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Flow-injection analysis of catecholamine secretion from bovine adrenal medulla cells on microbeads
ANALYTICAL
BIOCHEMISTRY
144,
Flow-Injection
MICHELLE
2 18-227 (1985)
Analysis of Catecholamine Secretion Adrenal Medulla Cells on Microbeads
from Bovine
HERRERA,? LUNGSEN KAO,*~' DAVID J. CURRAN,? ANDEDWARD W. WESTHEAD*
Departments of *Biochemistry and jfhemistry,
University of Massachusetts, Amherst, Massachusetts 01003
Received February 29, 1984 Bovine adrenal medullary cells have been cultured on microbeads which are placed in a lowvolume flow system for measurements of stimulation-response parameters. Electronically controlled stream switching allows stimulation of cells with pulse lengths from 1 s to many minutes; pulses may be repeated indefinitely. Catecholamines secreted are detected by an electrochemical detector downstream from the cells. This flow-injection analysis technique provides a new level of sensitivity and precision for measurement of kinetic parameters of secretion. A manual injection valve allows stimulation by higher levels of stimulant in the presence of constant low levels of stimulant. Such experiments show interesting differences between the effects of K+ and acetylcholine on cells partially desensitized to acetylcholine. 0 1985 Academic
Press. Inc.
KEY WORDS: catecholamine secretion; flow-injection electrochemical detection; adrenal medulla.
analysis (FIA); kinetics of secretion;
Cultured cells of bovine adrenal medulla have become widely used for the study of stimulated secretion of catecholamines (1). This system provides much closer control over the cellular environment than does the procedure of organ perfusion (2). The uniformity of stimulation and simplicity of sampling suggest that the kinetic parameters of secretion, inhibition, and desensitization might be studied advantageously with cultured cells. In 1980 Green and Perlman (3) introduced an on-line measurement of catecholamine secretion by isolated rat adrenal tumor (PC 12) cells placed on a membrane filter. Solution flowing over the cells passed through the filter directly to an electrochemical detector which measured the oxidation current due to secreted catecholamines. By stream switching from buffer to stimulant solution the authors obtained information on the kinetics of the secretory process.
This paper describes a new on-line system for measuring catecholamine released from chromaffin cells. The cells are allowed to attach to treated styrene-divinylbenzene beads which are then packed in a short column placed in the flow line. Because this system has a very low dead volume and uniform flow through the packed bed, it has a much lower dispersion than the system previously described. These properties allow analytical information to be obtained by injecting short pulses of stimulant through the cells, generating sharp peaks of catecholamine at the detector. This method is known to analytical chemists as flow-injection analysis (PIA)* and is widely used in process stream analysis and in applications where automation and rapid sample throughput are desired. The theory and applications of FIA have been extensively studied and reviewed (4-7), including application to immobilized enzyme reactors (8). In PIA the determination
’ Present address: Sloan Kettering Institute for Cancer Research, 1275 York Avenue, New York, N. Y. 10021.
2 Abbreviations used: FIA, flow-injection analysis; Ach, acetylcholine.
0003-2697185 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
218
FLOW-INJECTION
ANALYSIS
OF
of sample concentration can be made by calibration of peak height with standards. The effect of chemical kinetics on the FIA peak shape has been considered (8- 10) and the equations derived for these complex systems are relevant to the system we describe. This flow-through system allows precise measurements of secretion levels during desensitization of the cells to stimulant so that kinetic parameters may be calculated. The ability to choose injection pulses of varying durations and frequencies makes possible the examination of secretion and desensitization as a function of short times, and thus may approach the in viva time frame of stimulation. The purpose of this paper is to demonstrate the potential of this technique. The data presented have been selected to demonstrate the variety of experimental parameters that may be investigated and the versatility of the system. MATERIALS
AND
METHODS
Flow-line materials purchased from Rainin Instrument Company include Altex l/16-in.o.d., 0.3-mm-i.d. Teflon tubing, polypropylene tube end fittings and couplers, and Tefzel tube connectors used to connect the Teflon tubing to Tygon tubing from the solvent reservoirs. The manual injection valve was a Rheodyne Teflon sample injection valve. The Teflon three-way solenoid valve was a Series 1 valve from General Valve. Stainless-steel Swagelok fittings, l/8 and l/16 in., coupled to a stainless-steel column (1.8 cm X 2.5 mm) were used for the cell chamber. Polystyrene beads, 37- to 74-pm diameter, 12% crosslinked, were from Bio-Rad and were treated to produce a hydrophilic surface to promote cell adhesion ( 11,12). Chromaffin cells were cultured using the techniques and sources of media described in (13) based on the original procedures of Fenwick et al. (14). The electrochemical detection of standard and secreted catecholamines was performed with a Bioanalytical Systems LC-3 amperometric detector equipped with a glassy carbon
CATECHOLAMINE
SECRETION
219
working electrode, an Ag/AgCl reference electrode, a stainless-steel counter electrode, a Faraday shield, and background nulling circuitry. A spacer thickness of 0.005 in. was used. The oxidation current was recorded on an Omniscribe dual-pen recorder, which also monitored the injection signal next described. A Tektronix FG 501 function generator was used to generate a variable-frequency square wave which was fed through the valve driver (made by M. Conboy, Electronics Shop, Univ. of Mass. Chemistry Dept.) which controlled the valve position and allowed repeatable stream-switching times. It must be noted that the oxidation current recorded in all these experiments results from oxidation of both catecholamines and ascorbic acid, which is also secreted by the cells. Ascorbic acid is present in the vesicles of the adrenal medulla at a concentration about 10% that of catecholamines and under our conditions the detector sensitivity for ascorbate is about half that for catecholamines. In experiments in which the effluent from stimulated cells was received by a fraction collector, analysis by HPLC showed that less than 1% of the signal was due to ascorbic acid. All experiments were performed at room temperature, 26°C. The apparatus assembled for FIA of catecholamine secretion is shown in Fig. 1. Glass solvent reservoirs were filled with 500 ml each of (i) buffer composed of 154 mM NaCl, 2.2 mM CaC12, 5.6 mM KCI, 5.0 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes) buffer, and 10.0 mM glucose, pH 7.4, and (ii) stimulant solution containing 25 PM Ach in the buffer solution. The reservoirs were placed 2 m above the rest of the apparatus for gravity-fed delivery. All solutions were prepared in deionized distilled water and microfiltered (0.22 pm) to prevent clogging and disruption of the cell bed by particulates. The experiments were begun by equalizing the flow rates of the two solution reservoirs (since electrochemical detection is flow-rate dependent), allowing the background current to equilibrate at a potential of +0.65 V, and
220
HERRERA
flow-balancing
valve
ET AL.
