K+-ATPase in axonal growth cone particles isolated from fetal rat brain

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Int. J. Devl Neuroscience, Vol. 12, No. 5, pp. 485-489, 1994 Elsevier Science Ltd Copyright © 1994 ISDN 0736-5748(94)E0025-W Printed in Great Britain. All rights reserved 0736-5748/94 $7.00 +0.00

B I O C H E M I C A L P R O P E R T I E S OF Na+/K+-ATPase IN A X O N A L G R O W T H C O N E PARTICLES I S O L A T E D F R O M F E T A L R A T B R A I N R. MERCADO and J. HERNANDEZ-R. Laboratorio de Neurontogenia del Departamento de Fisiologia, Bioffsicay Neurociencias. Centro de Investigaci6n y de Estudios Avanzadosdel I.P.N. M6xico, D.F. M6xico (Received 21 September 1993;revised 22 February 1994;accepted 5 April 1994)

Ahstraet--Axonal growth cones (AGC) isolated from fetal rat brain have an important specific activity of N+/K+-ATPase. Kineticassays of the enzymein AGC showedthat Kmvaluesfor ATP or K+ are similar to those reported for the adult brain enzyme. For Na + the affinity (Kin) was lower. Vmaxfor the three substrates was several times lower in AGC as comparedto the adult value. We also observed two apparent inhibition constants of Na+/K+-ATPaseby ouabain, one of low affinity, possibly correspondingto the al isoform and another of high affinitywhich is different to that described for the et2 isoform of the enzyme. These results support an important role for the sodium pump in the mantainance of volume and cationic balance in neuronal differentiating structures. The functional differences observed also suggest that the enzymatic complex of Na+/K+-ATPasein AGC is in a transitional state towards the adult configuration. Key words: axogenesis, growth cones, sodium pump.

The axonal growth cone is a neuroblast structure that seeks out and contacts potential target cells during brain development, playing a critical role in axogenesis and synaptogenesis. 3,4A1,24,27 Most of the morphological studies of growth cones in vivo and in vitro have been generally related to cytoskeletal components, 14,15 but the advent of procedures to isolate axonal growth cone particles 8,2° ( A G C ) , has made it possible to study some of their biochemical properties. N a + / K + - A T P a s e (sodium pump, A T P phosphohydrolase, E C 3.6.1.3) is a tetrameric complex that spans the plasma membrane and serves to maintain the cellular cationic gradients and membrane potentials. 2,7 Messenger and Warner 19 have also reported that inhibition of the sodium pump by strophantidin reduced the final number of differentiated neurons in amphibian tissue cultures, suggesting that it has an important role in neurogenesis. We believe that N a + / K +-ATPase activity may be relevant in the functions of axonal growth cones, which are still poorly understood. We therefore decided to study the activity of this enzyme and its kinetics for ATP, Na + and K +, and to assess its ouabain inhibition pattern, in an enriched A G C fraction isolated from the fetal rat brain. Since an interaction between N a + / K + - A T P a s e and serotonin (5-HT) has been reported in the adult rat brain, 12,13 in which 5-HT increases the velocity of reaction of the enzyme, 5,18 we found it interesting to see whether the kinetics of N a + / K + - A T P a s e for A T P is also influenced by 5-HT in A G C , as a possible mechanism of regulating growth cone activity.

EXPERIMENTAL PROCEDURES A G C were isolated according to the method of Pfenninger et aL, 2° slightly modified as follows: fetal rat brains from the 17th day of gestation were homogenized in 10 volumes of 0.32 M sucrose, to which have been added in mM: MgCI2, 1 and T E S - N a O H , 1, p H 7.3, at 700 rpms, five strokes in a Thomas pestle glass homogenizer (0.25 m m clearance). To remove nuclei and cell debris the homogenate was centrifuged at 1660 g for 15 min. The low speed supernatant was loaded onto a discontinuous sucrose density gradient (2.66--0.75 M) then centrifuged at 240,000 g for 60 min using a Beckman 60 Ti vertical rotor. Three fractions were separated and removed. The fraction enriched with A G C was collected at the interface between the load and 0.75 M sucrose and diluted with 10 ml of ice-cold 0.1 M Tris-HCl buffer, p H 7.3. Then it was centrifuged at 220,000 g for 40 min. The final pellet was resuspended in cold 0.05 M Tris-HCl buffer, p H 7.3 and stored at - 2 0 ° C until used. *To whom all correspondence should be addressed at: Lab. of Neurontogeny, Dept. Physiology, Biophysics and Neurosciences, CINVESTAV-I.P.N.,Av. Instituto Polit6cnicoNacional 2508, 07000 M6xico, D.F., M6xico. Tel: 525-7-5402-00 Ext. 5124;Fax: 525-752-61-06. 485

