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Effect of denervation of the (Na+, K+)ATPase activity of Electrophorus electricus (L.) electrocyte
Comp. Biochem. PhysioL Vol. 103B,No. 3, pp. 623-628, 1992 Printed in Great Britain
0305-0491/92$5.00+ 0.00 © 1992PergamonPress Ltd
EFFECT OF DENERVATION ON THE (Na ÷, K+)ATPase ACTIVITY OF ELECTROPHORUS ELECTRICUS (L.) ELECTROCYTE E. GOMES-QuINTANA,G. AgAfJJO NLrN~ and A. HASS~N-VOLOCH Laborat6rio de Fisico-Quimica Biol6gica, Iustituto de Biofisica Carlos Chagas Fiiho da Universidade Federal do Rio de Janeiro, Centro de Ci6cias da Sa6de, 21949, Rio de Janeiro, ILl', Brasil (Tel.: 055-21-280-8193; Fax: 055-21-280-8193) (Received 7 April 1992; accepted 15 May 1992)
Alaaract--l. The effect of denervation on membrane-bound (Na +, K+)ATPase activity from the noninnervated (fraction P2) and innervated (fraction P3) membrane faces of Electrophorus electricus (L.) electrocyte is described. 2. After 20 days denervation both fractions presented a decreased (Na +, K+)Mg2+-dependent ATPase activity as compared with the normal electrocyte fractions. 3. When submitted to a discontinuous sucrose gradient, normal P2 membrane fraction exhibits a bimodal distribution of the enzyme which upon denervation changes to an unimodal distribution. P3 fraction showed a similar but less evident change of the enzyme distribution. 4. Sialic acid, which is described as a constituent of the enzyme beta-subunit, also showed a higher decrease (26.51%) in denervated P2 fraction as compared with P3 denervated membrane fraction (9%).
INTRODUCTION The electrocytes of the electric organs of Electrophorus electricus (L.) are highly specialized cells able to produce a synchronic electric discharge generating bioelectric potentials similar to those in nerve and muscle. Propagated action potentials in electrically excitable cells are known to result from transient changes in the permeability of the cell surface membrane to cations. Studies on the molecular mechanisms of active transport were initiated by Skou (1957) with (Na +, K+)Mg2+-dependent ATPase and furthered by Glynn and Karlish (1975) on the enzyme activity of electric organs. The electrogenic properties of the membrane was also described at that time by Karlin (1967), however, only recently has it been recognized that the membrane potential must influence the kinetics of transport phenomena (Lauger and Appel, 1988). Earlier studies of ion movements in isolated electrocytes showed that the flux of cations (Na + and K +) is higher across the non-innervated (anterior face) than across the innervated (posterior face) membrane (Schoffeniels, 1959) and also that only the innervated face of the electrocyte is chemically or electrically excitable (Auger and Fessard, 1939) presenting high acetylcholinesterase (ACHE) activity (Couceiro and Freire, 1953). Later on, cytochemical and biochemical analyses have shown that the (Na ÷, K+)ATPase activity is localized in both the anterior and posterior membranes (Soml6 et al., 1977). Since (Na +, K+)Mg2+-dependent ATPase is typically a membrane enzyme and since the excitability phenomenon and electric potential propagation are derived from ionic transport, we can presume that any alteration of synthesis or function of this enzyme will interfere with bioelectrogenesis.
Denervation techniques have been used by many authors to study the relationship between nerves and related structures as the denervated muscles undergo changes in permeability and electrical properties of the membrane. Reports on alteration in biochemical parameters of skeletal muscle and electric organs plasma membrane occurring after denervation have also increased in the last decades (Smith and Appel, 1977; Falcato-Ribeiro et al., 1980). "Ihe (Na +, K+)ATPase activity is known to be modulated by lipids in two major ways: (a) through binding of specific lipids involving lipid arrangement on the hydrophilic and hydrophobic regions of the protein-lipid binding sites, probably stabilizing the protein conformation (Kawakami et aL, 1985) and (b) through the physicochemical state of the lipids, which can be more or less fluid depending on the temperature (Schull et al., 1986). It is now well acknowledged that the (Na +, K +) ATPase consists of two subunits of integral membrane proteins, the alpha and beta subunits, whose MrS are of the order of 100,000 and 50,000, respectively (Jorgensen, 1982; Sweadner, 1985). The catalytic properties are confined to the alpha subunit and the beta subunit, a sialoglycoprotein, seems to play a role in the membrane insertion process (Sweadner, 1985). There is also evidence for the presence of at least two isoforms of (Na +, K+)ATPase that differ in their enzymatic properties (Sweadner, 1985). In this paper we describe the effect of denervation on the electroeyte (Na+,K+)Mg2+-dependent ATPase activity and the correspondent enzyme distribution on sucrose gradients. Both, innervated and non-innervated membrane fractions, present, in the normal electrocyte, two (Na +, K+)ATPase isoforms, while the denervated electrocyte presents a modified pattern with apparently only one isoform.