FIA injection pulse
___
3-woy
solenoid (teflon)volve
square wove generator
‘“““J$
zero deod- volume connectors
valve
_______
drover
3
dual -oen
1
recorder
measure catecholammes manual
injection valve
___ I +
I
I! 0.3
mm teflon
tubing
-------
%-l -
with
adrenal
cells
I
P
woste
electrochemical detector
FIG. 1. Diagram of experimental apparatus.
nulling the background current electronically. The beads with attached cells were pipetted from the culture dish into the Swagelok fitting to form a packed bed about 6 mm long X 2.5 mm wide secured at both ends by glass wool, so that the effective dead volume was minimal. This low-volume flow system minimizes dispersion (sample dilution), which in turn minimizes peak broadening, thereby optimizing sensitivity and detection limits. Perfusion of cells with buffer was begun immediately after they were packed and was not interrupted in the course of an experiment. Stream switching from buffer to stimulant solution was performed with a manual switch on the valve driver. This long-term stimulation of the cells was used to study the kinetics of desensitization of the cells to stimulant.
Injections of stimulant for FIA experiments were performed for various pulse durations using square-wave pulses of various frequencies (0.025 to 0.6 Hz) generated by the function generator to drive the valve between buffer and stimulant positions. The steady volumetric flow rate (0.70 or 1.1 ml/min) was later used to determine the volume of stimulant injected. Data were recorded at 1 to 100 nA full scale sensitivity at chart speeds of 25 cm/min (for precise time-axis measurements) and 2.5 cm/min. Standard catecholamine determinations were performed by replacing the stimulant solution reservoir with a solution 0.5 pM in both epinephrine and norepinephrine. The solutions were prepared within an hour of use by diluting 1 mM stock solution of catecholamines (stored frozen in 0.1 M HCl) with
.
FLOW-INJECTION
ANALYSIS
OF CATECHOLAMINE
microfiltered buffer. The same streamswitching and FIA experiments performed with the stimulant solution were repeated using the standard solution for concentration calibration purposes. Dispersion of the sample in the flow-through system was also studied with the standard solution. The 1 PM standard solutions were sufficiently concentrated so that uptake by a bed of cells was negligible. Standards run with and without cells in the line differed by only 6%. A manual injection valve (cf. Fig. 1) equipped with a 62-~1 sample loop was used to inject the standard solution periodically during the course of a stimulant or standard run in order to monitor the electrode sensitivity (by peak height) and the dispersion (by peak width). In addition, it provided a second means of injecting stimulant in a “doublestimulation” experiment. In this case, a stimulant (25 pM Ach) was introduced by stream switching with the solenoid valve and 250 pM Ach or 56 mM K+ was injected manually at various points before, during, and after the constant stimulation with 25 pM Ach. The manual injection valve thus serves a dual purpose but it could be removed to decrease dispersion even further. Upon completion of the experiments with a batch of cells, the beads were washed from the fitting with 8% trichloroacetic acid solution. This solution was stored at -5°C for residual catecholamine analysis. RESULTS AND DISCUSSION
SECRETION
221
odic manual injections of standard. Therefore, the secretion of protein by exocytosis did not appear to foul the electrode. The peak widths did not change either, indicating that flow rate and dispersion were constant throughout each experiment. Typical response curves of the standard and secreted catecholamines are shown in Fig. 2. FIA peaks for various pulse times (T) are indicated by solid lines and responses to long-term stream switching from buffer to stimulant are indicated by dashed lines. Measurements in both FIA and stream-switching experiments are described in Fig. 3. Stream-Switching
Experiments
Stream-switching experiments were performed with cells 5 and 12 days in culture (“young” and “old” cells). The desensitization of catecholamine secretion during constant stimulation can be measured as tl12, as pictured in Fig. 3. The desensitization can be characterized more accurately by plotting In f (current) vs t (time) at times greater than the maximum as shown in Figs. 4A and B. The linearity of the plots shows that densitization at this concentration of stimulant is characterized by a single exponential constant (k). These values are tabulated along with tl12 values in Table 1. The plot of In I vs t for the young cells (Fig. 4A) was linear over an order of magnitude drop in I during a period of about 6 min. The plot for the older cells (Fig. 4B) was linear after subtracting the final constant level of secretion ( 15% of maximum) from all the f values. Shutoff occurs at a substantially slower rate with the younger cells than with the older cells. This phenomenon will be discussed in the next section. The tl12 values are comparable to those of Green and Perlman (3), but a quantitative comparison cannot be justified because their cells were PC 12 cells from a rat tumor line.
The background current from a typical batch of unstimulated cells was initially 13 nA, and it decreased to 6.9 nA within 2 h. The final background is comparable to the 6.5-nA current of the flowing stream with the cells removed. The 13-nA current corresponds to a basal secretion rate of 1 pmol/s. The peak-to-peak noise level of the baseline was comparable to that without cells, or 20 FIA Measurements pA. Electrode sensitivity did not change appreciably through hours of flow analysis, as Measurements made on the FIA peaks are indicated by the constant height of the peri- described in Fig. 3. Peak dimensions are
222
HERRERA
ET AL.
TIME FIG. 2. Response curves for FIA and stream-switching experiments. Catecholamine response in flowing stream versus time for the different pulse times indicated (seconds). Dashed lines are for infinite pulse length produced by stream switching. Upper set of curves: 0.5 pM catecholamine standard added for 5 to 20 s. Lower set of curves: Catecholamines secreted in response to injection pulses of 25 pM acetylcholine lasting from 5 to 14 s.
