[2] Pancreatic lobules in the in vitro study of pancreatic acinar cell function

[2]

PANCREATIC LOBULES IN ACINAR CELL FUNCTION

[2] P a n c r e a t i c

17

Lobules in the in Vitro Study of Pancreatic Acinar Cell Function B y G E O R G E SCHEELE

In the past 15 years, in vitro preparations that have been developed to study intact pancreatic acinar cells have included pancreatic slices,~ pieces, 2 lobules, 3 dissociated single cells,4 and dispersed acini? Compared to pancreatic lobules, slices and pieces show considerably increased levels of mechanical damage to cells. Compared to dissociated acini, dissociated single cells show increased levels of damage due to the proteases, phospholipases, chelators, and mechanical shearing forces required for further tissue dissociation. When properly prepared, pancreatic lobules and dissociated acini show similar in vitro responses to varying levels of exogenously added secretagogues. The use of pancreatic lobules has two advantages over the use of dispersed acini: (a) ease of preparation; and (b) omission of degradative enzymes and chelators during tissue preparation. The use of dispersed acini, however, shows two advantages over the use of lobules: (a) increased access of labeled probes to the plasma membrane, and (b) apparent removal of nerve endings from isolated acini. Methods for the preparation and study of pancreatic lobules are presented in this chapter. Preparation of Pancreatic Lobules Pancreatic tissue excised from a small animal (e.g., mouse, rat, guinea pig, or rabbit) is placed in a petri dish containing ice-cold Krebs-Ringer bicarbonate (KRB) buffer (125 m M NaCl, 5 m M KCI, 2.53 m M CaCI2, I. 16 mMMgSO,, 25 mMNaHCO3). A 10-ml syringe with a No. 26 needle, I in. long, is used to inject KRB buffer directly into the gland at random sites. Injected buffer dissects along the connective tissue planes and separates macroscopic lobules (Fig. l a), which are then individually removed by simple excision using a pair of small curved ophthalmic scissors. This procedure minimizes damage to acinar cells, since most of the surgical trauma is limited to ducts and vessels. The excised lobules preserve the overall acinar architecture of the tissue (Fig. I b), and their small size J. D. Jamieson and G. E. Palade, J. Cell Biol. 34, 57 (1967). 2 L. Benz, B. Eckstein, E. K. Mathews, and J. A. Williams, Br. J. Pharmacol. 46, 66 (1972). 3 G. A. Scheele and G. E. Palade, J. Biol. Chem. 250, 2660 (1975). 4 A. Amsterdam and J. D. Jamieson, J. Cell Biol. 63, 1057 (1974). 5 G. S. Schultz, M. P. Sarras, G. R. Gunther, B. E. Hull, H. A. Alicea, F. S. Gorelick, and J. D. Jamieson, Exp. Cell Res. 130, 49 (1980). METHODS IN ENZYMOLOGY, VOL. 98

Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181998-1

18

SPECIALIZED METHODS

[9.]

FIG. 1. (a) Guinea pig pancreas in which "macroscopic" lobules have been spread out by injecting incubation medium (Krebs- Ringer bicarbonate) into the gland. The actual length of the distended pancreas was ~ 6 cm. Long arrows mark large interlobular ducts. Short arrows indicate well-separated lobules that are removed by excision with fine scissors for use in analytical in vitro incubation studies. For preparative studies larger segments of tissue, containing four to eight macroscopic lobules, can be taken. (b) Low-power light micrograph of two lobules used in the analytical in vitro incubation system. Black dots represent groups of zymogen granules located near central lumens in pancreatic acini. Note the structural integrity of the lobules. Transverse section, × 90. Taken from Scheele and Palade. 3

(~< 2.0 × 1.0 × 0.5 mm) allows for easy penetration of oxygen and solutes from the incubation medium. Individual lobules from the guinea pig pancreas are approximately 6 mg wet weight, contain ~ 16.6/tg of DNA, 0.5 mg of protein, and ~ 1.5 X 106 cells. Lobules prepared from pancreatic tissue taken from cats, dogs, and humans cannot be used for in vitro studies because they exceed the size limits required for efficient exchange of gas and

