ACTA NEUROBIOL. EXP. 1974, 34: 563-567

MODIFICATION OF THE BIOASSAY OF ACETYLCHOLINE

Demtment of Bfmhanhtry of <Nervou6 System and M M e Nencki Institute of Experimental Biology, Warszawa, Poland

Although many different methods like spectrofluorometry (1, 3, 8), gas chromatography (5, lo), and enzymic assay (9), have been developed for quantitative determination of acetylcholine, the most commonly em- ployed and sensitive method is still the bioassay (2, 4, 6, 7).

Pig. 1. Photograph of khe cuppma$us: a, h o b controlling the timer; b, indicator; c, scale of the tmkxempemmeter; d3, system for stopping the ilndicabor; e, micruram- pemmder; k, box containing the .&oboedement; 1, h o b for setting the base l w d measurement (see. text); m, plastic tube; n, diaphmgm; p, lnwcb bakh; s, double-arm

lever.

564 SHORT COMMUNICATION

The apparatus described in this paper is simple and inexpensive. It allows one to obtain more reliable and reproducible results than those from a smoked drum.

The apparatus is shown in Fig. 1. A beam of light falls on a photo- element. A movable diaphragm between the lamp and the photoelement is located. It is connected to the muscle by means of a double-arm lever.

The amount of light which falls on the photoelement depends on the state of the muscle. The diaphragm rises with the contraction of the muscle; when the muscle is only partly contracted less light falls on the photoelement. The reading of the microamperometer (in pa) is directly proportional to the size of the muscle contraction. The apparatus is suit- able for a dorsal muscle of the leech, a frog muscle (rectus abdominis), or a guinea-pig ileum. The bath containing the muscle is the same as that normally used in the smoked drum method. The muscle is connected to one arm of the lever. A free hanging aluminium diaphragm is attached to the second arm. The arm connected to the diaphragm was 17 times longer than that connected to the muscle.

The base level (OQ/O of extinction) may be set with knob 1 (Fig. 1)

Fig. 2. Conlttrucbon of the apporaltw: k, box containing the iphohelement; z, photo- element; s, doubls-amn lever; p, muxle baUh; o, muscle; n, diaphragm; f , light s o m e ; m, plastic tube, thwulph which the llght flbws from its source to the photo- element; u, rod connected to photoelement for setting lOO0/o of extinction (see text);

x, m e d i a n to minoampemmeter.

SHORT COMMUNICATION 565

which is used for changing the vertical position of the instrument box (Fig. 2), made up of the photoelement lei and the plastic tube m (Fig. 1 and 2). The photoelement k may be moved up and down by means of rod u (Fig. 2) for setting 100010 of extinction. In this case, the muscle bath and the diaphragm remains stationary.

Fig. 3. Sketch showing the basic mnstmetion of the modified microampemmeter: a, Ihob controlling timer; b, imdlcatior; c, wale of the m i m m p m m t w ; t, Oianer; dl, d, d , system which rnwhainiwlly stops the indicator (see text); w, scale of the

timer.

The slightly modified microamperometer is shown in Fig. 3. The in- dicator b is stopped mechanically after a preset time interval regulated

Fig. 4. Calibration curve for frog muscle.

I I I I I I

50 100 150 200 ng of acetylcholine per volume of the bofh

566 SHORT COMMUWCATION

by knob' a (Fig. 1 and 2). The system that stops the indicator consists of a frame which is pressing against the indicator d 3 (Fig. 1 and 3), a bar connecting the frame to the core bf the electromagnet d2 (Fig. 3), and the electromagnet dl connected to the timer t (Fig. 3). The electromagnet re- acts after a set time controlled by means of the knob a (Fig. 3). The indi- cator is then pressed down and held in place by the frame d3 . The con- traction time of the muscle may be fixed from 0.5 min (for very sensi- tive muscles) up to 3 min (for less sensitive muscles).