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Table 1. Distribution of (Na ÷, K+)ATPase and AChE activities in subcellular fractions from normal and denervated electric organ (Na +, K+)ATPase AChE Membrane Specific Total Specific Total fraction activity units activity units P2 Normal 91 4,034 2,513 111,401 Denervated 74 1,562 1,687 35,848 P3 Normal 60 3,302 6,821 375,482 Denervated 50 1,540 4,920 151,536 (Na +, K+)ATPase specific activity is given in gmol Pi liberated/hr/mg protein; AChE specific activity is given in/Jmol of ACh hydrolysed/hr/mg protein. Values express the mean from six experiments; SD + 10%. MATERIAL AND METHODS
All experiments were performed with specimens of the electric eel Electrophorus electricus (L.) obtained from "Museu Goeldi" (Belrm-Parfi) and kept at the aquarium facilities of the Instituto de Biofisica Carlos Chagas FilhoRJ. Membranes rich in (Na +, K+)ATPase and AChE were prepared as previously described by Soml6 et al. (1977) and a pool of these fractions, separated at 10,000g (P2) and 100,000g (P3), were stored at -70°C. Denervation The surgical procedure for denervation consists of a longitudinal incision, about 10cm long, on the dorsal anterior part of the fish. The incision shows the midline nerve and also the nerves which innervate the electric organ coming from the spinal cord. Ten to 20 nerves were sectioned through the muscle and 0.5 cm of each nerve was cut out in order to avoid regeneration. One side of the main electric organ was subjected to denervation and the contralateral side was used as a control. After 20 days, the animals were killed by excision at the central nervous system. Denervated and normal electric organs were dissected from recently killed eels, the main electric organs (normal and denervated) were removed, homogenized and used for membrane preparations. Sucrose gradient fractionation Membrane fractions (P2 and P3) from the normal and denervated electric organ were further subfractionated on a five step discontinuous sucrose gradient. Aliquots of P2 and P3 fractions, containing approximately 70 mg of protein, were applied to a 15-45% sucrose gradient, and centrifuged at 100,000g, in a Beckman SW50,1 rotor. Fractions were collected from top to bottom of the centrifuge tubes for the enzyme assays. Enzymes assays (a) (Na ÷, K÷)-ATPase activity was measured at pH 7.6, with the following substrate media: 2.5 mM ATP, 3.25 mM MgCI2, 0.2 mM EDTA, 14 mM KC1, 72 mM NaCI, 160 mM Tris-HC1 pH 7.2. Membrane fractions were added in concentrations of about 20/,g/ml protein and incubations carried out at 37°C for 15 min. The reaction was started by the addition of ATP and was stopped with 5% TCA. Enzyme blanks were included in each experiment and aliquots were transferred for inorganic phosphate (Pi) estimation by the method of Fiske and SubbaRow (1925). The specific activities were expressed as the amount of inorganic phosphate liberated/mg protein/hr. Protein concentration was estimated by the method of Lowry et al. (1951) using bovine serum albumin as a standard. Membranes were also exposed to the inhibitor ouabain 0.4 mM, in the same medium as for (Na +, K+)ATPase, for 10min, before incubation at 37°C and Pi estimated as described above. (b) Acetylcholinesterase (ACHE) activity in P2 and P3 was assayed according to Hass6n-Voloch and Liepin (1963). The enzyme activity was measured at pH 8.0. The acetic acid released from a substrate medium containing
0.28 M ACh in 0.1 M sodium acetate and 0.2 M MgC12 was titrated with 0.1 M NaOH. A Beckman Century SS-1 pH meter was used and 0.1 M NaOH was delivered from an AGLA micrometer-syringe outfit with continuous stirring, at 25°C. Readings were taken at 1 min intervals over a 4 min period. Specific activity is defined as the amount of acetylcholine hydrolysed/ mg protein/hr. (c) Sialic acid determination was performed in P2 and P3 fractions, according to the method of Warren (1963), using a spectrophotometric determination. SDS-PAGE SDS gel electrophoresis was performed essentially according to Laemmli (1970) using either homogeneous gels containing 12.5% polyacrylamide or gradient gels containing 5-15 % polyacrylamide. Protein was stained with Coomassie Brilliant Blue G-250. The following proteins served as Mr markers: myosin, /~-galactosidase, phosphorylase b, bovine serum albumin, ovalbumin and carbonic anhydrase (Sigma Chemical Co. MS-SDS-200 Kit).