[2]

PANCREATIC LOBULES IN ACINAR CELL FUNCTION

19

solutes. In these larger glands one is restricted to the use of pancreatic tissue slices (or dissociated acini). Lobules are incubated in either (a) KRB medium gassed with 95% 02-5% CO2 and supplemented with 1 mg of glucose and 100/tg of bovine serum albumin per milliliter and with essential and nonessential amino acids at 0.2 m M each; or (b) Krebs- Ringer HEPES medium (phosphate and bicarbonate replaced by 25raM HEPES, pH 7.4) gassed with 100% 02 and supplemented as above with glucose, albumin, and amino acids. The HEPES-buffered medium has two advantages: its pH value is less sensitive to temperature changes and gassing conditions; and divalent cations, particularly Ca 2+, can be elevated above physiological levels without precipitation of calcium salts. Guinea pig pancreatic lobules are stable in these media without trypsin inhibitors. However, since exocrine proteins from the mouse, rat, and rabbit show a tendency toward autoactivation, pancreatic lobules prepared from these species are incubated in medium supplemented (per milliliter) with 20/~g of soybean trypsin inhibitor (STI; Worthington Corp., Freehold, New Jersey,) and 100 KIU of Trasylol (FBA Pharmaceuticals, New York, New York). Applications

for Study of Pancreatic

Lobules

Preparative Isolation of Pancreatic Exocrine Proteins Purified preparations of secretory proteins can be extracted from a crude fraction of zymogen granules or obtained physiologically after carbamylcholine-induced secretion of proteins into the incubation medium. Zymogen Granule Lysate. Lobules prepared from one pancreatic gland ( ~ 1 g of tissue wet weight) are homogenized in 10 ml of 0.3 M sucrose containing 20/~g of soybean trypsin inhibitor and 100 KIU of Trasylol per milliliter, 1 m M DFP (Sigma Chemical Co., St. Louis, Missouri) and 1 m M benzamidine (Aldrich Chemical Co., Milwaukee, Wisconsin). Nuclei and cell debris are removed by sedimentation for 10 rain at 3 ° and 600 g. A crude zymogen granule pellet is obtained from the postnuclear supernatant by sedimentation for 10 rain at 3 ° and 1000 g.6 The granule pellet is lysed either in a 1.0-ml solution containing 1% Triton X-100, 25 m M Tris-HC1 (pH 9.0), 20/lg of STI and 100 KIU of Trasylol per milliliter, 1 mMDFP, and 1 m M benzamidine or in a 1.0-ml solution containing 0.2 M NaHCO3 (pH 8.5) and the protease inhibitors indicated above. The majority of membranes are removed by sedimentation in a Brinkmann microcentrifuge for 15 rain at 3 ° and 8000 g. Secreted Proteins. Pancreatic lobules from one gland ( ~ 1 g of tissue) are 6 A. M. Tartakoffand J. D. Jamieson, this series, Vol. 31, p. 57.

20

SPECIALIZED METHODS

[2]

incubated under physiological conditions for 3 hr in the presence of 10-5 M carbamylcholine (Sigma). After this incubation period, the medium is decanted and supplemented with 2 pg of soybean trypsin inhibitor and 10 KIU of Trasylol per milliliter, 1 m M diisopropyl fluorophosphate, and 1 m M benzamidine. Contaminating cells and loose debris are removed by sedimentation for 30 min at 105,000 g and 3 °. Secretory proteins obtained either from the incubation medium (1-2 mg/ml) or a zymogen granule lysate (5 - 10 mg/ml) are aliquoted in samples of 50- 100 pl and stored at - 8 0 ° after rapid freezing in liquid NE. Samples are thawed and used only once, since repeated freezing and thawing can result in autoactivation and degradation of samples.

Preparative Isolation of Endogenously Labeled Radioactive Proteins Radioactive proteins can be endogenously labeled by incubation of pancreatic lobules under physiological conditions for 3 hr in the presence of radioactive amino acids and the remaining complement of unlabeled amino acids at 0.2 mMeach. Highest levels of specific radioactivity can be obtained using [35S]methionine or [35S]cysteine. Approximately 95% of radioactivity incorporated into pancreatic lobules appears within secretory proteins. 7 Labeled secretory proteins can be isolated from the incubation medium or a zymogen granule extract as described above.