dependent on analyte concentration as well as on pulse duration, T. Since peak height, H, at a particular T is proportional to the concentration of injected analyte, the peak heights of the FIA of the standard were used to tentatively calculate the amount of catecholamine secreted from the cells upon injection of stimulant. The absolute quantities (picomoles) of catecholamines secreted were calculated by calibration with the standard. FIA was used to study the desensitization of a different batch of 5day-old cells to repeated pulses of stimulant. Fig. 5 shows the amount secreted (from peak height) vs time, upon injecting 4-s pulses (T = 4 s) of 100 /IM acetylcholine every 1 to 2 min. The response of the cells decreased sharply from the first to
the second injection and then more gradually with consecutive injections. After 50 min of this periodic stimulation, the cells were allowed to rest for about 15 min. The last point shows the dramatic increase in secretion response after recovery of the cells. FIA data from secretions of two different samples of cells (5 and 12 days old from the same preparation used for the data of Fig. 3) are shown in Table 2. The injection volumes are calculated by multiplying flow rate and pulse time. The amount secreted is obtained from calibration with the standard catecholamine response for a particular T. The Co values arc the amount secreted (pmol) divided by the injection volume. The amount secreted was normalized for injection volume in this
FLOW-INJECTION
ANALYSIS
Siandard
t=o
G/2
FIG. 3. Measurements on response curves. Solid lines are FIA curves. Dashed lines are stream-switching responses. T, pulse injection duration (s); C, and C,, concentration of injected standard and stimulant, respectively: t time axis (s); H and H’, peak heights of standard and secreted FIA peaks in nA, respectively; IV,,, and IV,,,, peak widths at half-height for standard and secreted peaks. H,,, the steady-state current (in nA) for C, (standard); D, the dispersion of the standard, or H,,/H; t,,?, the half-time for desensitization in the streamswitching experiments; M, maximum secretion.
manner so comparisons between different T values in terms of average amount secreted during pulse duration could be made. The older (12-day-old) batch of cells secreted 510% the amount of the younger cells (5 days
OF CATECHOLAMINE
old) but older cells contain less catecholamine than younger cells (13). Furthermore, at the time of these experiments, we were not quantitating the number of beads in the flow line so the difference in total secretion is not a relevant part of these results. The effect of pulse time on the average concentration secreted (Co) was studied in both batches of cells. The younger cells seemed to secrete a greater average concentration of catecholamines at shorter times than at longer times. An explanation for this trend is that since secretion decreases due to desensitization, the average concentration also decreases with increasing duration of stimulant pulse, 7’. However, this did not appear to be true of the older cells, since the concentration secreted was fairly constant and independent of T, with an average of 0.52 X lo-’ M and relative standard deviation of 6.5% (n = 25).
The difference in desensitization behavior of the two batches of cells may be compared in terms of tli2, k, and T dependency. The young cells appear to desensitize at a rate slower than that of the older cells as indicated by the lower k and greater tl,2 in Table 1. However, the T dependency of the amount secreted by the younger cells suggests that the younger cells desensitize very rapidly at short times, i.e., before the maximum secretion rate which occurs at about 12 s. Peak width is also dependent on the de-
-se.TIME (SEC)
223
SECRETION
/ 20
I
, 60
, TIME
, 100
,
I 140
I
180
I
1 2;
(SEC)
FIG. 4. Logarithmic plots for desensitization in stream-switching experiments with 25 (A) 5-Day-old cells. (B) 12-Day-old cells.
pM
acetylcholine.
224
HERRERA TABLE
1
Expt
Age of cell (days)
Maximum secretion rate (PmoVs)
h/2 (4
k (s-l)
1 2 3
5 12 12
5.9 0.52 0.43
120 28.3 27.6
0.007 0.022 0.023
Note. Experiment 3 is a repeat of experiment 2, to give some idea of the reproducibility of the constants.
sensitization of cells. Fig. 6 shows a plot of peak width vs T for the standards and secreted catecholamines. At small T the peak widths appear to be statistically equal. The increase in the difference of peak widths with increasing T is probably due to reaction band broadening (10) combined with the kinetics of cell response to the changing stimulant concentration. At even longer T, the peak widths again approach those of the standards, a result of the desensitization of the cells to the stimulant. The secretion curve therefore decays to baseline more rapidly than the standard curve and the peak width (which should be independent of peak height in standard FIA) is therefore smaller. It must be noted that the variability of the shape of the secreted peaks makes uncertain the determination of amount secreted using peak height of the standard curves. We are in the process of interfacing the FIA system to a computer that will measure peak areas for more accurate determinations of catecholamine using area calibration curves. Double-Stimulation
ET AL.
constant stimulation by 25 PM acetylcholine. The peak heights were not identical in each run (only one run of each is shown) but the following general trends were reproducible: (i) the peak caused by the 250 PM Ach did not change much when the background 25 PM Ach was switched from OFF to ON, but the K+ peak did increase considerably, (ii) both sets of peaks decreased gradually with the 25 PM Ach ON due to the desensitization of the cells, and (iii) the 250 PM Ach peaks recovered quickly after the 25 PM Ach was turned OFF while the K+ peaks went immediately back to the original value. These results are in accord with the previous suggestion (3) that there are separate mechanisms of catecholamine secretion for Ach and K+, i.e., receptor-dependent channels and voltagedependent channels that work independently of one another. Duration
of Experiments
At the conclusion of a 5-h experiment involving 100 or more stimulation pulses, the catecholamine left in the cells was 70 to 80% of the amount at the start of the experiment. At this point, cells that were rested for 15 min responded to stimulus very much as they had initially. The long-term stability of secretion may be due to constant removal of secretory products and stability of the cells in the packed bed, which has predominantly 70 t0
Experiments
In addition to low detection limits and good time resolution, the FIA system is also capable of great versatility for the study of any synergistic or desensitizing effects associated with secondary stimulation. Figure 7 shows an example of “double stimulation” in which manual injections (62 ~1) of either stimulant, 250 PM Ach, or 56 PM K+ are done before, during, and after a period of
TIME
(SEC
1
FIG. 5. Catecholamine secreted versus time for repeated pulsed stimulation. Each point is in response to a 4-s injection of 100 pM acetylcholine at a flow rate of 1.0 mlimin. ~~~~,
FLOW-INJECTION
ANALYSIS
OF
CATECHOLAMINE
TABLE
225
SECRETION
2
DETERMINATION OF CATECHOLAMINES Young cells (5 days old) Pulse time T 6) 20.0
Injection volume (t.4
Total secretion (pmol)
370
72 58
Old cells ( 12 days old)
G (M X lo’)
Total secretion (pm4
G (M X lo’)
240
5.0
0.21
14.3
172
9.4 8.5 8.5
0.55 0.50 0.50
11.1
133
7.7 7.0
0.58 0.53
120
6.1 6.1 6.0
0.5 1 0.51 0.50
6.7
80
4.3 3.9 4.0
0.53 0.48 0.49
5.0
60
3.2 3.0 3.1 2.9
0.53 0.50 0.52 0.49
1.5
0.51 0.51
10.