RESULTS ( N a + , K + ) A T P a s e - M g :+ d e p e n d e n t a n d acetylcholinesterase activities were m e a s u r e d in m e m b r a n e fractions P2 a n d P3, o b t a i n e d by differential centrifugation a n d the results are s h o w n in Table 1. As described by Soml6 et al. (1977), (Na ÷, K ÷ ) A T P a s e activity is present in b o t h fractions P2 a n d P3, p r e d o m i n a t i n g in fraction P2 (Table 1). ( N a ÷, K + ) A T P a s e from denervated o r g a n s presented specific activities lower t h a n the c o r r e s p o n d i n g activity o f a n o r m a l p r e p a r a t i o n , o f the o r d e r o f 18 a n d 16% for P2 a n d P3 d e n e r v a t e d fractions, respectively. A 10-fold decrease in specific activity was also observed w h e n o u a b a i n was a d d e d to the i n c u b a t i o n medium, confirming the identity o f the enzyme present in b o t h fractions P2 a n d P3, a n d d e m o n s t r a t i n g a similar response to the i n h i b i t o r which is independent of their actual c o n d i t i o n ( n o r m a l or denervated). C o n c e r n i n g acetylcholinesterase activity, which was used here mainly as a m e m b r a n e m a r k e r o f the post-synaptic m e m b r a n e (posterior i n n e r v a t e d electrocyte m e m b r a n e ) , higher activities were concentrated in P3 fraction a n d in the denervated condition. This enzyme showed a decreased activity as occurs with ( N a +, K +)ATPase. The activity profiles o b t a i n e d by m e a n s of a disc o n t i n u o u s sucrose gradient analysis are depicted in Figs 1 a n d 2. Figure 1 shows the distribution o f ( N a ÷, K ÷ ) A T P a s e a n d acetylcholinesterase f r o m n o r m a l a n d d e n e r v a t e d P2 m e m b r a n e fractions. Denervated P2 fractions exhibit a u n i m o d a l distribution
Denervation decreases (Na +, K+)ATPase activity
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of (Na +, K÷)ATPase with lower specific activities of the order of 60 #mol Pi liberated/hr/mg protein, in contrast with P2 normal fractions which have a bimodal distribution and higher specific activities (80 and 100/~mol Pi liberated/hr/mg protein). Figure 2 shows the profile of distribution of (Na +, K ÷)ATPase and acetylcholinesterase in normal and denervated P3 membrane fractions. Denervation, apparently, did not alter (Na ÷, K÷)ATPase distribution in this fraction, except for the lower specific activity observed when compared to the normal P3 preparation. As with acetylcholinesterase, the overall activity is also
diminished in comparison with the normal electric organ enzyme. P2 and P3 denervated membrane fractions also showed a decrease in sialic acid concentration and a discrete increase of the total protein concentrations when compared to normal membrane fragments (Table 2). Sialic acid concentration decreased 25.51% in P2 (denervated membrane fraction) and only 9% in P3 denervated membrane fractions. Figure 3 shows a typical polyacrylamide SDS gel electrophoresis pattern of P2 and P3 membrane proteins of normal and denervated electric organs.
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Fig. 2. Distribution of the (Na +, K+)Mg2+-dependent ATPase ( - - V - - ~ 7 - - ) and AChE ( - - O - - O - - ) specific activities from a discontinuous sucrose gradient of P3 fraction from normal and denervated electrocyte membranes as described in the legend for Fig. 1. ATPase inhibition by o ~ b a i n (0.4 mM) is also indicated ( - - & - - A - - ) .
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Table 2. Total protein and sialic acid composition of P2 (noninnervated) and P3 (innervated) subeellular fractions of normal and denervated electric organ
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Fractions
Proteins (g%)
Sialic acid (nmol/mg/protein)
Normal Denervated Normal Denervated
14.23 +_ 3.37 19.29 + 3.58 22.46 + 3,46 26.03 + 2.02
52.41 + 3.83 38.52 ___4.79 25.55 ___2.62 23.16 + 4.53
Values are expressed as means +_ SD (n = 3). Experimental conditions are described in Material and Methods.
The Mrs indicated were determined from m r markers. The appearance of several bands indicates that the P2 and P3, normal and denervated membrane fracfractions, are composed of a heterogenous population of proteins, as expected. Denervated electrocyte membranes (P2 and P3) show an apparent decrease in some of the proteins, when compared to normal membranes, which can be observed at the 45,000 and 115,000 regions. Figure 4 shows a polyacrylamide SDS gel electrophoresis pattern of the sucrose gradients of P2 and P3 normal and denervated membrane fractions. There is a major band occurring at the apparent Mr of 100,000 probably corresponding to the alpha subunit of (Na ÷, K+)ATPase. The beta-subunit known to be a carbohydrate containing protein stained positive with periodic acid-Schiff reagent for glycoproteins (unpublished observations).
DISCUSSION
The electric organ of the electric eel is a valuable source of excitable membranes for the study of the structural and biochemical alterations produced by denervation. After denervation, the electric organ of E. eleetrieus shows a late and slow atrophy, which permits the study of nerve control upon this structure, the modifications observed seem to be different when compared to what occurs with skeletal striated muscle (Smith and Appel, 1977). With normal and denervated electric organ membrane fractions vesicles are mainly produced, but in membrane preparations obtained from denervated tissues, disrupted vesicles are frequently observed, demonstrating a rather irregular organization of the membrane fragments (Quintana and Lobfio; 1985). In this paper, we present evidence that denervation exerts changes in the membranes (Na +, K+)ATPase activities, probably representing the readjustment of the different metabolism pathways to maintain the basic conditions of the denervated electric organ. The decreased content of sialic acid in our denervated membrane fragments points to a decrease of synthesis concomitant with a slight increase of total protein synthesis. Studies made by FalcatoRibeiro et al. (1975) and Torres da Matta et al. (1985) on denervated electric organs showed qualitative and quantitative modifications in the total protein profiles as well as of the glycolitic enzymes
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Fig. 3. Polyacrylamide SDS gel ¢lectrophoresis from normal (N) and denervated (D) P2 and P3 electrocyte membranes. The M, markers indicated are: (1) myosin, (2) galactosidase, (3) phosphorylase b, (4) bovine serum albumin, (5) ovalbumin and (6) carbonic anhydrase. The arrows correspond to proteins of M r + 50,000 and + 106,000. The gel was stained with Coomassie Blue as indicated in Materials and Methods.