Analysis of mRNA-Directed Protein Synthesis The high resolution achieved in the separation of exocrine pancreatic proteins by two-demensional isoelectric focusing/sodium dodecyl sulfate (IEF/SDS)-gel electrophoresis allows the precise measurement of biosynthetic rates of individual secretory products. We have made such measurements in the following manner. Guinea pig pancreatic lobules are incubated with [35S]methionine for 15 min, and the incorporation of radioactive methionine is terminated by rapid freezing in liquid nitrogen. The tissue is then homogenized in 1% Triton X-100 and 25 mMTris-HC1 (pH 9.0), and proteins contained in tissue homogenates are separated by two-dimensional IEF/SDS-gel electrophoresis. Coomassie blue-stained spots characterized as secretory proteins and unstained regions of the gel containing short-lived precursor forms (identified previously using fluorography) are removed from the second-dimension gel and dissolved in hydrogen peroxide, and solubilized radioactivity is quantitated by liquid scintillation spectrometry. These methods have allowed us to measure changes in bio7 G. A. Scheele, G. E. Palade, and A. M. Tartakoff, J. CellBiol. 78, 110 (1978).

[2]

PANCREATIC LOBULES IN ACINAR CELL FUNCTION

21

synthetic rates of individual exocrine proteins in the presence of hormonal stimulation. 8

Analysis of the Pathwayfor Intracellular Transport of Secretory Proteins In association with cell fractionation techniques and two-dimensional gel separation of proteins, pulse-chase studies carried out on pancreatic lobules allow a thorough analysis of the movements of individual secretory proteins through intracellular compartments and their eventual discharge into the extracellular medium. Previous studies utilizing [3H]leucine-labeled pulse-chase studies and cell fractionation techniques were unable, with certainty, to determine the route of intracellular transport for these proteins, largely owing to redistribution artifacts associated with tissue homogenization. Scheele et aL7used guinea pig pancreatic lobules pulse-labeled with ~4C amino acids and chased for varying periods of time with '2C amino acids, and they performed tissue homogenization in the presence of tracer amounts of 3H-labeled pancreatic proteins. Analysis of 3H/14Cratios in two-dimensional gel spots derived from individual cell fractions provided information on the extent of leakage of secretory proteins from individual membrane-enclosed compartments along the secretory pathway and the extent of redistribution of leaked molecules to membrane surfaces by nonspecific adsorption. These studies indicated that the appearance of exportable proteins in the postmicrosomal supernatant fraction could be accounted for by the uniform leakage of proteins during tissue homogenization and the preferential binding of positively charged molecules to negatively charged surfaces of membrane-bound organelles. Figure 2 shows the extent to which individual radioactively labeled exocrine proteins, when introduced into the homogenizing medium, will adsorb to the surfaces of subcellular organelles. The data indicate, for the majority of enzymes and zymogens, that adsorption to membrane surfaces is directly dependent on the isoelectric point of the protein. The majority of proteins adsorbed to individual subcellular fractions could be removed with a 100 mMKCI wash. Figure 3 shows in schematic form the extent of leakage and adsorption artifacts that occur when pulse-labeled proteins are located in the rough endoplasmic reticulum (RER) (Fig. 3b) and in zymogen granules (Fig. 3c). Figure 4 shows the improvement observed in intracellular transport kinetics when the data are corrected for redistribution artifacts. These studies provided important evidence in support of the hypothesis that secretory proteins remain segregated within a series ofinterconnectable membrane-bound compartments during their transport through the cell. They also provided the first clear demonstration, by cell fractionation 8 G. A. Scheele, Methods CellBiol. 23, 345 (1981).

22

SPECIALIZED METHODS

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PANCREATIC LOBULES IN ACINAR CELL FUNCTION

23

techniques, that exocrine pancreatic proteins are transported successively from the RER to the Golgi apparatus and from there to zymogen granules.