2.5
180
45
2.0 1.6
Injection volume w
31
1.7
21 20
6.0 4.5
1.25
1.00
18
26 19 18 17
14.3 10.5 10.0 9.5
0.83
laminar flow and sufficiently low pressure drop to create a favorable environment for the cells. Dispersion and Detection Limits
The dispersion (D) of the standard solution in a 62-~1 injection volume with a 0.70-ml/ min flow rate (T = 5 s) was measured as HO/
30
1.5 1.6
0.52
15
0.80 0.86 0.88
0.53 0.57 0.58
12
0.60
0.50
10
0.59 0.47 0.58
0.59 0.47 0.58
H. D was 4.3 with the fitting packed with beads and 5.3 with an equivalent length of 0.3-mm tubing replacing the packed fitting. The packed fitting actually decreases peak broadening, which is a well-known phenomenon in FIA (4) and is analytically advantageous in terms of detection limits. The dispersion is much lower than previously reported by Green and Perlman (3).
226
HERRERA
ET AL.
immediately detected in an elution volume which is even smaller than the elution volume in liquid chromatography. FIA detection limits of the amount secreted by the cells can therefore be a factor of lo-100 times better than those obtained by removing aliquots for chromatography. 11 0
I 4
II
/ 8
I 12
I
I 16
I
I 20
II
CONCLUSIONS
This work has demonstrated the use of flow-injection analysis to study the in vitro FIG. 6. Peak width versus pulse duration in FIA mode. secretion of catecholamines from adrenal cells. The advantages are very low detection limits, wide variability of stimulation times Their system had a dead volume of 300 ~1 down to about 1 s, accurate repeatability, and a time for 50% washout of 30 s. The long experiment time, and concentration decorresponding figures for our system were 13 termination by direct calibration with stan~1 and 5 s. The lower dispersion of our dards. Because washout of stimulant is rapid system greatly lowers distortion of the FIA and complete, kinetic analysis of parameters curves and improves detection limits in the affecting secretion can be conveniently studFIA experiments. ied. The phenomenon of desensitization, for Detection limits of catecholamines were example, can be studied as a function of derived from twice the value of the baseline stimulant concentration, pulse length, recovnoise, which was 10 pA peak to peak. This ery time, and frequency of repeated pulses. value was the same with or without the cells The ease of injection of short pulses of in the flow line. For the stream-switching stimulant against a constant low-level backexperiments on the 12-day-old cells, this corground of stimulation may also provide new responds to a detection limit of lo-” M, or information on the desensitization process. a secretion rate of lo-l5 mol/s (using a flow We anticipate that the effects of drugs and rate of 0.70 ml/min). The detection limit in the FIA experiments using the shortest pulse duration of 0.83 s was lo-l4 mol (total amount secreted). This detection limit was very low because of the stability of the detector and the cells and also because a gravityfed pump was used. Pump noise in the work of Green and Perlman limited their streamswitching detection to 0.4 nA, which is 20 times higher than our detection limit. FIA detection limits on that type of flow-through system would be relatively poor due to the broadened peaks resulting from high disperc sion. buffer I 25vMACh buffer Compared to liquid chromatographic deFIG. 7. Double-stimulation experiments showing verterminations of secretion in which, typically, satility of system. Five-second injection pulses of 250 a 20-~1 aliquot may be removed from 0.5 ml pM acetylcholine (A), or 56 mM K+ (B). ON and OFF of solution bathing cells on a dish, the total indicate beginning and end of perfusion with 25 pM secreted catecholamines in our system are acetylcholine. PULSE
DURATION,
T (SEC)
FLOW-INJECTION
ANALYSIS
OF CATECHOLAMINE
other modifiers of secretion can be resolved into effects on strength of initial response and rate of desensitization. We are in the process of improving FIA measurements by the use of a computer for data acquisition and analysis. Calculations of peak height and area for concentration determination and determination of the total amount secreted, the injection times, times to reach maximum and baseline,and peak symmetry will be routinely done by the Apple computer. Pulse times for stimulant injection can also be generated by the computer, so controlled pulse and delay times will be used in studying recovery time under varying durations of stimulation. Computer modeling can also be performed using FIA theory and chemical kinetics to generate theoretical response curves to compare with the experimental ones and to determine reaction orders. FIA parameters such as slopes of the curves, peak broadening, peak height, and time to reach maximum secretion can also be used to study the kinetics of secretion. ACKNOWLEDGMENTS This work supported by a grant from National Institutes of Health (GM24197) Biomedical Sciences Research Grant to the of Massachusetts. M. Herrera thanks A. D. for their sponsorship in a summer American
the U. S. and by a University Little, Inc. Chemical
SECRETION
227
Society Division of Analytical Chemistry fellowship, and the University of Massachusetts for the graduate fellowship. We thank Dr. B. Jacobson and Dr. J. Shiozawa for a supply of hydrophilic styrene beads and John Cryan for preparing some of the cell cultures. We thank Dr. D. Green and Dr. R. Perlman for sending us an unpublished manuscript.
REFERENCES Westhead, E. W., and Livett, B. G. (1983) Trends Neurosci. 6, 2954-255. Sorimachi, M., and Nishimura, S. (1982) Adv. Biosci. 36,ll. Green, D., and Perlman, R. (1981) Annl. Biochem. 110, 270. Ruzicka, J., and Hansen, E. (1981) Flow Injection Analysis, Wiley-Interscience, New York. Rocks, B., and Riley, C. (1982) Clin. Chem. 28 (3), 409. Ruzicka, J. (1983) Anal. Chem. 55, 1040A. Stewart, K. ( 1983) Anal. Chem. 55, 93 1A. Can, P., and Bowers, L. (1980) Immobilized Enzymes in Analytical and Clinical Chemistry, WileyInterscience, New York. 9. Mottola, H. (1981) Anal. Chem. 53, 1713. IO. Nondek, L., Brinkman, V., and Frei, K. (1983) Anal. Chem. 55, 1466. 11. Jacobson, B. S., and Ryan, U. S. (1982) Tissue and Cell14, 69-83. 12. Curtis, A. A. G., Forrester, J. V., Mclnnes, C., and Lawrie, F. (1983) .I. Cell Biol. 97, 1500-1506. 13. Kilpatrick, D. L., Ledbetter, F. H., Carson, K. A., Kirshner, A. G., Slepetis, R., and Kirshner, N. (1980) J. Neurochem. 35, 679-692. 14. Fenwick, E. M., Fajdiga, P. B., Howe, N. B. S., and Livett, B. G. ( 1978) J. Cell Biol. 76, 12-30.
