri2 plicate. The concentrations of various steroids were at 10-7 mol/ L. P < 0105 vs control ; P < 0101 vs control. Con represents control. SK2N2SH, Na + Cl -,

5 6 : SK2N2SH 607 [1 ],, [4, 0 min ], B2BSA [5 ],,,, SK2N2SH, Km,,, 50 min,, 10-9 mol/ L,, mol/ L, 10-6 mol/ L, Ca 2 +, Ca 2 +,, C, Ca 2 +, Ca 2 + ;, Ca 2 + [1 ] Bi G ( ), Zhu BG ( ), Chen YZ ( ). Rapid effects of steroids on glycine uptake in cerebral cortex synaptosomes. Chin J Neurosci ( ), 1998 ; 14 (4) : (in Chinese with English abstract). [2 ] Jones DG. Synapse and Synaptosomes ; Morphological Aspects, Chapman and Hall, 1975 ; [ ] Zafra F, Gimenez C. Characteristics and adaptive regulation of glycine transport in cultured glial cells. Biochem J, 1989 ; 258 (2) : [4 ] Brann DW, Hendry LB, Mahesh VB. Emerging diversities in the mechanism of action of steroid hormones. J steroid Biochem Mol Biol, 1995, 52 (2) : [5 ] Hua SY, Chen YZ. Membrane receptor2mediated electrophysiological effects of glucocorticoids on mammalian neurons. Endocrinology ; 124 (2) : Ca 2 +

6 608 Acta Physiologica Sinica Dec. 1999, 51 (6), THE RAPID EFFECTS OF STEROIDS ON GLYCINE UPTAKE IN NEUROBLASTOMA CELL STRAIN SK2N2SH CELLS BI GANG, CHEN YI2ZHANG ( Neuroscience Research Institute, Second Military Medical University, Shanghai 2004) ABSTRACT In the present study, glycine uptake in SK2N2SH cells was determined with liquid scintillation technique, and the rapid effects of steroids on glycine uptake in SK2N2SH cells were investigated. The results were as follows. High2affinity glycine uptake in SK2 N2SH cells was dependent on Na + and Cl -. Corticosterone (CORT), progesterone (P) and dexamethasone (DEX) had rapid effects on the glycine uptake. Since estradiol ( E2) and deoxycorticosterone (DOC) had no effects, it was suggested that the rapid effects of steroids were specific. The rapid effects of CORT were concentration2dependent in a range of mol/ L. The rapid effects were not affected by the inhibitor of protein syn2 thesis and persisted even when CORT was conjugated with bovine serum album, but at2 tenuated when Ca 2 + was absent in the external medium. The results suggest that the steroid effect on glycine uptake in SK2N2SH cells was nongenomicly mediated. Key words : steroids ; SK2N2SH cell ; glycine ; nongenomic ; mechanism ; uptake Supported by the National Natural Science Foundation of China (No190100, ) Present address : The Second Section, Naval Medical Research Institute, Shanghai 2004 Corresponding author. Tel : E2mail :