Neuroscience Let ters, 66 (1986) 159-162 159

Elsevier Scientific Publishers Ireland Ltd.

NSL 039119

O X Y H E M O G L O B I N INHIBITION OF A C E T Y L C H O L I N E S T E R A S E ACTIVITY

M A T T H E W D. LINNIK and T O N Y J-F. LEE*

Departnwnt Of Pharmacology, Southern Illinois University School o[" Medicine, P.O. Box 3926. Springfield.

IL 62707¢ ( U.S.A. )

I Received November 5th, 1985; Revised version received December 16th, 1985: Accepted December 18th,

1985)

Key wor&': oxyhemoglob in - methemoglobin acetylcholinesterase Electrophorus eh,ctrieus

The effects of human oxyhemoglobin (HbO:), human methemoglobin (MetHb), and porcine serum

albumin (PSA) on the activity of acetylcholinesterase (ACHE) isolated from Electrophorus electricus were

cxamined. HbO, produced a dose-dependent reduction in AChE activity. Fifty percent of activity was

obtained at 5 #M HbO> while 95% inhibition was obtained at 5(1 ~M. In this concentration range MetHb

and PSA had little effect on esterase activity.

Acetylcholinesterase (ACHE) is a seemingly ubiquitous enzyme with multiple func- tions. Its role in cholinergic neurotransmission is well documented, but there is also evidence for its involvement in dopaminergic neurotransmission [8], and hydrolysis of substance P [4] and enkephalins [5]. AChE may be membrane-bound or soluble and exists in multiple molecular forms [11].

We initiated this investigation into the effect of hemoglobin (Hb) on cholinergic function based on data indicating that Hb, in particular oxyhemoglobin (HbOe), was capable of altering responses to neurogenic stimulation in the bovine penile retractor muscle [3] and dog cerebral arteries [10]. The following study was designed to exam- ine the effect of HbO> methemoglobin (MetHb) and porcine serum albumin (PSA) on the activity of AChE extracted from the electric eel, Eh, ctrophorus electricus.

HbO: was prepared from the purchased Hb which contains up to 75% MetHb. In order to distinguish the effect of HbO2 from MetHb, it was necessary to reduce the Hb according to a modification of the method of Asakura [1]. Human Hb was dis- solved in water at a concentration of 19 mg/ml. Once solubilized, this was added to sodium dithionite (1 mg dithionite/15 mg prot.). A l-ml aliquot was immediately removed and passed over a Sephadex G-25 column to separate Hb from dithionite. Protein concentration of Hb was determined using the BioRad protein assay, 15 rain alter addition of reagent, using Hb as the standard.

*Author for correspondence.

0304-3940/86,$ 03.50 © 1986 Elsevier Sciuntitic Publishers Ircland Ltd.

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The method used for determination of AChE activity was a modification of the radiometric procedure of Johnson and Russell [9]. Units of AChE (4.8 x 10 -5) were placed into 6-ml glass vials on ice along with appropriate concentrations of Hb and PSA to a volume of 160 #1. [SH]Acetylcholine (ACh) iodide was the final item added to attain 20 pM [3H]ACh in 200/A. Vials were placed in a 37°C oscillating water bath for 15 min. The reaction was stopped by the addition of 200 #1 of the following sol- ution: chloroacetic acid, 1 M; NaOH, 0.5 M; NaC1, 2 M. After shaking this was fol- lowed by 4 ml liquid scintillation cocktail containing 0.51% PPO and 0.03% POPOP in toluene with 10% isoamyl alcohol. Vials were vortexed and phases were allowed to separate for 30 rain before counting. The addition of stopping mixture blocks esterase activity while protonating the [3H]acetate produced. This enhances [3H]ace- rate extraction into the cocktail, while non-hydrolyzed ACh remains predominantly in the aqueous layer. For each assay blanks with [3H]ACh, but no ACHE, were deter- mined and subtracted from all samples. Blanks accounted for less than 3% of the radioactivity in the vial. Total radioactivity was determined by combining [3H]ACh with Ready Solv EP, a liquid scintillation cocktail for aqueous samples. All assays were run in triplicate.

Due to the chromophoric nature of Hb, a quench curve was developed using Hb as the quenching agent. Radioactivity was counted in a Beckman LS 5800 liquid scin- tillation counter, utilizing the H number method of quench compensation. Counting efficiency varied from 21 to 46%, dependent upon the level of quench.