BIOCHEMISTRY
144,
Flow-Injection
MICHELLE
2 18-227 (1985)
Analysis of Catecholamine Secretion Adrenal Medulla Cells on Microbeads
from Bovine
HERRERA,? LUNGSEN KAO,*~' DAVID J. CURRAN,? ANDEDWARD W. WESTHEAD*
Departments of *Biochemistry and jfhemistry,
University of Massachusetts, Amherst, Massachusetts 01003
Received February 29, 1984 Bovine adrenal medullary cells have been cultured on microbeads which are placed in a lowvolume flow system for measurements of stimulation-response parameters. Electronically controlled stream switching allows stimulation of cells with pulse lengths from 1 s to many minutes; pulses may be repeated indefinitely. Catecholamines secreted are detected by an electrochemical detector downstream from the cells. This flow-injection analysis technique provides a new level of sensitivity and precision for measurement of kinetic parameters of secretion. A manual injection valve allows stimulation by higher levels of stimulant in the presence of constant low levels of stimulant. Such experiments show interesting differences between the effects of K+ and acetylcholine on cells partially desensitized to acetylcholine. 0 1985 Academic
Press. Inc.
KEY WORDS: catecholamine secretion; flow-injection electrochemical detection; adrenal medulla.
analysis (FIA); kinetics of secretion;
Cultured cells of bovine adrenal medulla have become widely used for the study of stimulated secretion of catecholamines (1). This system provides much closer control over the cellular environment than does the procedure of organ perfusion (2). The uniformity of stimulation and simplicity of sampling suggest that the kinetic parameters of secretion, inhibition, and desensitization might be studied advantageously with cultured cells. In 1980 Green and Perlman (3) introduced an on-line measurement of catecholamine secretion by isolated rat adrenal tumor (PC 12) cells placed on a membrane filter. Solution flowing over the cells passed through the filter directly to an electrochemical detector which measured the oxidation current due to secreted catecholamines. By stream switching from buffer to stimulant solution the authors obtained information on the kinetics of the secretory process.
This paper describes a new on-line system for measuring catecholamine released from chromaffin cells. The cells are allowed to attach to treated styrene-divinylbenzene beads which are then packed in a short column placed in the flow line. Because this system has a very low dead volume and uniform flow through the packed bed, it has a much lower dispersion than the system previously described. These properties allow analytical information to be obtained by injecting short pulses of stimulant through the cells, generating sharp peaks of catecholamine at the detector. This method is known to analytical chemists as flow-injection analysis (PIA)* and is widely used in process stream analysis and in applications where automation and rapid sample throughput are desired. The theory and applications of FIA have been extensively studied and reviewed (4-7), including application to immobilized enzyme reactors (8). In PIA the determination
’ Present address: Sloan Kettering Institute for Cancer Research, 1275 York Avenue, New York, N. Y. 10021.
2 Abbreviations used: FIA, flow-injection analysis; Ach, acetylcholine.
0003-2697185 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
218
FLOW-INJECTION
ANALYSIS
OF
of sample concentration can be made by calibration of peak height with standards. The effect of chemical kinetics on the FIA peak shape has been considered (8- 10) and the equations derived for these complex systems are relevant to the system we describe. This flow-through system allows precise measurements of secretion levels during desensitization of the cells to stimulant so that kinetic parameters may be calculated. The ability to choose injection pulses of varying durations and frequencies makes possible the examination of secretion and desensitization as a function of short times, and thus may approach the in viva time frame of stimulation. The purpose of this paper is to demonstrate the potential of this technique. The data presented have been selected to demonstrate the variety of experimental parameters that may be investigated and the versatility of the system. MATERIALS
AND
METHODS
Flow-line materials purchased from Rainin Instrument Company include Altex l/16-in.o.d., 0.3-mm-i.d. Teflon tubing, polypropylene tube end fittings and couplers, and Tefzel tube connectors used to connect the Teflon tubing to Tygon tubing from the solvent reservoirs. The manual injection valve was a Rheodyne Teflon sample injection valve. The Teflon three-way solenoid valve was a Series 1 valve from General Valve. Stainless-steel Swagelok fittings, l/8 and l/16 in., coupled to a stainless-steel column (1.8 cm X 2.5 mm) were used for the cell chamber. Polystyrene beads, 37- to 74-pm diameter, 12% crosslinked, were from Bio-Rad and were treated to produce a hydrophilic surface to promote cell adhesion ( 11,12). Chromaffin cells were cultured using the techniques and sources of media described in (13) based on the original procedures of Fenwick et al. (14). The electrochemical detection of standard and secreted catecholamines was performed with a Bioanalytical Systems LC-3 amperometric detector equipped with a glassy carbon
CATECHOLAMINE
SECRETION
219
working electrode, an Ag/AgCl reference electrode, a stainless-steel counter electrode, a Faraday shield, and background nulling circuitry. A spacer thickness of 0.005 in. was used. The oxidation current was recorded on an Omniscribe dual-pen recorder, which also monitored the injection signal next described. A Tektronix FG 501 function generator was used to generate a variable-frequency square wave which was fed through the valve driver (made by M. Conboy, Electronics Shop, Univ. of Mass. Chemistry Dept.) which controlled the valve position and allowed repeatable stream-switching times. It must be noted that the oxidation current recorded in all these experiments results from oxidation of both catecholamines and ascorbic acid, which is also secreted by the cells. Ascorbic acid is present in the vesicles of the adrenal medulla at a concentration about 10% that of catecholamines and under our conditions the detector sensitivity for ascorbate is about half that for catecholamines. In experiments in which the effluent from stimulated cells was received by a fraction collector, analysis by HPLC showed that less than 1% of the signal was due to ascorbic acid. All experiments were performed at room temperature, 26°C. The apparatus assembled for FIA of catecholamine secretion is shown in Fig. 1. Glass solvent reservoirs were filled with 500 ml each of (i) buffer composed of 154 mM NaCl, 2.2 mM CaC12, 5.6 mM KCI, 5.0 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes) buffer, and 10.0 mM glucose, pH 7.4, and (ii) stimulant solution containing 25 PM Ach in the buffer solution. The reservoirs were placed 2 m above the rest of the apparatus for gravity-fed delivery. All solutions were prepared in deionized distilled water and microfiltered (0.22 pm) to prevent clogging and disruption of the cell bed by particulates. The experiments were begun by equalizing the flow rates of the two solution reservoirs (since electrochemical detection is flow-rate dependent), allowing the background current to equilibrate at a potential of +0.65 V, and
220
HERRERA
flow-balancing
valve
ET AL.
FIA injection pulse
___
3-woy
solenoid (teflon)volve
square wove generator
‘“““J$
zero deod- volume connectors
valve
_______
drover
3
dual -oen
1
recorder
measure catecholammes manual
injection valve
___ I +
I
I! 0.3
mm teflon
tubing
-------
%-l -
with
adrenal
cells
I
P
woste
electrochemical detector
FIG. 1. Diagram of experimental apparatus.
nulling the background current electronically. The beads with attached cells were pipetted from the culture dish into the Swagelok fitting to form a packed bed about 6 mm long X 2.5 mm wide secured at both ends by glass wool, so that the effective dead volume was minimal. This low-volume flow system minimizes dispersion (sample dilution), which in turn minimizes peak broadening, thereby optimizing sensitivity and detection limits. Perfusion of cells with buffer was begun immediately after they were packed and was not interrupted in the course of an experiment. Stream switching from buffer to stimulant solution was performed with a manual switch on the valve driver. This long-term stimulation of the cells was used to study the kinetics of desensitization of the cells to stimulant.
Injections of stimulant for FIA experiments were performed for various pulse durations using square-wave pulses of various frequencies (0.025 to 0.6 Hz) generated by the function generator to drive the valve between buffer and stimulant positions. The steady volumetric flow rate (0.70 or 1.1 ml/min) was later used to determine the volume of stimulant injected. Data were recorded at 1 to 100 nA full scale sensitivity at chart speeds of 25 cm/min (for precise time-axis measurements) and 2.5 cm/min. Standard catecholamine determinations were performed by replacing the stimulant solution reservoir with a solution 0.5 pM in both epinephrine and norepinephrine. The solutions were prepared within an hour of use by diluting 1 mM stock solution of catecholamines (stored frozen in 0.1 M HCl) with
.
FLOW-INJECTION
ANALYSIS
OF CATECHOLAMINE
microfiltered buffer. The same streamswitching and FIA experiments performed with the stimulant solution were repeated using the standard solution for concentration calibration purposes. Dispersion of the sample in the flow-through system was also studied with the standard solution. The 1 PM standard solutions were sufficiently concentrated so that uptake by a bed of cells was negligible. Standards run with and without cells in the line differed by only 6%. A manual injection valve (cf. Fig. 1) equipped with a 62-~1 sample loop was used to inject the standard solution periodically during the course of a stimulant or standard run in order to monitor the electrode sensitivity (by peak height) and the dispersion (by peak width). In addition, it provided a second means of injecting stimulant in a “doublestimulation” experiment. In this case, a stimulant (25 pM Ach) was introduced by stream switching with the solenoid valve and 250 pM Ach or 56 mM K+ was injected manually at various points before, during, and after the constant stimulation with 25 pM Ach. The manual injection valve thus serves a dual purpose but it could be removed to decrease dispersion even further. Upon completion of the experiments with a batch of cells, the beads were washed from the fitting with 8% trichloroacetic acid solution. This solution was stored at -5°C for residual catecholamine analysis. RESULTS AND DISCUSSION
SECRETION
221
odic manual injections of standard. Therefore, the secretion of protein by exocytosis did not appear to foul the electrode. The peak widths did not change either, indicating that flow rate and dispersion were constant throughout each experiment. Typical response curves of the standard and secreted catecholamines are shown in Fig. 2. FIA peaks for various pulse times (T) are indicated by solid lines and responses to long-term stream switching from buffer to stimulant are indicated by dashed lines. Measurements in both FIA and stream-switching experiments are described in Fig. 3. Stream-Switching
Experiments
Stream-switching experiments were performed with cells 5 and 12 days in culture (“young” and “old” cells). The desensitization of catecholamine secretion during constant stimulation can be measured as tl12, as pictured in Fig. 3. The desensitization can be characterized more accurately by plotting In f (current) vs t (time) at times greater than the maximum as shown in Figs. 4A and B. The linearity of the plots shows that densitization at this concentration of stimulant is characterized by a single exponential constant (k). These values are tabulated along with tl12 values in Table 1. The plot of In I vs t for the young cells (Fig. 4A) was linear over an order of magnitude drop in I during a period of about 6 min. The plot for the older cells (Fig. 4B) was linear after subtracting the final constant level of secretion ( 15% of maximum) from all the f values. Shutoff occurs at a substantially slower rate with the younger cells than with the older cells. This phenomenon will be discussed in the next section. The tl12 values are comparable to those of Green and Perlman (3), but a quantitative comparison cannot be justified because their cells were PC 12 cells from a rat tumor line.
The background current from a typical batch of unstimulated cells was initially 13 nA, and it decreased to 6.9 nA within 2 h. The final background is comparable to the 6.5-nA current of the flowing stream with the cells removed. The 13-nA current corresponds to a basal secretion rate of 1 pmol/s. The peak-to-peak noise level of the baseline was comparable to that without cells, or 20 FIA Measurements pA. Electrode sensitivity did not change appreciably through hours of flow analysis, as Measurements made on the FIA peaks are indicated by the constant height of the peri- described in Fig. 3. Peak dimensions are
222
HERRERA
ET AL.
TIME FIG. 2. Response curves for FIA and stream-switching experiments. Catecholamine response in flowing stream versus time for the different pulse times indicated (seconds). Dashed lines are for infinite pulse length produced by stream switching. Upper set of curves: 0.5 pM catecholamine standard added for 5 to 20 s. Lower set of curves: Catecholamines secreted in response to injection pulses of 25 pM acetylcholine lasting from 5 to 14 s.