486

R. Mercado and J. Hernfindez-R.

N a + / K + - A T P a s e activity was determined as follows: an aliquot of the A G C equivalent to 150 I~g of protein was preincubated during 10 min at 37°C in a reaction mixture containing in mM: MgCI2, 6: NaCI, 120; KC1, 20; T r i s - H C ! buffer, 30, p H 7.4, for the total ATPase activity. T o inhibit the N a + / K + - A T P a s e , 0.1 m M ouabain was added. The reaction was triggered with 3 mM A T P (Vanadium free, Sigma), and at 10 rain was stopped with 50% (w/v) cold trichloroacetic acid. For the kinetics of N a + / K +-ATPase various concentrations of A T P (0.1-7 mM), Na + ( 10-140 mM) and K + (5- 30 mM) were employed. Choline chloride was used to maintain the tonicity of the reaction mixture. O u a b a i n inhibition curves were o b t a i n e d with concentrations ranging f r o m 10 9 to 10 -3 M. To see if 5-HT modifies the N a + / K + - A T P a s e kinetics with A T P a high (10 -3 M) and a low (6×10 -7 M) concentration of the amine was added in the presence of 0.1 m M pargyline, a M A O inhibitor. 23 Inorganic phosphate (Pi) was determined by the photometric method of Fiske and Subbarrow 6 in an aliquot of 600 g supernatant obtained from each sample. T h e N a + / K + - A T P a s e activity was considered to be the difference between total and ouabain resistant activity. Protein was determined according to the method of L o w r y et al. 16 using bovine serum albumin as standard. Best fit saturation curves and the kinetic constants (Kin and Vmax) were calculated using the R O S F I T program. 9 RESULTS N a + / K + - A T P a s e in A G C showed a specific activity of 1.7_+0.03 p.mol Pi/mg protein/hr. In Fig. 1 A - C we show the saturation curves of N a + / K + - A T P a s e for ATP, Na + and K ÷, respectively. The corresponding linearization plots according to Hanes 1° are also shown, from where the kinetic Y d

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Fig. 1. Kinetic parameters of Na+/K+-ATPase against ATP (A), Na + (B), and K + (C) in AGC. The corresponding linearization plots according to Hanes are shown as insets. Each point of the saturation curves corresponds to three experiments made in triplicate samples.

Sodium pump in axonal growth cones

487

Table 1. Kinetic constants of Na+/K+-ATPase against ATP, Na +, and K + in axonal growth cones from fetal rat brain. Vmax

Substrate ATP Na + K+

Km(mM)

(~mol Pi/mg protein/hr)

1.01±0.1 32.60±3.0t 1.60±0.1

2.14_+02 3.00_+0.2* 2.46±0.5

X-+S.D. from three experiments made in triplicate samples. *P<0.05 (Student's t-test). tP<0.0005 (Student's t-test).

