Transport of Acetylcholine in a Membrane

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1 APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY 9, (1984) Transprt f Acetylchline in a Membrane Laminate Mdel f the Neurmuscular Junctin SACHIO HIROSE* Central Research Labratries, Mitsubishi Petrchemical C., Ltd., 1315, Ami, Inashiki, Ibaraki 3003, Japan AND WOLF R. VIETH Department f Chemical and Bichemical Engineering, Rutgers University, Piscataway, NJ Received August 30, 1983; Accepted September 26, 1983 Abstract A laminate mdel f the cleft-plus-pstsynaptic membrane structure f the neurmuscular junctin was studied. In rder t prepare a mdel f the pstsynaptic membrane, the prperties f acetylchline (Ach) receptr-rich vesicles purified frm Trped fish were measured. Immbilizatin f vesicles was demnstrated by varius methds, in particular, by investigating cllagen and carrageenan matrices as mdels f the fluidfilled fibrus matrix f the cleft. It was fund that a laminated system emplying a liquid membrane-cntaining vesicle suspensin, tgether with a swllen cllagen membrane, is an apprpriate mdel fr examining imprtant transprt/receptin aspects f the cleft-plus-pstsynaptic membrane structure. Cmbined transprt with immbilizatin f Ach in the liquid membrane system was elucidated and effective diffusivities in the vesicle suspensin layer were calculated. Effective diffusivities f the cmpsite system simulating the cleft and the pstsynaptic membrane were evaluated as well. These data illustrate the imprtance f penetrant immbilizatin in retarding the diffusin prcess during neurtransmissin. *Authr t whm all crrespndence and reprint requests shuld be addressed by The Humana Press Inc. All rights f any nature whatsever reserved /84/ $

82 HIROSE AND VIETH Index Entries: Transprt, f acetylchline in a membrane; acetylchline, transprt in a membrane; membrane, acetylchline transprt in.

2 82 HIROSE AND VIETH Index Entries: Transprt, f acetylchline in a membrane; acetylchline, transprt in a membrane; membrane, acetylchline transprt in. Intrductin One f the essential bilgical membranus systems in animals mdulates the transmissin f nerve impulses at synapses r neureffectr junctins (1, 2). Recently, a cmprehensive review f the respnse t acetylchline in the neurtransmissin prcess has been prvided by Lester (3). Lester elucidates graphically that the fusin f vesicles cntaining acetylchline with the presynaptic membrane results in acetylchline release int the fluid-filled cleft between the terminal and the muscle cell. Then, the mlecules f acetylchline penetrate the synaptic cleft and bind t receptrs embedded in the muscle-cell membrane, allwing bth sdium and ptassium ins t cunterflw thrugh the membrane. Our ultimate gal is t characterize experimentally the basic phenmena f the transprt prcess acrss the synaptic cleft-pstsynaptic membrane cmbinatin in which the matrix is cmprised f cllagen and mucplysaccharide fibers cntaining immbilized acetylchlinesterase. Frm previus wrk, the sharpening effect f enzyme reactin n lcal-penetrant cncentratin gradients had already been islated. Therefre, we elected t mit the enzyme frm ur mdel systems in this wrk s as t be able t fcus mre directly n the rle f the receptr alne. In a recent paper (9), Ach idide (AchI) and cllagen were selected as mdels f the penetrant and the fluid-filled cllagenus matrix f the synaptic cleft, respectively. Experimental data n transprt and srptin under the cnditins f varius AchI cncentratins were measured. In additin, accumulatins f AchI at lw bulk cncentratins in the vesicles that cmprise Ach receptr-rich membrane purified frm Trped fish were measured, since the electrrgans f Trped fish are well-knwn as a surce f the Ach receptr prtein fr bichemical experiments (4-6). In this paper f ur series, in rder t demnstrate the transprt characteristics f mdel membranes fr the synaptic cleft and Ach receptr tward neurtransmitters such as Ach, varius immbilized preparatins f vesicles using cllagen, carrageenan, and prus membranes are studied. Transprt f Ach thrugh synaptic mdel membranes is investigated, elucidating the perfrmance f Ach and 22Na uptakes f vesicles purified frm Trped fish. These data are analyzed and discussed with reference t the behavir f actual nerve-muscle membrane systems. Materials Experimental Micrfibrillar cllagen hydrchlride (cllagen) and carrageenan were supplied by FMC, Inc. (Princetn, NJ). Acetylchline idide was purchased frm Kdak C.,