dependent on analyte concentration as well as on pulse duration, T. Since peak height, H, at a particular T is proportional to the concentration of injected analyte, the peak heights of the FIA of the standard were used to tentatively calculate the amount of catecholamine secreted from the cells upon injection of stimulant. The absolute quantities (picomoles) of catecholamines secreted were calculated by calibration with the standard. FIA was used to study the desensitization of a different batch of 5day-old cells to repeated pulses of stimulant. Fig. 5 shows the amount secreted (from peak height) vs time, upon injecting 4-s pulses (T = 4 s) of 100 /IM acetylcholine every 1 to 2 min. The response of the cells decreased sharply from the first to
the second injection and then more gradually with consecutive injections. After 50 min of this periodic stimulation, the cells were allowed to rest for about 15 min. The last point shows the dramatic increase in secretion response after recovery of the cells. FIA data from secretions of two different samples of cells (5 and 12 days old from the same preparation used for the data of Fig. 3) are shown in Table 2. The injection volumes are calculated by multiplying flow rate and pulse time. The amount secreted is obtained from calibration with the standard catecholamine response for a particular T. The Co values arc the amount secreted (pmol) divided by the injection volume. The amount secreted was normalized for injection volume in this
FLOW-INJECTION
ANALYSIS
Siandard
t=o
G/2
FIG. 3. Measurements on response curves. Solid lines are FIA curves. Dashed lines are stream-switching responses. T, pulse injection duration (s); C, and C,, concentration of injected standard and stimulant, respectively: t time axis (s); H and H’, peak heights of standard and secreted FIA peaks in nA, respectively; IV,,, and IV,,,, peak widths at half-height for standard and secreted peaks. H,,, the steady-state current (in nA) for C, (standard); D, the dispersion of the standard, or H,,/H; t,,?, the half-time for desensitization in the streamswitching experiments; M, maximum secretion.
manner so comparisons between different T values in terms of average amount secreted during pulse duration could be made. The older (12-day-old) batch of cells secreted 510% the amount of the younger cells (5 days
OF CATECHOLAMINE
old) but older cells contain less catecholamine than younger cells (13). Furthermore, at the time of these experiments, we were not quantitating the number of beads in the flow line so the difference in total secretion is not a relevant part of these results. The effect of pulse time on the average concentration secreted (Co) was studied in both batches of cells. The younger cells seemed to secrete a greater average concentration of catecholamines at shorter times than at longer times. An explanation for this trend is that since secretion decreases due to desensitization, the average concentration also decreases with increasing duration of stimulant pulse, 7’. However, this did not appear to be true of the older cells, since the concentration secreted was fairly constant and independent of T, with an average of 0.52 X lo-’ M and relative standard deviation of 6.5% (n = 25).
The difference in desensitization behavior of the two batches of cells may be compared in terms of tli2, k, and T dependency. The young cells appear to desensitize at a rate slower than that of the older cells as indicated by the lower k and greater tl,2 in Table 1. However, the T dependency of the amount secreted by the younger cells suggests that the younger cells desensitize very rapidly at short times, i.e., before the maximum secretion rate which occurs at about 12 s. Peak width is also dependent on the de-
-se.TIME (SEC)
223
SECRETION
/ 20
I
, 60
, TIME
, 100
,
I 140
I
180
I
1 2;
(SEC)
FIG. 4. Logarithmic plots for desensitization in stream-switching experiments with 25 (A) 5-Day-old cells. (B) 12-Day-old cells.
pM
acetylcholine.
224
HERRERA TABLE
1
Expt
Age of cell (days)
Maximum secretion rate (PmoVs)
h/2 (4
k (s-l)
1 2 3
5 12 12
5.9 0.52 0.43
120 28.3 27.6
0.007 0.022 0.023
Note. Experiment 3 is a repeat of experiment 2, to give some idea of the reproducibility of the constants.
sensitization of cells. Fig. 6 shows a plot of peak width vs T for the standards and secreted catecholamines. At small T the peak widths appear to be statistically equal. The increase in the difference of peak widths with increasing T is probably due to reaction band broadening (10) combined with the kinetics of cell response to the changing stimulant concentration. At even longer T, the peak widths again approach those of the standards, a result of the desensitization of the cells to the stimulant. The secretion curve therefore decays to baseline more rapidly than the standard curve and the peak width (which should be independent of peak height in standard FIA) is therefore smaller. It must be noted that the variability of the shape of the secreted peaks makes uncertain the determination of amount secreted using peak height of the standard curves. We are in the process of interfacing the FIA system to a computer that will measure peak areas for more accurate determinations of catecholamine using area calibration curves. Double-Stimulation
ET AL.
constant stimulation by 25 PM acetylcholine. The peak heights were not identical in each run (only one run of each is shown) but the following general trends were reproducible: (i) the peak caused by the 250 PM Ach did not change much when the background 25 PM Ach was switched from OFF to ON, but the K+ peak did increase considerably, (ii) both sets of peaks decreased gradually with the 25 PM Ach ON due to the desensitization of the cells, and (iii) the 250 PM Ach peaks recovered quickly after the 25 PM Ach was turned OFF while the K+ peaks went immediately back to the original value. These results are in accord with the previous suggestion (3) that there are separate mechanisms of catecholamine secretion for Ach and K+, i.e., receptor-dependent channels and voltagedependent channels that work independently of one another. Duration
of Experiments
At the conclusion of a 5-h experiment involving 100 or more stimulation pulses, the catecholamine left in the cells was 70 to 80% of the amount at the start of the experiment. At this point, cells that were rested for 15 min responded to stimulus very much as they had initially. The long-term stability of secretion may be due to constant removal of secretory products and stability of the cells in the packed bed, which has predominantly 70 t0
Experiments
In addition to low detection limits and good time resolution, the FIA system is also capable of great versatility for the study of any synergistic or desensitizing effects associated with secondary stimulation. Figure 7 shows an example of “double stimulation” in which manual injections (62 ~1) of either stimulant, 250 PM Ach, or 56 PM K+ are done before, during, and after a period of
TIME
(SEC
1
FIG. 5. Catecholamine secreted versus time for repeated pulsed stimulation. Each point is in response to a 4-s injection of 100 pM acetylcholine at a flow rate of 1.0 mlimin. ~~~~,
FLOW-INJECTION
ANALYSIS
OF
CATECHOLAMINE
TABLE
225
SECRETION
2
DETERMINATION OF CATECHOLAMINES Young cells (5 days old) Pulse time T 6) 20.0
Injection volume (t.4
Total secretion (pmol)
370
72 58
Old cells ( 12 days old)
G (M X lo’)
Total secretion (pm4
G (M X lo’)
240
5.0
0.21
14.3
172
9.4 8.5 8.5
0.55 0.50 0.50
11.1
133
7.7 7.0
0.58 0.53
120
6.1 6.1 6.0
0.5 1 0.51 0.50
6.7
80
4.3 3.9 4.0
0.53 0.48 0.49
5.0
60
3.2 3.0 3.1 2.9
0.53 0.50 0.52 0.49
1.5
0.51 0.51
10.