The procedure involved here is very much the same as that with standard assays using the smoked drum method. Doses of acetylcholine are given at regular time intervals (1 min) followed by periods of wash- ing the muscle with Ringer solution (2 min); thus, one measurement lasts 3 min. Since the sensitivity of the muscle to acetylcholine changes with time every dose of the unknown sample must be preceded and fol- lowed by a matching amount of acetylcholine. As shown in Fig. 4 and Fig. 5, linearity for frog muscle is obtained for the range from 50 ng to 200 ng- of acetylcholine per volume of bath (in the described apparatus 2.5 ml), and for the leech muscle for the range from 5 ng to 20 ng, re- spectively. The results are calculated by comparing the value of pa ob- tained by a known amount of standard acetylcholine solution with the value of pa obtained by the unknown sample.

The authors wish t~ thank Wfes&or Stella Niemiei-ho for her i n . t m t and encouragement amd Mns. Bozena Heiler-Gd~biew&a for her skilled lteahnical assis- tance. Thiis wmk was supported by Pmject 00.4~1 af the Polish Academy of Sciences.

SHORT COMMUNICATION

1. BROWNING, E. T. 10'72 Rudmetziic enzyme assay for chloline and acetylcho- Elne. Anal. Biochem. 46: 6%4-018.

2. CHENG, S. C. 1WrL. Pyruvak r n e t a h h in the h ~ b t e r nerve as affected by the partial pressure of ofh dioxide: obemat iom on me tab l i c oampad- mentation J. N e m h e m . 19: 481--47U.

3. FELLMANN, J. H. L W . A chemfcd rnethlod for determination of acetylcholine: its application in la study of presynaptic release and a chokine acetyltans- ferase away. J. Neumhem. 116: 1136-143.

4. GOMEZ, M. V., DAI, M. E. M. mid DINIZ, C. R. 1973. Effect of sampikm v e m , tityustoxh on the release of acetylcholine from incubated slices of rat. brain. J. Neumhem. 20: 105111061.

5. HAMMAR, C. G., HANIN, I., HOLMSTEIYT, B., KITZ, R. J., JENDEN, D. J. and KARLEN, B. 1968. Ldent i f ica~m of aceS.lcho2ine in fresh ra t brain by combined gas chmnato@aphy-mass spech'ometry. Nature 220: 615-917.

6. JENDEN, D. J. and CAMPBELL, L. B. 1971. Meawrement of c h d n e esters. Meth. Biochem. Anal., Suppl.: 1183-216.

7. LANCASTER, R. 1191'1. Meawrement of rate d acetylcblirlze cliffusion thmugh a brain slice and its significance hn studies of the cellular ddistrilbution of acetylchwlhesterase. J. Neumchem. 18: 21329-23134.

8. O'NEILL, J. J, and SAKAMOTO, T. 1970. Enzymatic fluorimetric 1deknn5mtion of acetylcholjme in biological exDracts. J. N e u w h m . '17: 1 1 Y I r l M O .

9. REID, W. D., HAUBRICH, D. R. and KRISHNA, G. Ilsfll. Ehuymic rdioa&?ay tor wetylcholilne and choline bn brain. Amal. Biochm. 41: 890-397.

10. SBILAGYI, P. I. A., GREEXV, I. P., BROWN, 0. M. and MARGOLIS, S. 19'72. The measurement of 'namgram i m u n t s of a c e t y l c b ~ e 5n tissue by pyro- lysis gas chmmatography. J. Neumhem. 19: 2555-2l566.

Received 20 November 1973

Jerzy KALIRSKI, Department of Cell Biology, Nencki Institute of Expendmental Biology, Pasteura 3, 00-973 Wairszawa, Poland. Andrze) WIERASZKO, Department of Biochemistry of Nervous System and Muscle, Nencki Institute of Experimental Bidogy, Pasteura 3, 00-973 Warszawa, Polacnd.