ACETYLCHOLINE TRANSPORT IN A MEMBRANE 83 Ltd. (Rchester, NY) and radiactive AchI [14C(U)] (NEC-350, 4.8 m Ci/mml, 47.1 mg) and radiactive 22Na+ (0.2 m Ci/mL), frm New England Nuclear C. (Bstn, Mass.). Liquid scintillatin ccktail (Ready-Slv~HP) was purchased frm Beckman Instruments, Inc.

3 ACETYLCHOLINE TRANSPORT IN A MEMBRANE 83 Ltd. (Rchester, NY) and radiactive AchI [14C(U)] (NEC-350, 4.8 m Ci/mml, 47.1 mg) and radiactive 22Na+ (0.2 m Ci/mL), frm New England Nuclear C. (Bstn, Mass.). Liquid scintillatin ccktail (Ready-Slv~HP) was purchased frm Beckman Instruments, Inc. (Fullertn, CA). Trped Califrnia electrplax rgans (Pacific Bimarine C., CA) btained frm freshly killed animals were frzen in liquid nitrgen and stred at -90~ until used. Micrprus cellulse membranes (Grade GA-1, mean pre size 5 ~xm) were purchased frm Gelman Sciences, Inc. (Michigan, IL) and dialysis membranes (mlecular weight cutff; 12,000-14,000) frm Spectrum Medical Industries, Inc. (Ls Angeles, CA). A Beckman liquid scintillatin cunter (Mdel LS-133) was used fr radiactive AchI measurements. Glass distilled water was used in all prcedures. Other reagents were cmmercially available, i.e., analytical reagents r labratry-grade chemicals. Purificatin and Recnstitutin f Vesicles frm Trped Fish Standard prcedures used t purify vesicles, including the functinally active acetylchline receptr frm the electrrgan f Trped fish, are described in detail elsewhere (4). The methd used in these experiments after purificatin f vesicles is utlined as fllws: vesicles were resuspended in flux buffer r recnstitutin buffer (2% Na chlate, Sigma; 25 mg/ml f sybean L-a-phsphatidylchline, Sigma; 100 mm phsphate buffer, ph 7.5; 10 mm NAN3) at cncentratins frm 0.02 t 1.0 g vesicles/ml. Vesicle suspensins were used directly fr AchI uptake studies and were used as well in diffusin studies with liquid membrane mdels f the pstsynaptic membrane. AchI-(14C) and Carbamylchline-lnduced 22Na+ Uptake f Recnstituted Vesicles AchI-(14C) uptake was cnducted at rm temperature. In micrfuge tubes, 430 ~L f AchI slutin (10-4 tl/l), 20 p~l f AchI-(14C) slutin (20,000 cpm/0.1 ml), and 45 p,l f flux buffer (10 mm Na phsphate, ph 7.8; 400 mm NaC1, 5 mm EDTA, 0.02% NAN3, 5 mm idacetamide) were mixed. The assay was initiated by pipeting in 5 p~l f vesicles slutin (ca., 0.05 g/ml) in flux buffer with an Eppendrf pipeter. After mixing by five up-and-dwn strkes f the pipeter, the mixture was transferred t a vial in which 0.5 g f wet Dwex 50W-X8-100 (Sigma) treated with Trizma base (Sigma) was added t inically adsrb excess AchI in the mixture. And then, sucrse slutin (3 ml f 175 mm) was added, and the mixture was gently shaken fr 1 min. One-half milliliter f this mixture was pipeted int a liquid scintillatin ccktail (10 ml), and AchI cncentratins in vesicles were measured by the methd f radiassay. The 22Na+ uptake f recnstituted vesicles was carried ut by almst the same methd as abve. The detailed methd is described in a previus paper (5). Preparatin f the Cllagen Membrane A suspensin (30 g) cntaining 0.5% (w/w) f cllagen was grund fr fur perids f 30 s each at high speed in a cmmercial Waring blender and then deaerated