Denervation decreases (Na +, K+)ATPase activity
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Fig. 4. Polyacrylamide SDS gel electrophoresis of (Na +, K +) ATPase more active peaks derived from the discontinuous sucrose gradient fractions (1)2 and P3) of normal and denervated electrocyte membranes. The arrows correspond to proteins of Mr + 50,000 and + 106,00. activities which we have also observed for (Na +, K+)ATPase. The electrophoretic pattern shows that some proteins diminish or simply disappears after denervation. However, most of these are not yet identified. The results of the enzymes distribution after centrifugation in sucrose gradients indicate the presence of the three distinct forms of AChE in electrocytes of the main electric organ, as already observed by Da Matta and Hasstn-Voloch (1975). Within 20 days after denervation we observed a decrease in AChE specific activity of about 35%, which is in agreement with other authors (Rosenberg et al., 1964). As for the (Na +, K+)ATPase, which couples the hydrolysis of ATP with the transport of Na + and K + ions across the cell membrane, we observe that denervation promotes a decrease of the enzyme activity and a different profile of enzyme distribution in the P2 and P3 membrane fractions. Hayashi et al. (1977) studying the (Na +, K+)ATPase from a microsomal fraction of canine kidney obtained two peaks of this enzyme, which was attributed to different concentrations of the annulus lipids. The two peaks of (Na +, K +)ATPase that appears normally in our P2 and P3 preparations when submitted to a sucrose
gradient disappears after denervation, leading to one broad single peak. The difference in density of these fractions may also be due to the different composition and content of total lipids bound to the enzyme. In a previous work we observed a slight increase of the total lipids (14%) and cholesterol (8%) in the extracts of denervated electrocytes membrane fractions (Quintana et al., 1988). As it occurs with ACHE, which has three distinct forms in the electrocytes of the main electric organ (Da Matta and Hass6nVoloch, 1975), there may be different molecular forms of (Na +, K+)ATPase in the electrocyte membrane. Studies of mammalian brain enzymes by Wayama et al. (1989) also presented evidence of three molecular forms for (Na +, K+)ATPase, but other authors believe that only two molecular forms exists in mammalian heart (Sweadner, 1985). Thus we can consider that the decreased enzymatic activity observed with (Na +, K+)ATPase and AChE may be an adaptation of the denervated tissue to the new situation. Biochemical modifications have been observed in many studies of denervated organs in the last few years. Changes inducing alterations in receptor activity (Almon and Appel, 1976) enzyme activities (Smith and Appel (1977); Torres da Matta et al.,
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1985) c a r b o h y d r a t e c o m p o n e n t s (Jeffrey a n d Appel, 1978) a n d p h o s p h o l i p i d m e t a b o l i s m (Appel et al., 1974; Q u i n t a n a et al., 1988) strongly suggest drastic changes in m e m b r a n e o r g a n i z a t i o n a n d function. REFERENCES
Almon R. R. and Appel S. H. (1976) Cholinergic sites in skeletal muscle I. Denervation effects. Biochemistry 15, 3662-3667. Appel, S. H., Andrew C. G. and Almon R. R. (1974) Phosphatidylinositol turnover in muscle membranes following denervation. J. Neurochem. 23, 1077-1080. Auger D. and Fessard A. (1939) In Hypotheses sur le mechanisme de l'electrogenese chez les poissons electriques (Edited by Oz6rio de Almeida A. and M. eds), pp. 25-34. Rio de Janeiro. Couceiro A. and Freire J. R. C. (1953) Localization histochimique de l'acetylcholinesterase dans le tissu electrique de l'Electrophorus electricus (L.). An Acad. Brasil. Cien. 25, 205-214. Da Matta N. A. and Hass6n-Voloch A. (1975) Molecular forms of acetylchotinesterase from the three electric organs of Electrophorus electricus (L.). Comp. Biochem. Physiol. 50C, 5-8. Falcato-Ribeiro A. F. and Chagas C. (1975) Effect de la denervation sur le metabolisme macromoleeulaire chez le tissu electrique de l'Electrophorus electricus (L.). C.R. Acad. Sci. Paris, serie D 281, 1425-1439. Falcato-Ribeiro A. F., Chagas C. and Couceiro A. (1980) Histoenzymological evidence for the modification in the activity of some enzymes in the denervated electrogenic tissue of Electrophorus electrieus (L.). IRCS Med. Sci. 8, 776-777. Fiske C. H. and SubbaRow J. (1925) The colorimetric determination of phosphorus. J. biol. Chem. 66, 375-400. Glynn I. M. and Karlish S. J. D. (1975) The sodium pump. Annu. Rev. Physiol. 37, 13-55. Hass6n A. and Liepin L. L. (1963) Reversible inhibition of electric organ cholinesterase by curare and curare-like agents. Biochim. biophys. Acta 75, 397-401. Hayashi Y., Kimimura M., Homareda H. and Matsuy H. (1977) Purification and characteristics of N a + , K +ATPase from canine kidney by zonal centrifugation in sucrose density gradient. Biochim biophys. Acta. 482, 185-196. Jeffrey L. P. and Appel H. (1978) Denervation alterations in surface membrane glyeoprotein glycosyltransferases of mammalian skeletal muscle. Exp. Neurology 61, 432-441. Jorgensen P. L. (1982) Mechanisms of the (Na ÷, K +) pump protein structure and conformations of the pure (Na +, K+)ATPase. Biochim. biophys. Acta. 694, 27-68. Karlin A. (1967) Permeability and internal concentration of ions during depolarization of the electroplaques. Proc. natn. Acad. Sci. U.S.A. 58, 1162-1167.
