ACTA NEUROBIOL. EXP. 1974, 34: 563-567 MODIFICATION OF THE BIOASSAY OF ACETYLCHOLINE Demtment o Bfmhanhtry o <Nervou6System and M M e f f 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 developedfor quantitative determination of acetylcholine, the most commonly em-ployed and sensitive method is still the bioassay (2, 4, 6, 7).P g 1. Photograph of khe cuppma$us: a, h o b controlling the timer; b, indicator; c, i.scale of the tmkxempemmeter; d3, system for stopping the ilndicabor; e, micruram-pemmder; k, box containing the .&oboedement; 1, h o b f r setting the base l w d o 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. Itallows one to obtain more reliable and reproducible results than thosefrom 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 photoelementis 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 thestate of the muscle. The diaphragm rises with the contraction of themuscle; when the muscle is only partly contracted less light falls on thephotoelement. The reading of the microamperometer (in pa) is directlyproportional to the size of the muscle contraction. The apparatus is suit-able for a dorsal muscle of the leech, a frog muscle (rectus abdominis), ora guinea-pig ileum. The bath containing the muscle is the same as thatnormally used in the smoked drum method. The muscle is connected toone arm of the lever. A free hanging aluminium diaphragm is attachedto the second arm. The arm connected to the diaphragm was 17 timeslonger than that connected to the muscle. The base level (OQ/O of extinction) may be set with knob 1 (Fig. 1)Fig. 2. Conlttrucbon o the apporaltw: k, box containing the iphohelement; z, photo- felement; s, doubls-amn lever; p, m u x l e baUh; o, muscle; n, diaphragm; f , lights o m e ; m, plastic tube, thwulph which the llght flbws from its source t the photo- oelement; u, rod connected to photoelement for setting lOO0/o o extinction (see text); f x, m e d i a n to minoampemmeter.
SHORT COMMUNICATION 565which 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. 1and 2). The photoelement k may be moved up and down by means ofrod u (Fig. 2) for setting 100010 of extinction. In this case, the muscle bathand 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 50 I 100 I 150 I 200 I ng of acetylcholine per volume of the bofh
566 SHORT COMMUWCATIONby knob a (Fig. 1 and 2). The system that stops the indicator consists ofa frame which is pressing against the indicator d 3 (Fig. 1 and 3), a barconnecting the frame to the core bf the electromagnet d2 (Fig. 3), and theelectromagnet 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 d 3 . 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 withstandard assays using the smoked drum method. Doses of acetylcholineare given at regular time intervals (1 min) followed by periods of wash-ing the muscle with Ringer solution (2 min); thus, one measurementlasts 3 min. Since the sensitivity of the muscle to acetylcholine changeswith time every dose of the unknown sample must be preceded and fol-lowed by a matching amount of acetylcholine. As shown in Fig. 4 andFig. 5, linearity for frog muscle is obtained for the range from 50 ng to200 ng- of acetylcholine per volume of bath (in the described apparatus2.5 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 thevalue of pa obtained by the unknown sample. The authors wish t~ thank Wfes&or Stella Niemiei-ho f o r her i n . t m t andencouragement amd Mns. Bozena Heiler-Gd~biew&a for her skilled lteahnical assis-tance. Thiis wmk was supported by Pmject 00.4~1 f the Polish Academy of Sciences. a
SHORT COMMUNICATION 1. BROWNING, E. T. 1072 Rudmetziic enzyme assay for chloline and acetylcho- Elne. Anal. Biochem. 46: 6%4-018. 2. CHENG, S. C. 1WrL. P y r u v a k r n e t a h h in t h e h ~ b t e rnerve as affected by the partial pressure of o hdioxide: o b e m a t i o m o n m e t a b l i c oampad- f 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 o presynaptic release and a chokine acetyltans- f ferase away. J. N e u m h e m . 116: 1136-143. 4. GOMEZ, M. V., DAI, M. E. M. m DINIZ, C. R. 1973. Effect of sampikm v e m , i d tityustoxh o n 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. L d e n t i f i c a ~ m aceS.lcho2ine in fresh r a t brain by of combined gas chmnato@aphy-mass spechometry. 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. 1 9 .Meawrement of rate d acetylcblirlze cliffusion thmugh 111 a brain slice and is significance hn studies of the cellular ddistrilbution of t acetylchwlhesterase. J. Neumchem. 18: 21329-23134. 8. ONEILL, J. J, and SAKAMOTO, T 1970. Enzymatic fluorimetric 1deknn5mtion . o acetylcholjme i biological exDracts. J. N e u w h m . 17: 1 1 Y I r l M O . f n 9. REID, W. D., HAUBRICH, D. R. and KRISHNA, G. Ilsfll. Ehuymic rdioa&?ay t o r wetylcholilne and choline bn brain. Amal. Biochm. 41: 890-397.10. SBILAGYI, P. I. A., GREEXV, I. P., BROWN, 0.M. and MARGOLIS, S. 1972. The measurement o namgram i m u n t s of a c e t y l c b ~ e n tissue by pyro- f 5 lysis gas chmmatography. J. N e u m h e m . 19: 2555-2l566.Received 20 November 1973Jerzy KALIRSKI, Department o Cell Biology, Nencki Institute of Expendmental Biology, fPasteura 3, 00-973 Wairszawa, Poland.Andrze) WIERASZKO, Department of Biochemistry of Nervous System and Muscle, NenckiInstitute of Experimental Bidogy, Pasteura 3, 00-973 Warszawa, Polacnd.