4 84 HIROSE AND VIETH fr 30 min by a vacuum pump. The dispersin (i.e., 5 g) btained was cast in Petri dishes (60 mm diam.) made f plystyrene. After evapratin fr a few days, dry cllagen membrane was aged in an incubatr at 55~ fr 10 d. Cllagen membranes btained had a degree f swelling f clse t 3.0. Several Vesicle Immbilizatin Methds fr Pstsynaptic Mdel Membranes Cllagen-Vesicle Membranes Preparatin f cllagen-immbilized vesicles is described in the utline as fllws: Suspended cllagen (0.5 w/w%) was dialyzed against distilled water t reach ph f 6.5 t 7.0 fr a few days.after dialysis, vesicles ( g) and dialyzed cllagen (1.0 g) were mixed by a spatula at 4~ The mixture was pured int Petri dishes (18 mm diameter) and dried at 4~ fr a week. Carrageenan-Vesicle Membranes This methd is als generally useful fr immbilizatin f enzymes and can be carried ut under "wet" cnditins. Carrageenan slutins (1-4 w/w%) in flux buffer were prepared at 60~ t disslve carrageenan cmpletely and were cled dwn t 30~ just befre gelatin f carrageenan slutin. Pellets f vesicles ( g) were suspended int cled carrageenan slutins ( g). The mixture was immediately cast int the space (1-3 mm) between tw glass plates at 30~ t avid denaturing, because this temperature was relatively high fr vesicles f Trped fish t be kept stable. Then, this mixture was kept in a refrigeratr at 4~ fr a few hurs and immersed int 2M ptassium phsphate flux buffer instead f sdium phsphate t cagulate the carrageenan. Liquid Membranes Liquid membranes cnsisting f vesicle suspensins in prus membranes were applied as mdels f the pstsynaptic membranes in neurtransmissin. Pellets f vesicles (1.0 g) were suspended int flux buffer (4.0 ml) and made up t 5 ml as basic vesicle suspensins. Then, vlume fractin f vesicles in the suspensin was in all cases. A micrprus cellulse membrane was dipped in the vesicles suspensin t be impregnated with the Ach receptr-rich vesicles. Bth sides f the wet micrprus membrane cntaining the vesicle suspensin were attached t cllagen membranes previusly swllen in flux buffer. Figure 1 shws bth a schematic diagram f the membrane system and a graphical depictin f the pstsynaptic membrane (10). These membrane laminates were inserted int diffusin cells fr subsequent studies. Time-Lag Experiments Previus time-lag experiments (7) were scaled dwn because f the relatively small quantity f available vesicles and the use f radiactive AchI in this study. The cntinuus flw system is shwn in Fig. 2. A flw rate (q, 1.55 ml/min) f recycled "ht" AchI slutin and Na-phsphate buffer (0.05M, ph 7.0) was pumped int cells by a Masterflex pump (Mdel 7014, Cle-Parmer Inst. C., Chicag, IL). Ht AchI slutins were prepared t permit additin f 0.5 ml f radiactive AchI slutin (20,000 cprn/0.1 ml) int 10 ml f cld AchI slutin at a given cncentratin. The effective membrane area (A) was cm 2 (11 mm