Analysis of Posttranslational Modification of Pancreatic Gene Products Since there exist multiple forms of enzymes and zymogens among pancreatic proteins, it is necessary to determine which forms represent separate gene products and which represent modified forms of single gene products. Characterizations of this type are vital to the quantitation of mRNA-directed protein synthesis described above (see Analysis of mRNADirected Protein Synthesis). Pancreatic lobules are given a 10-min pulse with [35S]methionine followed by a varying chase period with [3ZS]methionine. Chase intervals are terminated by rapid freezing in liquid nitrogen, lobules are homogenized in 1% Triton X- 100 and 25 mMTris-HC 1, pH 9.0, and radioactive proteins contained in tissue homogenates are analyzed by fluorography after their separation by two-dimensional IEF/SDS-gel electrophoresis. Radioactive proteins that change positions in the two-dimensional pattern of protein spots after varying intervals of chase represent posttranslational modifications. Among guinea pig pancreatic proteins isolated from pancreatic lobules, both short-lived and long-lived intermediate forms have been identified.8

Secretagogue-Induced Discharge of Exocrine Proteins Functional discharge of exocrine proteins is studied by incubation of 5-10 pancreatic lobules in 5 ml of KRB solution, supplemented as de-

FIG. 2. Differential adsorption of exogenous gH-labeled exocrine pancreatic proteins on subcellular fractions. Radioactive proteins associated with individual fractions from a typical cell fractionation experiment were separated by two-dimensional isoelectric focusing/SDS-gel electrophoresis, and the distribution of radioactivity among protein spots was expressed relative to the distribution of radioactivity found in the postnuclear supernate. The lower abscissa indicates the isoelectric point of individual exocrine proteins. The identity of these spots is given in the upper abscissa as follows: PA, procarboxypeptidase A; L, lipase; PB, procarboxypeptidase B; PE, proelastase; A, amylase; T, trypsinogen; C, chymotrypsinogen. Spots 3 and/or 4 are ribonuclease. Spots 1, 2, and 6 are unidentified. Individual enzyme forms are numbered as given by G. A. Scheele [J. BioL Chem. 250, 5375 (1975)]. (A) Comparison of individualexocrine proteins in the postmicrosomal supernate (PMS) with those in the postnuclear supernate. The symbols are as follows: O, 3H pancreatic proteins (exogenous label) in pancreatic PMS; A, ~2 pancreatic proteins (endogenous label, 10 min pulse, 10 min chase) in pancreatic PMS; O, 3H pancreatic proteins (exogenous label) in liver PMS. (B) Comparison of the distribution of individualproteins in the exogenous tracer in four peUetablefractions with the distribution of those in the postnuclear supernatant fraction. The symbols represent the following cell fractions; ©, free ribosomes; O, rough microsomes; A, smooth microsomes; A, zymogen granules. Taken from Scheele et al. 7

SPECIALIZEDMETHODS

24

_]

~" ._..1~ -

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ExocrineProteins (exogenouslabel) in PMS 45% 6% J

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[9.]

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FIG. 3. Diagrammatic representationof leakageand relocation by adsorption of exocrine proteins during homogenization and subcellular fractionation of guinea pig pancreatic lobules. (a) Distribution of exogenously labeled exocrine proteins (added to the homogenizing medium) among each of the subcellular fractions derived from the secretory pathway. (b) Leakage of exocrine proteins from the rough endoplasmic reticulum during homogenization and their relocation by adsorption to subcellular fractions derived from the secretory pathway. (c) Leakage of exocrine proteins from zymogen granules and relocation as described in (b). Each cell fraction is represented diagrammatically by its major component. Data for nuclear and mitochondrial fractions are not included; this explains the apparent underrecoveries. Taken from Scheele et al. 7

PANCREATIC LOBULES IN ACINAR CELL FUNCTION

[2]