Sodium dithionite, human Hb type IV, human MetHb type III, PSA fraction V, and AChE type VI-S from Electrophorus eleetricus were obtained from Sigma. [3H]Acetylcholine iodide (50 mCi/mmol) was from New England Nuclear.

The results indicate that addition of sodium dithionite to Hb and separation on a Sephadex G-25 column produced characteristically cherry-red HbO2. Spectropho- tometric analysis identified absorption maxima at 540 and 577 nm. If stored on ice, HbO2 was stable for at least 7 h, although at 37°C oxidation was accelerated.

AChE was added to the vials in order to obtain 16-18% hydrolysis of total ACh in 15 min. Hydrolysis never exceeded 20% of the substrate. The effects of HbO2, MetHb, and PSA were then determined. As shown in Fig. 1, MetHb and PSA, at concentrations ranging from 0.1 gM to 50 ktM, had little effect on esterase activity, and the influence exerted was not dose-dependent. Prepared HbO2, however, caused significant, dose-dependent reductions in esterase activity. Fifty percent inhibition of activity was obtained with 5 ~M HbO2, while 50/~M HbO2 caused almost total in- hibition of esterase activity.

In vertebrates there exist multiple forms of ACHE. These can be categorized according to their quaternary structure, asymmetric or globular, and according to the number of catalytic subunits present [11]. Although combination of catalytic subunits into multiple quaternay structures may result in alterations of some physio- chemical properties, their catalytic properties appear essentially identical [2, 15]. Furthermore, there are several observations suggesting structural homology between mammalian and Electrophorus AChE for both the assymetric and globular forms of the enzyme (for an in-depth discussion, see ref. 1l). It is this apparent homology

161

- 2 0 - >

0 O -

l- (,.) 2 0 - ,< it,,.

o 4 0 - C o

6 0 - . .0

¢,.

- - 8 0 -

1 O0 I I T i I i i i i OW_7 i i ~ I ! I I I

1 0 - 6 1 0 - 5 1 0 - 7 1 0 - 6 1 0 - 5 1 0 - 7 1 0 - 6 1 0 - 5

H b 0 2 M e t H b A l b u m i n

Fig. I. Effect of HbO2. MetHb and PSA on AChE activity. Zero inhibition (---) refers to total activity observed in 15 rain.

which allows for examination of mammalian protein effects in Electrophorus ACHE. Due to erythrocyte containment and the closed-circuit nature of the cardiovascular

system, Hb is not normally exposed to ACHE. Although human erythrocytes possess a membrane-bound, dimeric ACHE, this AChE is highly hydrophobic [12, 13] and maintenance of activity requires a hydrophobic environment [7, 16]. Furthermore, the catalytic activity is directed entirely towards the outside of the membrane [14]. Although in close proximity, one would expect little interaction between Hb and erythrocyte membrane-bound ACHE.

It is entirely possible, however, that Hb may interact with AChE in angiopathic conditions involving rupture of vascular walls. As seen in Fig. 1, there is a 50% reduc- tion in AChE activity at 5/2M HbO2. Almost total inhibition is obtained at 50/~M HbO:. Using normal human blood values of 5 x 106 erythrocytes per mm 3, and a

mean corpuscular Hb of 30 pg, it is calculated that 10 /2M Hb could be obtained in 1 ml by the addition of less than 5/,1 of whole blood. Excluding the effects of com- ponents other than Hb, this volume of blood would be expected to produce a 70 80% reduction of esterase activity.

Fig. 1 also demonstrates that AChE activity is minimally affected by MetHb or PSA. This suggests that the ferrous form of Hb is involved in the mechanism of inhi- bition whereas the oxidized state of Hb is nearly impotent. Since circulating Hb is almost exclusively in the reduced state, its exposure to AChE would result in inhibi- tion of the activity of this enzyme. Due to the high concentration of Hb in the blood. even a small rupture of a blood vessel, large enough to release erythrocytes, would thereby result in significant AChE inhibition.