5 ACETYLCHOLINE TRANSPORT IN A MEMBRANE 85 Upstream Ach Slutin,._.~...Q~ Synaptic //// Vesicles Presynaptic U / / MembraneC~ Membrane Fluid.filled ~ - - ~ ~ Vesicle Cleft Suspensin / / /// (Ach ase) O,.,e. ".t,.o, Membrane II Membrane ~ ~ ~ Muscle Ceil Dwnstream Ach Slutin Fig. 1. brane (b). (a) Schematic diagram f the liquid membrane system (a) and pstsynaptic mere- (b) diameter). Cell vlume (V) f the diffusin-cell (dwnstream side) was ml. In rder t avid establishing a cncentratin-plarized unstirred layer at the surface f the membrane, magnetic stirrers in bth cells were used. The ttal apparatus was kept in cld water with crushed ice (3~ The dwnstream AchI slutins were cllected fr 1 rain t measure the cncentratin f AchI by radiassay. The ttal amunt (Qt) f penetrant (AchI) that permeates thrugh the membrane in time (t) can be calculated as fllws: Q, = f~ JA at = f6 (Vdx/dt]t + qx)dt (1) where J, flux f Achl, is the number f mles f AchI transprted per unit area and time, and x is the dwnstream cncentratin f AchI. Q, calculated by Eq. (1) against time displays a nnlinear transitin regin asympttically tending t a steady state, represented by a straight line. It intercepts the time axis at a pint 0, called the time-lag. The diffusivity (D) f the penetrant can be determined as fllws: 8 D = L2/ l 0 Fig. 2. Apparatus fr measuring diffusivities f liquid membrane systems: (1) liquid membrane system (I1 mm diameter); (2) diffusin cell; (3) Masterflex pump (1.55 ml/ min); (4) recycled radiactive Ach slutin (10 ml) (upstream); (5) flux buffer (ph 7.5); (6) micrfuges (1.5 ml) fr sampling; (7) stirrer; and (8) water bath (3~

6 86 HIROSE AND VIETH where L is wet membrane thickness r ttal thickness f the liquid membrane laminate. Results Prperties f Vesicles Using Acetylchline and Carbamylchline Induced 2eNa+ Uptake The ph prfiles f vesicles suspensins and recnstituted vesicles were examined by acetylchline (Ach) uptake and carbamylchline (Cch) induced 22Na+ uptake, respectively, and the results btained are shwn in Fig. 3. Optimum ph range f Ach uptake was frm 7.5 t 7.8, brader than that f Cch induced 22Na+ uptakes f recnstituted vesicles. Figure 4 shws the thermstability f the vesicle suspensin (0.050 g/ml) fr 15 min at a given temperature. Ach uptake f vesicles decreased remarkably at mre than 30~ and cmplete inactivity f vesicles was bserved at 60~ Meanwhile, Ach uptake f vesicles retained 80% f its initial activity under the cnditins f thermstability at 25~ fr 120 min. Inactive vesicles which were kept fr 15 min as shwn in Fig. 4 were n lnger regenerated by reincubating at 4~ fr a few minutes. Figure 5 shws lifetime f vesicles and recnstituted vesicles at 4~ Ach uptakes f vesicles were cnstant within 2 mnths in the case f bth vesicles in flux buffer and thse in recnstitutin buffer. Cch (10-3 ml/l) induced 22Na+ uptakes abruptly decreased within a few weeks and n activity f recnstituted vesicles remained after a mnth. Vesicles in flux buffer withut recnstitutin by sybean lipids indicated n activity f Cch induced a2na+ uptakes A m 0 Fig. 3. Ach; x Na. Q. 7. < 0 m ee / Y I t t ph I O. 5O Z O 25 = (u ph Prfiles f vesicles fr Ach uptake and Cch-induced 22Na+ uptake, (D m

7 ACETYLCHOLINE TRANSPORT IN A MEMBRANE 87 loo -0 -~ "/5 m 11 ~ 50 < O ~ 25 ID 0 I I 0 20 Temperature (cl Fig. 4. Thermstability f vesicles in flux buffer after 15 min expsure. Artificial Mdel f the Pstsynaptic Membrane Using Vesicles Different trials t immbilize vesicles were carried ut t btain mdel pstsynaptic membranes. The results fr varius immbilizatin methds f vesicles are summarized in Table 1. Cllagen and carrageenan were individually selected and examined as hst matrixes fr immbilized vesicles, since Ach receptrs are embedded and bund in viv n the uter edge f a cllagenus matrix cntaining looq 0 -~ 75 < Ig E \ \ x\ x \ 2s \ 0 ()-- "-0- ~ x ~ Time (weeks) 100 ~ 75 ~ q 50 +~ Z 25 ~ E Fig. 5. Life time f vesicles fr Ach uptake and Cch-induced 22Na+ uptake at 4~ O Ach; x Na. 0

88 HIROSE AND VIETH TABLE 1 Vesicle Immbilizatin Activity vesicles after Materials immbilizatin Methds Cllagen N Entrapment Carrageenan N Entrapment LMS ~ Yes Impregnatin f micrpres ~ Membrane System.

