Neuroscience Letters, 117 (1990) 341-346 Elsevier Scientific Publishers Ireland Ltd.
341
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Cell modulation of hydrophobic tailed 16S acetylcholinesterase by intracellular calcium in rat superior cervical ganglion neurons Martine Verdi6re-Sahuque 2, Dani61e Goudou 1, Luis Garcia 1, Marc Nicolet 3 a n d Franqois Rieger 1 I Unit~ de Dbveloppement, Pathologie, ROgOn&ation du Syst&ne Neuromusculaire, I N S E R M U 153 and CNRS UA 614, Paris (France), 2Laboratoire de Cytologie, Paris (France) and 3Laboratoire de Physiologie Animale, Universitd de Franche-ComtO, Besan¢on (France) (Received 1 December 1989; Revised version received 29 May 1990; Accepted 1 June 1990) Key word~: Acetylcholinesterase; Asymmetric molecular form: Rat superior cervical ganglia; Primary cell culture In primary cell cultures of rat superior cervical ganglia (SCG) the tailed asymmetric 16S molecular form of acetylcholinesterase (ACHE) possesses hydrophilic (high-salt soluble, HSS) and hydrophobic (detergent extracted, DE) variants. Hydrophobic tailed acetylcholinesterase is associated with membranes through a glycolipid anchor. In the presence of tunicamycin, an antibiotic which inhibits protein glycosylation, the cellular amount of the hydrophobic DE 16S AChE is increased. Exposure of the cells to the calcium ionophore A 23187 leads to a decrease in DE 16S AChE and a correlated increase in hydrophilic HSS 16S ACHE. These results suggest the existence of an endogenous processing of tailed ACHE, transforming the hydrophobic variant into an hydrophilic one controlled through glycosylation and intracellular calcium.
Acetylcholinesterase (ACHE, EC 3.1.1.7,), is resolved in two groups of discrete molecular forms, the globular and asymmetric species [9, 20] in extracts from newborn rat superior cervical ganglia (SCG) neurons in culture. The asymmetric AChE form is a complex association of catalytic subunits with a collagen-like multistranded tail [1, 4, 13]. It is accumulated in the neuromuscular junction intersynaptic cleft [2] and requires high salt conditions for extraction [1] suggesting an association with extracellular matrix components (ECM). It is referred to as hydrophilic/high salt soluble (HSS) form [14, 19]. However, a variant of tailed asymmetric AChE can be extracted by non-ionic detergent from mammalian or frog muscle and neuronal cells [3, 6, 8, 15] suggesting a direct association with membranes. It is then referred to as hydrophobic/detergent-extracted (DE) form. We previously demonstrated that in Correspondence: M. Verdi6re-Sahuque, Unit6 de D~veloppement, Pathologie, R6g6n6ration du Syst6me Neuromusculaire, INSERM U 153 and CNRS UA 614, 17 rue du Fer-fi-Moulin, F-75005 Paris, France. 0304-3940/90/$ 03.50 (C' 1990 Elsevier Scientific Publishers Ireland Ltd.
342
SCG primary neuronal cultures the cell content in total tailed asymmetric AChE (sedimentation coefficient 16S) is regulated by electrical membrane activity and Ca 2 fluxes [21]. The present work focuses on the cellular regulation of a fraction of the tailed asymmetric 16S form i.e. the membranous DE 16S AChE through basic processes involving glycosylation and changes in intracellular Ca 2+. Increased Ca 2~ fluxes leading to elevated intracellular Ca z+ concentrations influence the proportion of hydrophobic versus hydrophilic forms and transform the hydrophobic membrane attached variant into the hydrophilic ECM bound one. We show here the influence of inhibiting glycosylation and found no inhibition of the synthesis of the membrane anchored DE 16S AChE but its accumulation suggesting that this may be a precursor ofHSS 16S ACHE. Primary SCG neuronal cultures from Wistar rat embryos at embryonic day (E) 21 [20] were treated by different drugs 8 days after plating. For biochemical determinations, cells or ganglia were submitted to a sequential extraction in the presence of, first, a low salt buffer and then detergent (1% Triton X-100) followed by high salt (t M NaC1) leading to 3 different pools of ACHE: the low salt soluble (LSS), the detergent extracted (DE) and the high salt soluble (HSS) AChE [3]. We performed for each fraction a sucrose gradient