The reversible nature of this inhibition and the full mechanism of inhibition have

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yet to be addressed . In l ight o f the d e m o n s t r a t e d cho l ine rg ic d y s f u n c t i o n in ce r t a in

pa tho log i c c o n d i t i o n s [6], however , resul ts o f the p re sen t s t udy m a y be i m p o r t a n t in

a t t e m p t i n g to e luc ida te the m e c h a n i s m s u n d e r l y i n g these diseases.

Th i s w o r k was s u p p o r t e d by g r a n t s f r o m N I H ( H L 27763) a n d a G r a n t - i n - A i d

f rom A m e r i c a n H e a r t A s s o c i a t i o n ( A M A 83-1040) wi th f u n d s c o n t r i b u t e d in pa r t

by I l l inois H e a r t A s s o c i a t i o n , a n d fu n d s f rom S I U School o f Medic ine . The a u t h o r s

t h a n k Ms. Salie F l u c k i g e r for p r e p a r a t i o n o f the m a n u s c r i p t .

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2 Barnett, P. and Rosenberry, T.L., Functional identity of catalytic subunits of acetylcholinesterase, Bio- chem. Biophys. Acta, 567 (1979) 154-160.

3 Bowman, A., Gillespie, J.S. and Pollock, D., Oxyhaemoglobin blocks non-adrenergic non-cholinergic inhibition in the bovine penile retractor penis muscle, Eur: J. Pharmacol., 85 (1982) 221-224.

4 Chubb, I.W., Hodgson, A.J. and White, G,H., Acetylcholinesterase hydrolyzes substance P, Neuro- sciences, 5 (1980) 2065-2072.

5 Chubb, I.W., Ranieri, E., Hodgson, A.J. and White, G.H., The hydrolysis of Leu- and Met-enkephalin by acetylcholinesterase, Neurosci. Lett., Suppl. 8 (1982) $39.

6 Coyle, J.T., Price, D.L. and DeLong, M.R., Alzheimer's disease: a disorder of cortical cholinergic innervation, Science, 219 (1983) 1184-1190.

7 Frenkel, F.J., Roelofsen, B., Brodbeck, U., Deenen, L.L.M. and Ott, P., Lipid-protein interactions in human erythrocyte-membrane acetylcholinesterase. Modulation of enzyme activity by lipids, Eur. J. Biochem., 109 (1980) 377-382.

8 Greenfield, S.A. and Shaw, S.G., Release of acetylcholinesterase and aminopeptidase in vivo following infusion of amphetamine into the substantia nigra, Neuroscience, 7 (1982) 2883-2893.

9 Johnson, C.D. and Russell, R.L., A rapid, simple radiometric assay for cholinesterase, suitable for multiple determinations, Anal. Biochem., 64 (1975) 229-238.

10 Lee, T.J-F., MclIhany, M.P. and Sarwinski, S., Erythrocyte extracts enhance neurogenic vasoconstric- tion of dog cerebral arteries in vitro, J. Cereb. Blood Flow Metab., 4 (1984) 474-476.

11 Massouli6, J. and Bon, S., The molecular forms of cholinesterase and acetylcholinesterase in verte- brates, Annu. Rev. Neurosci., 5 (1982) 57-106.

12 Ott, P., Jenny, B. and Brodbeck, U., Multiple molecular forms of purified human erythrocyte acetyl- cholinesterase, Eur. J. Biochem., 88 (1975) 119-125.

13 Ott, P. and Brodbeck, U., Multiple molecular forms of acetylcholinesterase from human erythrocyte membranes; interconversion and subunit composition of forms separated by density gradient centrifu- gation in zonal rotor, Eur. J. Biochem., 88 (1978) 119-125.

14 Steck, T.L., Preparation of impermeable inside-out and right-side-out vesicles from erythrocyte mem- branes. In E.D. Korn (Ed.), Methods in Membrane Biology, Plenum Press, New York, 1974, pp. 245- 281.

15 Vigny, M., Bon, S., Massouli~, J. and Leterrier, F., Active side catalytic efficiency of acetylcholines~er- ase molecular forms in Electrophorus. Torpedo, rat, and chicken, Eur. J. Biochem., 85 (1978) 317-323.

16 Wiedmer, T., DiFrancesco, C. and Brodbeck, U., Effects of amphiphiles on structure and activity of human erythrocyte membrane acetylcholinesterase, Eur. J. Biochem., 102 (1979) 59-64.