8 88 HIROSE AND VIETH TABLE 1 Vesicle Immbilizatin Activity vesicles after Materials immbilizatin Methds Cllagen N Entrapment Carrageenan N Entrapment LMS ~ Yes Impregnatin f micrpres ~ Membrane System. plysaccharide, as shwn by Lester (10). Pelleted vesicles were immbilized nt cllagen by the methd f a previus paper (5). Hwever, the lw ph ( ) f the suspended cllagen t which the pelleted vesicles were added, apparently resulted in critical denaturing f vesicles. Therefre, suspended cllagen was dialyzed against water r flux buffer (ph 7.8). Stable dialyzed cllagen suspensin reached ph 6.5 in a few days. The ph stability f vesicles in the cllagen suspensin under the cnditins f ph 6.5 fr 24 h gave 20% f initial activity f Ach uptake. N active cllagen-vesicle membranes were btained by the methd using dialyzed cllagen described in the experimental sectin, thugh the activity f vesicles in the cllagen suspensin (ph 6.5) was still retained. Further dialysis raised the ph f the cllagen suspensin t 7.0. Hwever, cllagen was cagulated and, cnsequently, n mechanically strng cllagen-vesicle membrane was btained. Dehydratin during the preparatin f cllagen membranes might have resulted in the denaturing f the vesicles. In the next step, carrageenan was used fr immbilizatin f vesicles. This methd has advantages in that "wet" preparatin can be used withut the need t make a dehydrated membrane. Carrageenan membrane is basically frmed in a sl-gel transfrmatin that depends n carrageenan cncentratin, temperature, and ptassium in cncentratin. Balanced cnditins, with carrageenan cncentratins f 2% (just high enugh fr the vesicle-membranes t be strng), and a preparatin temperature f 30~ (in which the denaturatin f vesicles culd be partially avided) were experimentally selected. The effect f ptassium ins (using ptassium phsphate) n activity f Ach uptake f vesicles was examined. This effect is shwn in Fig. 6. Activity f vesicles still remained, even in the high inic strength envirnment. Therefre, the mixture f vesicles and carrageenan in flux buffer was immersed in a 2M ptassium phsphate (flux buffer) instead f sdium phsphate t cagulate the carrageenan macrmlecules. Althugh vesicles immbilized in carrageenan were cmpletely inactive, vesicles suspended in a flux buffer including lw cntent f carrageenan were nevertheless active. A small amunt f calcium is an essential requirement fr carrageenan gel rigidity. Catin cntents f carrageenan are characterized as fllws: Na (6.0%), K (0.98%), Ca (0.23%), and Mg (0.22%). It is thught that strng interactins between active sites f vesicles and catins, especially calcium, cause site inhibitin r denaturatin f vesicles. Vesicle suspensins were then impregnated in prefrmed micrprus membranes f cllagen r cellulse (dialysis membrane). This liquid membrane type f