sedimentation analysis of the molecular forms of AChE (legends of Table I). TABLE l EXTRACTION PROPERTIES OF TAILED ASYMMETRIC 16S AChE FROM EMBRYONIC RAT SCG (E 14-E21) AND PRIMARY CULTURES Superior cervical ganglia (SCG)were dissected from Wistar rat (IFFA Credo) embryos at embryonic day 14 (El4) or 21 (E21). Primary neuronal cultures from E21 were obtained as previously described [20]. Homogeneizations were performed in a 3-step sequential procedure. The first extraction was carded out in a low-salt medium (phosphate buffer saline, PBS:0.13 M NaC1, 0.01 M Na2HPO4/NaH2PO4, pH 7). After centrifugation, the pellet (including membrane fraction) was extracted in a detergent medium 0% Triton X-100, 0.01 M Tris-HCl, pH 7.2). After centrifugation the second pellet was resuspended in highsalt medium (l M NaCl, 0.001 M EGTA, 0.01 M Tris-HC1, pH 7.2). All centrifugations were carded out for 6 min at 415,000 g at 4°C. Total AChE activity was estimated using the Ellman's method [5]. The multiple molecular forms of AChE were analyzed by velocity sedimentation on a 5-20% linear sucrose gradient (1% Triton X-100; 1 M NaCl; 0.001 M EGTA, 0.01 M Tris-HC1, pH 7.2). Gradients were centrifuged for 15 h at 38,000 rpm (g~v= 176,500 g) in a SW41 rotor. Results are mean± S.E.M. of 3-4 independent experiments. Student's t-test: b.c significantly different from "(P< 0.005). AChE activity is expressed in optical density variation (A) at 412 nm/h per ganglion or per dish. LSS fraction, low-salt buffer extraction; DE fraction, detergent buffer extraction; HSS fraction, high-salt buffer extraction. Extraction step
LSS% DE% HSS% Ratio DE/HSS los AChE activity
Embryonic SCG
Primary culture
El4
E21
5.6 +1.2 49.5 +4.2 44.9 +3.1 1.1 _+0.09" 0.04-+0.001
14.7 _+4.1 31.1 ___7.1 54.2 _+7.8 0.55 +0.07 b 0.33_+0.03
7.8 _+2.0 31.5 _+3.0 60.4 +4.8 0.52_+0.05 ~ 2.1 _+0.7
343 TABLE II EFFECT OF TUNICAMYCIN ON TAILED ASYMMETRIC 16S AChE IN PRIMARY CULTURES OF RAT SCG Tunicamycin (0.2/tg/ml) was applied for 24 h on cells cultured for 8 days. Its action was compared to control cultures at 10 days. Cultures were homogenized and AChE activity extracted in a three-step extraction procedure as described in Table I. Results are mean_+S.E.M, of 4 experiments. Student's t-test: ~P< 0.005, bp< 0.005, cp< 0.05, as compared to their controls. AChE activity is expressed as indicated in Table I. Extraction steps
Control
Tunicamycin
LSS% DE% HSS% Ratio DE/HSS 16S AChE activity
7.8 +2.0 31.5 _+3.0 60.4 +4.8 0.52 _+0.05 2.1 -+0.7
5.5 _+2.8 44.7 _+5.7 (+41%) a 49.6 _+5.5 0.90 _+0.09b 3.0 _+0.5 (+42%) c
T a b l e I shows that the LSS c o m p o n e n t o f 16S A C h E represents a b o u t 8% o f the total 16S A C h E activity in p r i m a r y s y m p a t h e t i c n e u r o n a l cell cultures, 6% in E 14 emb r y o an d 15% in E21 n e a r - t e r m e m b r y o . T h e f r a c t i o n o f 16S A C h E which is recovered after d e t e r g e n t e x t r a c t i o n represents a b o u t 30% o f the total 16S in tissues at E21 a nd in vitro an d reaches 50% at E l 4 . T h e fraction o f A C h E which is extracted in high-salt c o n d i t i o n s represents a b o u t 55% o f total A C h E in all tissues ex am i n ed . T h e ratios o f D E versus H S S 16S A C h E activities for near-term, p r i m a r y cultures are 0.55 + 0.07 and 0.52 + 0 . 0 5 , respectively, and significantly lower as c o m p a r e d to E l 4 (1.1 _+0.09, P < 0 . 0 0 5 ) . This D E c o m p o n e n t o f 16S A C h E constitutes a significant fraction o f total 16S A C h E associated to m e m b r a n e s t h r o u g h a glycolipid anc h o r [22]. In o r d e r to establish w h e t h e r glycosylation c o u l d influence h y d r o p h o b i c / h y d r o p h i lic a m o u n t s an d p r o p o r t i o n s , S C G p r i m a r y cultures were i n cu b at ed in the presence TABLE III EFFECT OF INTRACELLULAR Ca 2+ LEVELS ON THE EXTRACTION PROPERTIES OF TAILED ASYMMETRIC 16S AChE IN PRIMARY CULTURES OF RAT SCG Eight-day-old cultures were incubated in the presence of the ionophore A 23187 (10 6 M for 18 h). Cultures were homogenized and AChE activity extracted in a three-step extraction procedure as previously described in Table I. Results are mean+S.E.M, of 4 experiments. Student's t-test; P