Kawakami K., Noguchi, S., Noda M., Takahashi H., Ohta T., Kawamura, M., Nojima H., Nogano K., Hirose T., Inayama S., Hayashida H., Miyata T. and Numa S. (1985) Primary structure of the a-subunit of Torpedo californica (Na+,K+)ATPase deduced from cDNA sequence. Nature 316, 733-736. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. Lauger P. and Appel H. J. (1988) Transient behaviour of the Na +, K+-pump: Microscopic analysis of nonstationary ion-translocation. Biochim. biophys. Acta. 946, 1-10. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin-phenol reagent. J. biol. Chem. 193, 265-275. Quintana E. G. and Lobfio H. N. (1985) Influence of denervation on the cholinergie membrane of the electric tissue of Electrophorus electricus (L.). Comp. Biochem. Physiol. 81C, 461-467. Quintana E. G., Soml6 C. and Hasson-Voloch A. (1988) Modification of electrocyte membrane lipid composition induced by denervation. Braz. J. med. biol. Res. 221, 1163-1171. Rosenberg L., Mackey E. A., Higman H. B. and Dettbarn W. D. (1964) Choline acetylase and cholinesterase activity in denervated electroplax. Biochim. biophys. Acta. 988, 185-220. Schoffeniels E. (1959) Ion movements with single isolated electroplaques. Ann. N.Y. Acad. Sci. 81, 285-306. Shull G. E., Lane L. K. and Lingrel J. B. (1986) Amino acid sequence of the B subunit of the (Na +,K+)ATPase deduced from a eDNA. Nature 321, 429-431. Skou J. C. (1957) The influence of some cations on an adenosine triphosphatase from peripheral nerves. Biochim. biophys. Acta. 23, 394-491. Smith P. B. and Appel S. H. (1977) Development of denervation alterations in surface membranes of mammalian skeletal muscle. Exp. neurol. 56, 102-114. Soml6 C., de Souza W., Machado R. D. and Hass6nVoloch A. (1977) Biochemical and cytochemical localization of ATPases on the membranes of the electrocyte of Electrophorus eleetricus (L.). Cell Tiss. Res. 185, t 15 128. Sweadner K. J. (1985) Enzymatic properties of separated isozymes of the (Na +, K+)ATPase. J. biol. Chem. 260, 11503 11513. Torres-da-Matta J., Silva B. C., Da Matta N. A. and Hass6n-Voloch A. (1985) Effect of denervation on the glycolytic metabolism of the main electric organ of Electrophorus electricus (L.). Comp. Biochem. Physiol. 81B, 969-973. Warren L. (1963) Thiobarbituric acid assay of sialic acids. In Methods in Enzymology, Vol. VI, p. 463 (Edited by S. P. Colowick and N. O. Kaplan). Academic Press, New York. Urayama O., Shutt H. and Sweadner K. J. (1989) Identification of three isozyme proteins of the catalytic subunit of the Na +, K+-ATPase in rat brain. J. biol. Chem. 2,64, 8250-8270.
0305-0491/92$5.00+ 0.00 © 1992PergamonPress Ltd
EFFECT OF DENERVATION ON THE (Na ÷, K+)ATPase ACTIVITY OF ELECTROPHORUS ELECTRICUS (L.) ELECTROCYTE E. GOMES-QuINTANA,G. AgAfJJO NLrN~ and A. HASS~N-VOLOCH Laborat6rio de Fisico-Quimica Biol6gica, Iustituto de Biofisica Carlos Chagas Fiiho da Universidade Federal do Rio de Janeiro, Centro de Ci6cias da Sa6de, 21949, Rio de Janeiro, ILl', Brasil (Tel.: 055-21-280-8193; Fax: 055-21-280-8193) (Received 7 April 1992; accepted 15 May 1992)
Alaaract--l. The effect of denervation on membrane-bound (Na +, K+)ATPase activity from the noninnervated (fraction P2) and innervated (fraction P3) membrane faces of Electrophorus electricus (L.) electrocyte is described. 2. After 20 days denervation both fractions presented a decreased (Na +, K+)Mg2+-dependent ATPase activity as compared with the normal electrocyte fractions. 3. When submitted to a discontinuous sucrose gradient, normal P2 membrane fraction exhibits a bimodal distribution of the enzyme which upon denervation changes to an unimodal distribution. P3 fraction showed a similar but less evident change of the enzyme distribution. 4. Sialic acid, which is described as a constituent of the enzyme beta-subunit, also showed a higher decrease (26.51%) in denervated P2 fraction as compared with P3 denervated membrane fraction (9%).