2.5
180
45
2.0 1.6
Injection volume w
31
1.7
21 20
6.0 4.5
1.25
1.00
18
26 19 18 17
14.3 10.5 10.0 9.5
0.83
laminar flow and sufficiently low pressure drop to create a favorable environment for the cells. Dispersion and Detection Limits
The dispersion (D) of the standard solution in a 62-~1 injection volume with a 0.70-ml/ min flow rate (T = 5 s) was measured as HO/
30
1.5 1.6
0.52
15
0.80 0.86 0.88
0.53 0.57 0.58
12
0.60
0.50
10
0.59 0.47 0.58
0.59 0.47 0.58
H. D was 4.3 with the fitting packed with beads and 5.3 with an equivalent length of 0.3-mm tubing replacing the packed fitting. The packed fitting actually decreases peak broadening, which is a well-known phenomenon in FIA (4) and is analytically advantageous in terms of detection limits. The dispersion is much lower than previously reported by Green and Perlman (3).
226
HERRERA
ET AL.
immediately detected in an elution volume which is even smaller than the elution volume in liquid chromatography. FIA detection limits of the amount secreted by the cells can therefore be a factor of lo-100 times better than those obtained by removing aliquots for chromatography. 11 0
I 4
II
/ 8
I 12
I
I 16
I
I 20
II
CONCLUSIONS
This work has demonstrated the use of flow-injection analysis to study the in vitro FIG. 6. Peak width versus pulse duration in FIA mode. secretion of catecholamines from adrenal cells. The advantages are very low detection limits, wide variability of stimulation times Their system had a dead volume of 300 ~1 down to about 1 s, accurate repeatability, and a time for 50% washout of 30 s. The long experiment time, and concentration decorresponding figures for our system were 13 termination by direct calibration with stan~1 and 5 s. The lower dispersion of our dards. Because washout of stimulant is rapid system greatly lowers distortion of the FIA and complete, kinetic analysis of parameters curves and improves detection limits in the affecting secretion can be conveniently studFIA experiments. ied. The phenomenon of desensitization, for Detection limits of catecholamines were example, can be studied as a function of derived from twice the value of the baseline stimulant concentration, pulse length, recovnoise, which was 10 pA peak to peak. This ery time, and frequency of repeated pulses. value was the same with or without the cells The ease of injection of short pulses of in the flow line. For the stream-switching stimulant against a constant low-level backexperiments on the 12-day-old cells, this corground of stimulation may also provide new responds to a detection limit of lo-” M, or information on the desensitization process. a secretion rate of lo-l5 mol/s (using a flow We anticipate that the effects of drugs and rate of 0.70 ml/min). The detection limit in the FIA experiments using the shortest pulse duration of 0.83 s was lo-l4 mol (total amount secreted). This detection limit was very low because of the stability of the detector and the cells and also because a gravityfed pump was used. Pump noise in the work of Green and Perlman limited their streamswitching detection to 0.4 nA, which is 20 times higher than our detection limit. FIA detection limits on that type of flow-through system would be relatively poor due to the broadened peaks resulting from high disperc sion. buffer I 25vMACh buffer Compared to liquid chromatographic deFIG. 7. Double-stimulation experiments showing verterminations of secretion in which, typically, satility of system. Five-second injection pulses of 250 a 20-~1 aliquot may be removed from 0.5 ml pM acetylcholine (A), or 56 mM K+ (B). ON and OFF of solution bathing cells on a dish, the total indicate beginning and end of perfusion with 25 pM secreted catecholamines in our system are acetylcholine. PULSE
DURATION,
T (SEC)
FLOW-INJECTION
ANALYSIS
OF CATECHOLAMINE
other modifiers of secretion can be resolved into effects on strength of initial response and rate of desensitization. We are in the process of improving FIA measurements by the use of a computer for data acquisition and analysis. Calculations of peak height and area for concentration determination and determination of the total amount secreted, the injection times, times to reach maximum and baseline,and peak symmetry will be routinely done by the Apple computer. Pulse times for stimulant injection can also be generated by the computer, so controlled pulse and delay times will be used in studying recovery time under varying durations of stimulation. Computer modeling can also be performed using FIA theory and chemical kinetics to generate theoretical response curves to compare with the experimental ones and to determine reaction orders. FIA parameters such as slopes of the curves, peak broadening, peak height, and time to reach maximum secretion can also be used to study the kinetics of secretion. ACKNOWLEDGMENTS This work supported by a grant from National Institutes of Health (GM24197) Biomedical Sciences Research Grant to the of Massachusetts. M. Herrera thanks A. D. for their sponsorship in a summer American
the U. S. and by a University Little, Inc. Chemical
SECRETION
227
Society Division of Analytical Chemistry fellowship, and the University of Massachusetts for the graduate fellowship. We thank Dr. B. Jacobson and Dr. J. Shiozawa for a supply of hydrophilic styrene beads and John Cryan for preparing some of the cell cultures. We thank Dr. D. Green and Dr. R. Perlman for sending us an unpublished manuscript.
REFERENCES Westhead, E. W., and Livett, B. G. (1983) Trends Neurosci. 6, 2954-255. Sorimachi, M., and Nishimura, S. (1982) Adv. Biosci. 36,ll. Green, D., and Perlman, R. (1981) Annl. Biochem. 110, 270. Ruzicka, J., and Hansen, E. (1981) Flow Injection Analysis, Wiley-Interscience, New York. Rocks, B., and Riley, C. (1982) Clin. Chem. 28 (3), 409. Ruzicka, J. (1983) Anal. Chem. 55, 1040A. Stewart, K. ( 1983) Anal. Chem. 55, 93 1A. Can, P., and Bowers, L. (1980) Immobilized Enzymes in Analytical and Clinical Chemistry, WileyInterscience, New York. 9. Mottola, H. (1981) Anal. Chem. 53, 1713. IO. Nondek, L., Brinkman, V., and Frei, K. (1983) Anal. Chem. 55, 1466. 11. Jacobson, B. S., and Ryan, U. S. (1982) Tissue and Cell14, 69-83. 12. Curtis, A. A. G., Forrester, J. V., Mclnnes, C., and Lawrie, F. (1983) .I. Cell Biol. 97, 1500-1506. 13. Kilpatrick, D. L., Ledbetter, F. H., Carson, K. A., Kirshner, A. G., Slepetis, R., and Kirshner, N. (1980) J. Neurochem. 35, 679-692. 14. Fenwick, E. M., Fajdiga, P. B., Howe, N. B. S., and Livett, B. G. ( 1978) J. Cell Biol. 76, 12-30.