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Fig. 2. Inhibition of Na+/K+-ATPase by ouabain relative to basal specific activity, in AGC. Kiapp.1 = 10-4 M, Kiapp.2=5×10 -6 M. Each point of the inhibition curve corresponds to three experiments made in triplicate samples. Table 2. Kinetic constants of Na+/K+-ATPase against ATP in presence and absence of 5-HT in axonal growth cones from fetal rat brain Vnlax

Group Control 5-HT (0.6 ixM) 5-HT (1.0 mM)

Km (mM)

(txmol Pi/mg protein/hr)

1.01 ±0.1" 1.15_+0.3 1.16±0.5

2.14± 0.2t 2.25_+0.3 2.32±0.6

x±S.D, from three experiments made in triplicate samples. * tP=0.05 (Student's t-test).

c o n s t a n t s g m a n d Vmax w e r e o b t a i n e d ( T a b l e 1). T h e Vmax was s i m i l a r for A T P a n d K + s u b s t r a t e s a n d h i g h e r for N a + ( T a b l e 1). T h e affinity for N a + was also significantly l o w e r as c o m p a r e d to t h e affinity f o r A T P a n d K + ( T a b l e 1). F i g u r e 2 s h o w s t h e i n h i b i t i o n c u r v e o f N a + / K + - A T P a s e b y o u a b a i n . It is a b i m o d a l c u r v e with t w o a p p a r e n t i n h i b i t i o n c o n s t a n t s , o n e o f l x l 0 - 4 M a n d the o t h e r o f 5 × 1 0 - 6 M. T h e k i n e t i c c o n s t a n t s o f N a + / K + - A T P a s e f o r A T P in the p r e s e n c e a n d a b s e n c e o f 5 - H T a r e s h o w n in T a b l e 2, n o statistical d i f f e r e n c e s w e r e o b s e r v e d .

DISCUSSION It is i m p o r t a n t t o n o t e t h a t t h e b a s a l activity o f N a + / K + - A T P a s e o b s e r v e d in A G C c o r r e s p o n d s t o o n e - f o u r t h a n d o n e - s i x t h o f t h a t o b s e r v e d in h o m o g e n a t e a n d s y n a p t o s o m e s , r e s p e c t i v e l y , d e r i v e d f r o m t h e a d u l t r a t b r a i n , 18 suggesting t h a t in t h e s e e a r l y a x o n a l s t r u c t u r e s t h e activity o f t h e

488

R. Mercado and J. Hernfindez-R.

sodium pump may be functionally relevant in the growth process, which is in line with Messenger and Warner's 19 results in cultured neurons. In relation to Km values for A T P and K +, they are similar to those reported in other preparations of adult cerebral tissue. 18,22,25The differences observed concern mainly the handling of sodium by the enzyme in AGC. It seems that it handles sodium with less affinity when compared to A T P and K + , but with a higher reaction rate (Table 1). Km values reported for the adult brain 17"22'25 are in the range of 10-18 mM, therefore, according to the present results, in AGC, there is a slightly less affinity for sodium. In our laboratory the Vmax obtained for sodium in the adult brain (unpublished results) seems to be 40% higher as compared to the Vmax in AGC. Three molecular forms of the catalytic subunit of Na+/K +-ATPase have been described with different inhibition constants for glycosides, c,l, ~2 and oL3.21"26 The ouabain inhibition constants observed in A G C suggest that the a l and the c~2 isozymes are already present. The ~2 isozyme probably is in a transitional state, because its affinity for ouabain is lower as compared to the adult isoform, l We do not believe that this isozyme corresponds to the ~3 described by Urayama and Sweadner 26 because of its different sensitivity to ouabain. 5-HT did not modify the Na+/K + -ATPase kinetics for ATP in A G C as it does in the adult brain, I'~ suggesting that the regulation of the sodium pump by 5-HT may not be present in these early structures. Our results show a sodium pump in differentiating neuronal structures ( A G C ) with an important basal activity of 25% of that observed in adult whole brain preparations. This suggests that some functions of the sodium pump, such as volume regulation, ionic balance, and calcium exchange, may be of importance for the growth cone and for axon formation and growth. The behavior of Na+/K+-ATPase in A G C seems to be different than in the adult brain with distinct kinetics for sodium and a different sensitivity to ouabain, suggesting that this enzymatic complex, at this stage of development, is in a transitional period compared to the adult configuration. Acknowledgements: Technical assistance from Mrs Graciela Chagoya and Mr lgnacio Vargas is gratefully acknowledged.

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