INTRODUCTION The electrocytes of the electric organs of Electrophorus electricus (L.) are highly specialized cells able to produce a synchronic electric discharge generating bioelectric potentials similar to those in nerve and muscle. Propagated action potentials in electrically excitable cells are known to result from transient changes in the permeability of the cell surface membrane to cations. Studies on the molecular mechanisms of active transport were initiated by Skou (1957) with (Na +, K+)Mg2+-dependent ATPase and furthered by Glynn and Karlish (1975) on the enzyme activity of electric organs. The electrogenic properties of the membrane was also described at that time by Karlin (1967), however, only recently has it been recognized that the membrane potential must influence the kinetics of transport phenomena (Lauger and Appel, 1988). Earlier studies of ion movements in isolated electrocytes showed that the flux of cations (Na + and K +) is higher across the non-innervated (anterior face) than across the innervated (posterior face) membrane (Schoffeniels, 1959) and also that only the innervated face of the electrocyte is chemically or electrically excitable (Auger and Fessard, 1939) presenting high acetylcholinesterase (ACHE) activity (Couceiro and Freire, 1953). Later on, cytochemical and biochemical analyses have shown that the (Na ÷, K+)ATPase activity is localized in both the anterior and posterior membranes (Soml6 et al., 1977). Since (Na +, K+)Mg2+-dependent ATPase is typically a membrane enzyme and since the excitability phenomenon and electric potential propagation are derived from ionic transport, we can presume that any alteration of synthesis or function of this enzyme will interfere with bioelectrogenesis.
Denervation techniques have been used by many authors to study the relationship between nerves and related structures as the denervated muscles undergo changes in permeability and electrical properties of the membrane. Reports on alteration in biochemical parameters of skeletal muscle and electric organs plasma membrane occurring after denervation have also increased in the last decades (Smith and Appel, 1977; Falcato-Ribeiro et al., 1980). "Ihe (Na +, K+)ATPase activity is known to be modulated by lipids in two major ways: (a) through binding of specific lipids involving lipid arrangement on the hydrophilic and hydrophobic regions of the protein-lipid binding sites, probably stabilizing the protein conformation (Kawakami et aL, 1985) and (b) through the physicochemical state of the lipids, which can be more or less fluid depending on the temperature (Schull et al., 1986). It is now well acknowledged that the (Na +, K +) ATPase consists of two subunits of integral membrane proteins, the alpha and beta subunits, whose MrS are of the order of 100,000 and 50,000, respectively (Jorgensen, 1982; Sweadner, 1985). The catalytic properties are confined to the alpha subunit and the beta subunit, a sialoglycoprotein, seems to play a role in the membrane insertion process (Sweadner, 1985). There is also evidence for the presence of at least two isoforms of (Na +, K+)ATPase that differ in their enzymatic properties (Sweadner, 1985). In this paper we describe the effect of denervation on the electroeyte (Na+,K+)Mg2+-dependent ATPase activity and the correspondent enzyme distribution on sucrose gradients. Both, innervated and non-innervated membrane fractions, present, in the normal electrocyte, two (Na +, K+)ATPase isoforms, while the denervated electrocyte presents a modified pattern with apparently only one isoform.
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Table 1. Distribution of (Na ÷, K+)ATPase and AChE activities in subcellular fractions from normal and denervated electric organ (Na +, K+)ATPase AChE Membrane Specific Total Specific Total fraction activity units activity units P2 Normal 91 4,034 2,513 111,401 Denervated 74 1,562 1,687 35,848 P3 Normal 60 3,302 6,821 375,482 Denervated 50 1,540 4,920 151,536 (Na +, K+)ATPase specific activity is given in gmol Pi liberated/hr/mg protein; AChE specific activity is given in/Jmol of ACh hydrolysed/hr/mg protein. Values express the mean from six experiments; SD + 10%. MATERIAL AND METHODS
All experiments were performed with specimens of the electric eel Electrophorus electricus (L.) obtained from "Museu Goeldi" (Belrm-Parfi) and kept at the aquarium facilities of the Instituto de Biofisica Carlos Chagas FilhoRJ. Membranes rich in (Na +, K+)ATPase and AChE were prepared as previously described by Soml6 et al. (1977) and a pool of these fractions, separated at 10,000g (P2) and 100,000g (P3), were stored at -70°C. Denervation The surgical procedure for denervation consists of a longitudinal incision, about 10cm long, on the dorsal anterior part of the fish. The incision shows the midline nerve and also the nerves which innervate the electric organ coming from the spinal cord. Ten to 20 nerves were sectioned through the muscle and 0.5 cm of each nerve was cut out in order to avoid regeneration. One side of the main electric organ was subjected to denervation and the contralateral side was used as a control. After 20 days, the animals were killed by excision at the central nervous system. Denervated and normal electric organs were dissected from recently killed eels, the main electric organs (normal and denervated) were removed, homogenized and used for membrane preparations. Sucrose gradient fractionation Membrane fractions (P2 and P3) from the normal and denervated electric organ were further subfractionated on a five step discontinuous sucrose gradient. Aliquots of P2 and P3 fractions, containing approximately 70 mg of protein, were applied to a 15-45% sucrose gradient, and centrifuged at 100,000g, in a Beckman SW50,1 rotor. Fractions were collected from top to bottom of the centrifuge tubes for the enzyme assays. Enzymes assays (a) (Na ÷, K÷)-ATPase activity was measured at pH 7.6, with the following substrate media: 2.5 mM ATP, 3.25 mM MgCI2, 0.2 mM EDTA, 14 mM KC1, 72 mM NaCI, 160 mM Tris-HC1 pH 7.2. Membrane fractions were added in concentrations of about 20/,g/ml protein and incubations carried out at 37°C for 15 min. The reaction was started by the addition of ATP and was stopped with 5% TCA. Enzyme blanks were included in each experiment and aliquots were transferred for inorganic phosphate (Pi) estimation by the method of Fiske and SubbaRow (1925). The specific activities were expressed as the amount of inorganic phosphate liberated/mg protein/hr. Protein concentration was estimated by the method of Lowry et al. (1951) using bovine serum albumin as a standard. Membranes were also exposed to the inhibitor ouabain 0.4 mM, in the same medium as for (Na +, K+)ATPase, for 10min, before incubation at 37°C and Pi estimated as described above. (b) Acetylcholinesterase (ACHE) activity in P2 and P3 was assayed according to Hass6n-Voloch and Liepin (1963). The enzyme activity was measured at pH 8.0. The acetic acid released from a substrate medium containing
0.28 M ACh in 0.1 M sodium acetate and 0.2 M MgC12 was titrated with 0.1 M NaOH. A Beckman Century SS-1 pH meter was used and 0.1 M NaOH was delivered from an AGLA micrometer-syringe outfit with continuous stirring, at 25°C. Readings were taken at 1 min intervals over a 4 min period. Specific activity is defined as the amount of acetylcholine hydrolysed/ mg protein/hr. (c) Sialic acid determination was performed in P2 and P3 fractions, according to the method of Warren (1963), using a spectrophotometric determination. SDS-PAGE SDS gel electrophoresis was performed essentially according to Laemmli (1970) using either homogeneous gels containing 12.5% polyacrylamide or gradient gels containing 5-15 % polyacrylamide. Protein was stained with Coomassie Brilliant Blue G-250. The following proteins served as Mr markers: myosin, /~-galactosidase, phosphorylase b, bovine serum albumin, ovalbumin and carbonic anhydrase (Sigma Chemical Co. MS-SDS-200 Kit).
RESULTS ( N a + , K + ) A T P a s e - M g :+ d e p e n d e n t a n d acetylcholinesterase activities were m e a s u r e d in m e m b r a n e fractions P2 a n d P3, o b t a i n e d by differential centrifugation a n d the results are s h o w n in Table 1. As described by Soml6 et al. (1977), (Na ÷, K ÷ ) A T P a s e activity is present in b o t h fractions P2 a n d P3, p r e d o m i n a t i n g in fraction P2 (Table 1). ( N a ÷, K + ) A T P a s e from denervated o r g a n s presented specific activities lower t h a n the c o r r e s p o n d i n g activity o f a n o r m a l p r e p a r a t i o n , o f the o r d e r o f 18 a n d 16% for P2 a n d P3 d e n e r v a t e d fractions, respectively. A 10-fold decrease in specific activity was also observed w h e n o u a b a i n was a d d e d to the i n c u b a t i o n medium, confirming the identity o f the enzyme present in b o t h fractions P2 a n d P3, a n d d e m o n s t r a t i n g a similar response to the i n h i b i t o r which is independent of their actual c o n d i t i o n ( n o r m a l or denervated). C o n c e r n i n g acetylcholinesterase activity, which was used here mainly as a m e m b r a n e m a r k e r o f the post-synaptic m e m b r a n e (posterior i n n e r v a t e d electrocyte m e m b r a n e ) , higher activities were concentrated in P3 fraction a n d in the denervated condition. This enzyme showed a decreased activity as occurs with ( N a +, K +)ATPase. The activity profiles o b t a i n e d by m e a n s of a disc o n t i n u o u s sucrose gradient analysis are depicted in Figs 1 a n d 2. Figure 1 shows the distribution o f ( N a ÷, K ÷ ) A T P a s e a n d acetylcholinesterase f r o m n o r m a l a n d d e n e r v a t e d P2 m e m b r a n e fractions. Denervated P2 fractions exhibit a u n i m o d a l distribution
Denervation decreases (Na +, K+)ATPase activity
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of (Na +, K÷)ATPase with lower specific activities of the order of 60 #mol Pi liberated/hr/mg protein, in contrast with P2 normal fractions which have a bimodal distribution and higher specific activities (80 and 100/~mol Pi liberated/hr/mg protein). Figure 2 shows the profile of distribution of (Na +, K ÷)ATPase and acetylcholinesterase in normal and denervated P3 membrane fractions. Denervation, apparently, did not alter (Na ÷, K÷)ATPase distribution in this fraction, except for the lower specific activity observed when compared to the normal P3 preparation. As with acetylcholinesterase, the overall activity is also
diminished in comparison with the normal electric organ enzyme. P2 and P3 denervated membrane fractions also showed a decrease in sialic acid concentration and a discrete increase of the total protein concentrations when compared to normal membrane fragments (Table 2). Sialic acid concentration decreased 25.51% in P2 (denervated membrane fraction) and only 9% in P3 denervated membrane fractions. Figure 3 shows a typical polyacrylamide SDS gel electrophoresis pattern of P2 and P3 membrane proteins of normal and denervated electric organs.