341
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Cell modulation of hydrophobic tailed 16S acetylcholinesterase by intracellular calcium in rat superior cervical ganglion neurons Martine Verdi6re-Sahuque 2, Dani61e Goudou 1, Luis Garcia 1, Marc Nicolet 3 a n d Franqois Rieger 1 I Unit~ de Dbveloppement, Pathologie, ROgOn&ation du Syst&ne Neuromusculaire, I N S E R M U 153 and CNRS UA 614, Paris (France), 2Laboratoire de Cytologie, Paris (France) and 3Laboratoire de Physiologie Animale, Universitd de Franche-ComtO, Besan¢on (France) (Received 1 December 1989; Revised version received 29 May 1990; Accepted 1 June 1990) Key word~: Acetylcholinesterase; Asymmetric molecular form: Rat superior cervical ganglia; Primary cell culture In primary cell cultures of rat superior cervical ganglia (SCG) the tailed asymmetric 16S molecular form of acetylcholinesterase (ACHE) possesses hydrophilic (high-salt soluble, HSS) and hydrophobic (detergent extracted, DE) variants. Hydrophobic tailed acetylcholinesterase is associated with membranes through a glycolipid anchor. In the presence of tunicamycin, an antibiotic which inhibits protein glycosylation, the cellular amount of the hydrophobic DE 16S AChE is increased. Exposure of the cells to the calcium ionophore A 23187 leads to a decrease in DE 16S AChE and a correlated increase in hydrophilic HSS 16S ACHE. These results suggest the existence of an endogenous processing of tailed ACHE, transforming the hydrophobic variant into an hydrophilic one controlled through glycosylation and intracellular calcium.
Acetylcholinesterase (ACHE, EC 3.1.1.7,), is resolved in two groups of discrete molecular forms, the globular and asymmetric species [9, 20] in extracts from newborn rat superior cervical ganglia (SCG) neurons in culture. The asymmetric AChE form is a complex association of catalytic subunits with a collagen-like multistranded tail [1, 4, 13]. It is accumulated in the neuromuscular junction intersynaptic cleft [2] and requires high salt conditions for extraction [1] suggesting an association with extracellular matrix components (ECM). It is referred to as hydrophilic/high salt soluble (HSS) form [14, 19]. However, a variant of tailed asymmetric AChE can be extracted by non-ionic detergent from mammalian or frog muscle and neuronal cells [3, 6, 8, 15] suggesting a direct association with membranes. It is then referred to as hydrophobic/detergent-extracted (DE) form. We previously demonstrated that in Correspondence: M. Verdi6re-Sahuque, Unit6 de D~veloppement, Pathologie, R6g6n6ration du Syst6me Neuromusculaire, INSERM U 153 and CNRS UA 614, 17 rue du Fer-fi-Moulin, F-75005 Paris, France. 0304-3940/90/$ 03.50 (C' 1990 Elsevier Scientific Publishers Ireland Ltd.
342
SCG primary neuronal cultures the cell content in total tailed asymmetric AChE (sedimentation coefficient 16S) is regulated by electrical membrane activity and Ca 2 fluxes [21]. The present work focuses on the cellular regulation of a fraction of the tailed asymmetric 16S form i.e. the membranous DE 16S AChE through basic processes involving glycosylation and changes in intracellular Ca 2+. Increased Ca 2~ fluxes leading to elevated intracellular Ca z+ concentrations influence the proportion of hydrophobic versus hydrophilic forms and transform the hydrophobic membrane attached variant into the hydrophilic ECM bound one. We show here the influence of inhibiting glycosylation and found no inhibition of the synthesis of the membrane anchored DE 16S AChE but its accumulation suggesting that this may be a precursor ofHSS 16S ACHE. Primary SCG neuronal cultures from Wistar rat embryos at embryonic day (E) 21 [20] were treated by different drugs 8 days after plating. For biochemical determinations, cells or ganglia were submitted to a sequential extraction in the presence of, first, a low salt buffer and then detergent (1% Triton X-100) followed by high salt (t M NaC1) leading to 3 different pools of ACHE: the low salt soluble (LSS), the detergent extracted (DE) and the high salt soluble (HSS) AChE [3]. We performed for each fraction a sucrose gradient sedimentation analysis of the molecular forms