25

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• RM

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FIG. 4. Intracellular transport kinetics for pulse-labeled proteins in the exocrine pancreas presented (A) as raw data (uncorrected) (B) after correction for adsorption artifact, and (C) after correction for both adsorption and leakage artifacts. A curve for the smooth microsomal fraction (SM) cannot be given in (C) because data on leakage from this fraction are not available at present. RM, rough microsomal fraction; ZG, zymogen granules. Taken from Scheele e t al. 7

26

SPECIALIZED METHODS

[2]

scribed above with glucose, amino acids, and albumin, and in the presence or the absence of known secretagogues and experimental agents. The addition of a circular piece of nylon mesh [Nitex, 118 #m (Tetko Co., Elmsford, New York)] cut 1.5-2.0 cm in diameter allows rapid transfer of lobules from one flask to another, since lobules adhere to the mesh. Discharge of exocrine proteins can be quantified by two methods: Method a. Radioactive Protein Assay. Secretory proteins are labeled by the following pulse-chase protocol. Pancreatic lobules are pulsed with 0.44 /tM L-[4, 5-3H]leucine (20/tCi/ml) for 15 min, during which time approximately 95% of trichloroacetic acid-insoluble radioactivity is incorporated into secretory proteins. Pancreatic lobules are then briefly washed in KRB solution and incubated in a second flask containing KRB solution with physiological quantities of nonradioactive essential and nonessential amino acids for 90 min; during this time the majority of radiolabeled secretory proteins are transported to the zymogen granule pool. The lobules are then transferred to the experimental flask, which contains additional chase medium (5 ml) with the appropriate experimental agents under study. Sequential aliquots, usually 100/d, are withdrawn during the incubation period without readdition of flesh medium. After the incubation period, pancreatic lobules containing undischarged protein are homogenized in 5 ml of 1% Triton X-100 and 25 mM Tris-HC 1, pH 9.0. Aliquots (100/tl) of medium and tissue homogenate (250-4000 cpm per aliquot) are applied to Whatman 3MM filter disks, immersed in ice-cold 10% trichloroacetic acid and processed for trichloroacetic acid-insoluble radioactivity by the procedure of Mans and Novelli. 9 The data are expressed as cumulative discharge of labeled protein into the incubation medium as percentage of total labeled protein (tissue plus medium) as a function of time. In control studies, data derived from this pulse-chase protocol closely agreed with those derived from assay of amylase. Method b. Assay for Enzyme and Potential Enzyme Activity. Quantitation of secretory activity can also be achieved by measuring pancreatic enzymes (amylase, lipase, and ribonuclease) or zymogens (trypsinogen, after its activation by enterokinase or chymotrypsinogen;procarboxypeptidase A and procarboxypeptidase B, after their activation by trypsin). Optimal conditions for activation of guinea pig pancreatic zymogens, including time, temperature, and protein (zymogen) to activator ratios, are given by Scheele and Palade. 3 Using these conditions, we obtained linear relationships between the amounts of protein-activated and enzyme activity elicited by activation. Assay conditions for measurement of resulting enzyme activities are also summarized.3 Triton X-100 is added to tissue samples to ensure the release of enzymes and zymogens from membrane-bound compartments. All samples of homogenate and incubation medium are therefore adjusted 9 R. J. Mans and G. D. Novelli, Arch. Biochem. Biophys. 94, 48 (1961).

[9.]

P A N C R E A T I C LOBULES IN A C I N A R CELL F U N C T I O N

7O

CorbocholI0-5M / ~- 60 o

-- 50 E o

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--~ 20

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c~ io / ~ l b ~ , ~ J~"ge~ ' ' ' ' - ~

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Control I I 90 120 (rain)