9 ACETYLCHOLINE TRANSPORT IN A MEMBRANE 89 ~ 100( m 75 " 50 < O.>. 25 E 0 I I i Cnc. OF K Phsphate(M) Fig. 6. Effects f inic strength f ptassium n Ach uptake f vesicles. These experiments were carried ut at a variety f cncentratins f ptassium phsphate instead f 10 mm Na phsphate in flux buffer. system avided the denaturing steps alluded t earlier, while retaining the desired structural features we were seeking. Further experiments were carried ut by the use f the liquid membrane system, which was cmpsed f vesicle suspensins impregnated int micrprus cellulse and laminated t cllagen membranes. In rder t estimate the diffusin rate f neurtransmitter in the pstsynaptic membrane, transprt f Ach thrugh the liquid membrane mdel was measured. The effects f Ach cncentratins in upstream slutins n time-lag in the transprt prcess, under the cnditins f cnstant vlume fractins f vesicle suspensins (0.05), cllagen membrane thickness (ca. 150 ~xm), and temperature (3.0~ are shwn in Fig. 7. The time-lags asympttically decreased with increasing upstream Ach cncentratins, appraching 24 min when using vesicles and 20 min fr the vesicle-free cntrl. Effects f vlume fractins f vesicles in micrprus membranes n time-lags f the liquid membranes are als shwn in Fig. 8. These experiments were carried ut under standard cnditins: 10-3 ml/l Ach cncentratin f upstream slutins, 3.0~ temperature, ca. 150 Ixm cllagen membrane thickness, 100 Ixm micrprus cellulse membrane (5 txm pre size), and a range f vesicle vlume fractins frm 0.02 t Time-lags gradually increased with increasing vlume fractins f vesicles in micrprus membranes. Discussin Prperties f vesicles purified frm Trped fish were measured, in rder t prepare a mdel f the pstsynaptic membrane that cmprised, as the cleft, the cllagenus matrix and the Ach receptr impregnated as a liquid membrane int a micrprus cellulse hst membrane. Under the cnditins fr active vesicles de-

10 90 HIROSE AND VIETH m 2OO0 m i I \ x\ ~.. ~ 9 0 I I I I I I Cn. Of Achl (ml/i) Fig. 7. Effects f Ach cncentratins f upstream slutins n time-lags in the liquid membrane system; vlume fractin, 0.05 ((3) vesicle suspensins in the liquid layer, (x) flux buffer in the liquid layer. rivable frm Figs. 3-5, vesicles were immbilized as liquid membranes. These membranes were laminated t cllagen membranes n ne r bth sides. As already mentined, these were jined tgether t mdel the cleft and pstsynaptic membrane cmbinatin. In this manner, the liquid membrane system was made available fr investigatin f transprt f Ach. The time lags (0121) f the liquid m 2000 ~g m _ C 9 0 I I I I Vlume Fractin Of Vesicles Fig. 8. Effects f vlume fractins f vesicles in micrprus membranes n timelags in the liquid membrane system; Ach cncentratins upstream, 10-3 ml/l.

11 ACETYLCHOLINE TRANSPORT IN A MEMBRANE 91 membrane systems were measured under cnditins f varius Ach cncentratins in upstream Ach slutins and varius vlume fractins f vesicles in micrprus membranes. In general, fr a three-layer cmpsite (1) (2) (1), it is shwn that (8) ~12 \3DTK1( 4ll + 12 ) + 12 ( l, + 12 ) K2 DzK2 ~ ~ 6D2K2 (DIK1) D ~K1 D2K2 (3) where 0121 dentes the time-lag f the three layer cmpsite, and 11, D1, and K~ are the thickness, diffusivity, and distributin cefficient f the first layer, respectively. Subscripts f I and 2 dente the kind f membrane, that is, whether cllagen layer r vesicle suspensin layer. Fr this we had already determined that D1 = 2 x 10-6 cm2/s; K1 is likewise btained frm previus wrk (9). Fr the distributin cefficient f the vesicle suspensin layer, K2, we have K 2 = [(1 - ~) + ~K,.]/[(1 - ~) + ~] = 1 + r 1) (4) where K,, is the distributin cefficient f pelleted vesicles purified frm Trped fish (9) and r is the vlume fractin f pelleted vesicles in the vesicle suspensin layer. Experimental data f l~, 12, and r were btained frm this wrk. Therefre, values f D2 can be calculated frm Eq. (3); they are summarized in Table 2. The diffusivities f vesicle suspensin layers decreased with decreasing Ach cncentratins f upstream Ach slutins and increasing vlume fractins f vesicles. Furthermre, it is thught that Ach in neurtransmissin passes thrugh the cleft (cllagenus membrane) and Ach receptr-rich membrane, being regulated by the gradient f Ach cncentratins between upstream and dwnstream slutins. The neurtransmissin system fr the cleft plus pstsynaptic membrane cmbinatin is cnsidered t be a tw-layer cmpsite f (1) and (2). Thus, ll2/dl2kl2 = ll/d1k1 + 12/D2K2 (5) where Therefre, Dl/D12 can be written as KI2 = (llkl + 12K2)/112 D1 ll --- ll (6) O ll O2 K2 Taking K2 = Kv at ~ = 1.0, and DI and KI, 10-6 cmz/s and 10, respectively, t allw fr penetrant immbilizatin by the plysaccharide mieties, D1/D12 can be calculated, fr the case where l~ and 12 are taken as 900 and 100,~, respectively. The calculated rati is clse t fifteen, s that D~2 is estimated at abut 6 x 10-8 cmz/s. These results clearly shw the retardatin f the diffusin prcess (DI2 < DI) that ccurs because a fractin f the penetrant ppulatin is immbilized (K2) and