of AChE (legends of Table I). TABLE l EXTRACTION PROPERTIES OF TAILED ASYMMETRIC 16S AChE FROM EMBRYONIC RAT SCG (E 14-E21) AND PRIMARY CULTURES Superior cervical ganglia (SCG)were dissected from Wistar rat (IFFA Credo) embryos at embryonic day 14 (El4) or 21 (E21). Primary neuronal cultures from E21 were obtained as previously described [20]. Homogeneizations were performed in a 3-step sequential procedure. The first extraction was carded out in a low-salt medium (phosphate buffer saline, PBS:0.13 M NaC1, 0.01 M Na2HPO4/NaH2PO4, pH 7). After centrifugation, the pellet (including membrane fraction) was extracted in a detergent medium 0% Triton X-100, 0.01 M Tris-HCl, pH 7.2). After centrifugation the second pellet was resuspended in highsalt medium (l M NaCl, 0.001 M EGTA, 0.01 M Tris-HC1, pH 7.2). All centrifugations were carded out for 6 min at 415,000 g at 4°C. Total AChE activity was estimated using the Ellman's method [5]. The multiple molecular forms of AChE were analyzed by velocity sedimentation on a 5-20% linear sucrose gradient (1% Triton X-100; 1 M NaCl; 0.001 M EGTA, 0.01 M Tris-HC1, pH 7.2). Gradients were centrifuged for 15 h at 38,000 rpm (g~v= 176,500 g) in a SW41 rotor. Results are mean± S.E.M. of 3-4 independent experiments. Student's t-test: b.c significantly different from "(P< 0.005). AChE activity is expressed in optical density variation (A) at 412 nm/h per ganglion or per dish. LSS fraction, low-salt buffer extraction; DE fraction, detergent buffer extraction; HSS fraction, high-salt buffer extraction. Extraction step
LSS% DE% HSS% Ratio DE/HSS los AChE activity
Embryonic SCG
Primary culture
El4
E21
5.6 +1.2 49.5 +4.2 44.9 +3.1 1.1 _+0.09" 0.04-+0.001
14.7 _+4.1 31.1 ___7.1 54.2 _+7.8 0.55 +0.07 b 0.33_+0.03
7.8 _+2.0 31.5 _+3.0 60.4 +4.8 0.52_+0.05 ~ 2.1 _+0.7
343 TABLE II EFFECT OF TUNICAMYCIN ON TAILED ASYMMETRIC 16S AChE IN PRIMARY CULTURES OF RAT SCG Tunicamycin (0.2/tg/ml) was applied for 24 h on cells cultured for 8 days. Its action was compared to control cultures at 10 days. Cultures were homogenized and AChE activity extracted in a three-step extraction procedure as described in Table I. Results are mean_+S.E.M, of 4 experiments. Student's t-test: ~P< 0.005, bp< 0.005, cp< 0.05, as compared to their controls. AChE activity is expressed as indicated in Table I. Extraction steps
Control
Tunicamycin
LSS% DE% HSS% Ratio DE/HSS 16S AChE activity
7.8 +2.0 31.5 _+3.0 60.4 +4.8 0.52 _+0.05 2.1 -+0.7
5.5 _+2.8 44.7 _+5.7 (+41%) a 49.6 _+5.5 0.90 _+0.09b 3.0 _+0.5 (+42%) c
T a b l e I shows that the LSS c o m p o n e n t o f 16S A C h E represents a b o u t 8% o f the total 16S A C h E activity in p r i m a r y s y m p a t h e t i c n e u r o n a l cell cultures, 6% in E 14 emb r y o an d 15% in E21 n e a r - t e r m e m b r y o . T h e f r a c t i o n o f 16S A C h E which is recovered after d e t e r g e n t e x t r a c t i o n represents a b o u t 30% o f the total 16S in tissues at E21 a nd in vitro an d reaches 50% at E l 4 . T h e fraction o f A C h E which is extracted in high-salt c o n d i t i o n s represents a b o u t 55% o f total A C h E in all tissues ex am i n ed . T h e ratios o f D E versus H S S 16S A C h E activities for near-term, p r i m a r y cultures are 0.55 + 0.07 and 0.52 + 0 . 0 5 , respectively, and significantly lower as c o m p a r e d to E l 4 (1.1 _+0.09, P < 0 . 0 0 5 ) . This D E c o m p o n e n t o f 16S A C h E constitutes a significant fraction o f total 16S A C h E associated to m e m b r a n e s t h r o u g h a glycolipid anc h o r [22]. In o r d e r to establish w h e t h e r glycosylation c o u l d influence h y d r o p h o b i c / h y d r o p h i lic a m o u n t s an d p r o p o r t i o n s , S C G p r i m a r y cultures were i n cu b at ed in the presence TABLE III EFFECT OF INTRACELLULAR Ca 2+ LEVELS ON THE EXTRACTION PROPERTIES OF TAILED ASYMMETRIC 16S AChE IN PRIMARY CULTURES OF RAT SCG Eight-day-old cultures were incubated in the presence of the ionophore A 23187 (10 6 M for 18 h). Cultures were homogenized and AChE activity extracted in a three-step extraction procedure as previously described in Table I. Results are mean+S.E.M, of 4 experiments. Student's t-test; P