27

FIG. 5. Kinetics of discharge of seven secretory protein activities from guinea pig pancreatic lobules incubated in the presence and in the absence of 10-5 M carbamylcholine. Eight lobules were incubated for 2 hr in 5 ml of incubation medium. In a first experiment (open symbols) discharge of amylase activity (O) was compared to discharge of chymotrypsinogen (/k), trypsinogen (V), procarboxypeptidase A (c~), and procarboxypeptidase B ( ~ ) . The values given for the four zymogens represent the corresponding enzyme activities assayed after activation. In a second experiment (filled symbols) discharge of amylase activity (O) was compared to that of lipase (A) and ribonuclease (~') activity. The results are expressed as percentage of activities released into the medium at each time point relative to the sum of activities retained in the tissue and discharged into the medium at the end of the incubation period. Note that under these conditions the seven activities were discharged in synchrony and in constant proportions during rest or secretagogue stimulation. Taken from Scheele and Palade.~

to 0.1% Triton X-100 (v/v) prior to enzyme assay or activation. Data are expressed as described for discharge of radioactively labeled proteins. Figure 5 shows the kinetics of discharge of three enzymes (amylase, lipase, and ribonuclease) and four zymogens (trypsinogen, chymotrypsinogen, procarboxypeptidase A, and procarboxypeptidase B) from pancreatic lobules incubated in vitro for 2 hr in the presence and in the absence of optimal doses of carbamylcholine. Two-dimensional IEF/SDS-gel electrophoresis can be used also to quantitate exocrine proteins discharged from pancreatic lobules into the incubation medium under conditions of rest and secretagogue stimulation. Proteins separated by the two-dimensional procedure are quantitated after Coomassie Blue staining by two-dimensional spectrophotometric scanning and computer analysis of the scanning data. 8 Scanning was performed on photographic reproductions of stained gels with an Optronics two-dimensional gel scanner with optical density measurements taken at 100-pm intervals. Measurements recorded on magnetic tape were analyzed on a Digital Equipment Corporation PDP-11170 computer using programs thai determined the cumulative densities within and fractional densities among the two-dimensional spots. By use of the methods described in this section, we have (a) measured the effects of secretagogues, 3 ions, '°,'~ cyclic nucleotides, '2 and ATP stores 3 on L0G. A. Scheele and A. Haymovits, J. Biol. Chem. 254, 10346 (1979). ~' G. A. Scheele and A. Haymovits, J. Biol. Chem. 255, 4918 (1980). ,2 A. Haymovits and G. A. Scheele, Proc. Natl. Acad. Sci. U.S.A. 73, 156 (1976).

28

SPECIALIZED

METHODS

[3]

protein discharge; (b) defined the role of intracellular and extracellular calcium during stimulus-secretion coupling10; (c) identified changes in cellular cyclic nucleotide levels during cholinergic and hormonal stimulation~2; and (d) outlined mechanisms for the appearance of both parallel and nonparallel discharge of secretory proteins in the exocrine pancreas. 8,13 ~3 G. A. Scheele, Am. J. Physiol. 238, 6467 (1980).

[3] H i g h - V o l t a g e T e c h n i q u e s f o r G a i n i n g A c c e s s t o t h e Interior of Cells: Application to the Study of Exocytosis and Membrane Turnover

By P. F. BAKER and D. E. KNIGHT The plasma membrane barrier greatly hinders experimental manipulation of the cytosolic environment in which all intracellular events take place. Various techniques are available to overcome this problem. These can be divided very roughly into two groups: those that permit the introduction of substances into cells, but not their removal, and those that permit more complete control over the intracellular environment. The first group includes both microinjection and fusion with a suitable membrane-bound carrier, and the second group includes internal perfusion and dialysis, partial or complete destruction of the plasma membrane by detergents, and more restricted breakdown of the plasma membrane permeability barrier by treatment with complement or brief exposure either to hypotonic solutions or to intense electric fields. Each has its advantages and disadvantages. The techniques of perfusion and dialysis offer the greatest experimental control but can be applied only to single relatively large cells. For smaller cells most of the available methods involve exposure to foreign chemicals (detergents, complement) with all the uncertainties that attend the use of unphysiological and often highly reactive reagents. Exposure to brief, intense electric fields provides a simple, rapid, and chemically clean method for gaining access to the interior of cells. Under certain conditions high-voltage techniques can be used to render the plasma membrane transiently permeable, whereas under other conditions the plasma membrane remains permeable for prolonged periods. This chapter describes the main features of the high-voltage technique and its application in particular to the problem of membrane turnover by exocytosis and endocytosis. METHODS IN ENZYMOLOGY, VOL. 98

Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181998-I