12 92 HIROSE AND VIETH i2 9 r162 d d d d d d d d d 0'3 "O eq >- L) < ~ O [...,?T??T?????? O

ACETYLCHOLINE TRANSPORT IN A MEMBRANE 93 unavailable fr diffusin at any instant. This effect will be nly partially ffset by the gradient-sharpening effect f the enzyme reactin.

13 ACETYLCHOLINE TRANSPORT IN A MEMBRANE 93 unavailable fr diffusin at any instant. This effect will be nly partially ffset by the gradient-sharpening effect f the enzyme reactin. The rise time f the neurtransmissin prcess is knwn t be abut 100 p~s frm experimental data. Previus calculatins had indicated (10) that the prcess wuld require nly 20 ~s, cnsidering diffusin in the cleft alne. Thus, it was cncluded that the diffusin prcess in the cleft was nt cntrlling. Hwever, fr the cmpsite structure f the cleft plus pstsynaptic membrane, the effective diffusivity, D~2, is substantially reduced belw D1 because f retardatin caused by receptr binding. Therefre, we cnclude that the neurtransmissin prcess f Ach can in fact be cntrlled by a mdified diffusin prcess in the laminate structure. References 1. Nback, C. R., and Demarest, R. J., eds. (1981), The Human Nervus System, McGraw-Hill, New Yrk. 2. O'Brien, R. D. (ed.) (1980), The Receptrs, Plenum, New Yrk. 3. Lester, H. A. (1977), Sci. Amer. 236, Lindstrm, J., Anhlt, R., Einarsn, B., Engel, A., Osame, M., and Mnfal, M. (1980), J. Bil. Chem. 225, Anhlt, R., Lindstrm, J., and Mntal, M. (1980), Eur. J. Bichem. 109, Ellitt, J., Blanchard, S. G., Wu, W., Miller, J., Strader, C. D., Hartig, P., Mre, H. P., Racs, J., and Raftery, M. A. (1980), Bichem. J. 185, Hirse, S., Yasukawa, E., Hayashi, M., and Vieth, W. R. (1982), J. Membrane Sci. 11(2), Barrie, J. A., Levine, J. D., Michaels, A. S., and Wng, P. (1963), Trans. Faraday Sc. 59, Hirse, S., Vieth, W. R., and Taka, M. (1983), J. Mlec. Catal. 18, 11. I0. Eccles, J. C., and Jaeger, J. C. (1958), Prc. R. Sc. B148, 38.

The demonstration of lysosomes by the controlled temperature freezing-sectioning method By LUCILLE BITENSKY

The demonstration of lysosomes by the controlled temperature freezing-sectioning method By LUCILLE BITENSKY 205 The demnstratin f lyssmes by the cntrlled temperature freezing-sectining methd By LUCILLE BITESKY (Frm the Department f Pathlgy, Ryal Cllege f Surgens f England, Lincln's Inn Fields, Lndn, W.C. 2)