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Table 2. Total protein and sialic acid composition of P2 (noninnervated) and P3 (innervated) subeellular fractions of normal and denervated electric organ
P2 P3
Fractions
Proteins (g%)
Sialic acid (nmol/mg/protein)
Normal Denervated Normal Denervated
14.23 +_ 3.37 19.29 + 3.58 22.46 + 3,46 26.03 + 2.02
52.41 + 3.83 38.52 ___4.79 25.55 ___2.62 23.16 + 4.53
Values are expressed as means +_ SD (n = 3). Experimental conditions are described in Material and Methods.
The Mrs indicated were determined from m r markers. The appearance of several bands indicates that the P2 and P3, normal and denervated membrane fracfractions, are composed of a heterogenous population of proteins, as expected. Denervated electrocyte membranes (P2 and P3) show an apparent decrease in some of the proteins, when compared to normal membranes, which can be observed at the 45,000 and 115,000 regions. Figure 4 shows a polyacrylamide SDS gel electrophoresis pattern of the sucrose gradients of P2 and P3 normal and denervated membrane fractions. There is a major band occurring at the apparent Mr of 100,000 probably corresponding to the alpha subunit of (Na ÷, K+)ATPase. The beta-subunit known to be a carbohydrate containing protein stained positive with periodic acid-Schiff reagent for glycoproteins (unpublished observations).
DISCUSSION
The electric organ of the electric eel is a valuable source of excitable membranes for the study of the structural and biochemical alterations produced by denervation. After denervation, the electric organ of E. eleetrieus shows a late and slow atrophy, which permits the study of nerve control upon this structure, the modifications observed seem to be different when compared to what occurs with skeletal striated muscle (Smith and Appel, 1977). With normal and denervated electric organ membrane fractions vesicles are mainly produced, but in membrane preparations obtained from denervated tissues, disrupted vesicles are frequently observed, demonstrating a rather irregular organization of the membrane fragments (Quintana and Lobfio; 1985). In this paper, we present evidence that denervation exerts changes in the membranes (Na +, K+)ATPase activities, probably representing the readjustment of the different metabolism pathways to maintain the basic conditions of the denervated electric organ. The decreased content of sialic acid in our denervated membrane fragments points to a decrease of synthesis concomitant with a slight increase of total protein synthesis. Studies made by FalcatoRibeiro et al. (1975) and Torres da Matta et al. (1985) on denervated electric organs showed qualitative and quantitative modifications in the total protein profiles as well as of the glycolitic enzymes
205 116 97 66
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Denervation decreases (Na +, K+)ATPase activity
627
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Fig. 4. Polyacrylamide SDS gel electrophoresis of (Na +, K +) ATPase more active peaks derived from the discontinuous sucrose gradient fractions (1)2 and P3) of normal and denervated electrocyte membranes. The arrows correspond to proteins of Mr + 50,000 and + 106,00. activities which we have also observed for (Na +, K+)ATPase. The electrophoretic pattern shows that some proteins diminish or simply disappears after denervation. However, most of these are not yet identified. The results of the enzymes distribution after centrifugation in sucrose gradients indicate the presence of the three distinct forms of AChE in electrocytes of the main electric organ, as already observed by Da Matta and Hasstn-Voloch (1975). Within 20 days after denervation we observed a decrease in AChE specific activity of about 35%, which is in agreement with other authors (Rosenberg et al., 1964). As for the (Na +, K+)ATPase, which couples the hydrolysis of ATP with the transport of Na + and K + ions across the cell membrane, we observe that denervation promotes a decrease of the enzyme activity and a different profile of enzyme distribution in the P2 and P3 membrane fractions. Hayashi et al. (1977) studying the (Na +, K+)ATPase from a microsomal fraction of canine kidney obtained two peaks of this enzyme, which was attributed to different concentrations of the annulus lipids. The two peaks of (Na +, K +)ATPase that appears normally in our P2 and P3 preparations when submitted to a sucrose
gradient disappears after denervation, leading to one broad single peak. The difference in density of these fractions may also be due to the different composition and content of total lipids bound to the enzyme. In a previous work we observed a slight increase of the total lipids (14%) and cholesterol (8%) in the extracts of denervated electrocytes membrane fractions (Quintana et al., 1988). As it occurs with ACHE, which has three distinct forms in the electrocytes of the main electric organ (Da Matta and Hass6nVoloch, 1975), there may be different molecular forms of (Na +, K+)ATPase in the electrocyte membrane. Studies of mammalian brain enzymes by Wayama et al. (1989) also presented evidence of three molecular forms for (Na +, K+)ATPase, but other authors believe that only two molecular forms exists in mammalian heart (Sweadner, 1985). Thus we can consider that the decreased enzymatic activity observed with (Na +, K+)ATPase and AChE may be an adaptation of the denervated tissue to the new situation. Biochemical modifications have been observed in many studies of denervated organs in the last few years. Changes inducing alterations in receptor activity (Almon and Appel, 1976) enzyme activities (Smith and Appel (1977); Torres da Matta et al.,
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1985) c a r b o h y d r a t e c o m p o n e n t s (Jeffrey a n d Appel, 1978) a n d p h o s p h o l i p i d m e t a b o l i s m (Appel et al., 1974; Q u i n t a n a et al., 1988) strongly suggest drastic changes in m e m b r a n e o r g a n i z a t i o n a n d function. REFERENCES
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