Zymogen Granules of the Pancreas and the Parotid Gland and Their Role in Cell Secretion

Zymogen Granules of the Pancreas and the Parotid Gland and Their Role in Cell Secretion Adrien R. Beaudoin and Giiles Grondin Departement de Biologie, Faculte des Sciences, Universite de Sherbrooke, Sherbrooke, Qukbec J l K 2R1, Canada

I. Introduction

The exocrine acinar cells from the pancreas and the parotid gland are adapted for the synthesis and secretion of digestive proteins. The secretory mechanisms of these acinar cells, particularly those of the pancreas, have been the subject of extensive studies. As early as 1898, Pavlov and associates (Pavlov, 1910) demonstrated the significance of the nervous mechanism of stimulation. The regulation of secretion was clarified to some extent by the discovery of peptide hormones such as secretin (Baylis and Starling, 1902) and pancreozymin (Harper and Raper, 1943). Microscopic studies had previously shown that cells of the exocrine glands, including those of the pancreas (Heidenhain, 1875), contain characteristic granules and that the number of these granules decreases when secretion is stimulated. By means of differential centrifugation of homogenate from dog pancreas, Hokin (1955) was able to isolate a fraction that contained chiefly zymogen granules (ZGs) and showed that this organelle had a higher concentration of proteolytic activity, amylase, and lipase than the whole homogenate. This observation was definitive in that it clarified the role of ZGs as storage sites of digestive enzymes in exocrine glands. With the advent of electron microscopy, autoradiography, and new cell fractionation, immunological, and biochemical techniques, these organelles could be studied in great detail. In this review we summarize some of the knowledge acquired on the pancreas and the parotid gland ZGs in the past two decades. Two other aspects, closely related to this subject, have been recently reviewed: One deals with the transport and secretion of digestive proteins (Beaudoin and Grondin, 1991 ;Grossman, 1988),whereas the other focuses on the composition of pancreatic juice (Beaudoin et al., 1989). Inremarional Review of Cvtology. Val. 132

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In Section I1 of this review, we consider the overall variations of the ZG compartment and the variations of the granule size under a variety of physiological conditions. In this task we were greatly assisted by the excellent review of Cope (1983). Then we examine the origin and fate of the ZGs as observed by cytochemical methods. In this section some of the concepts proposed by Farquhar and Palade (1981) have been outlined. In Section V we summarize the contribution of freeze-fracture techniques to the definition of the ZG membrane architecture. In Section VI the content of ZG is analyzed by immunocytochemical methods and the localization of secretory proteins is related to some current views on secretion. In the two following sections the electrophysiological properties of ZGs and the influence of the cytoskeleton on secretion are discussed. Finally, the observations presented in this review are integrated into a scheme of ZG maturation.

II. ZG Morphometry: Influence of Various Physiological Parameters on Granule Size

Like many other glandular tissues, the pancreas and the parotid gland process their macromolecular products by cisternal packaging and exocytosis (Cope, 1983; Case, 1978; Grossman, 1988; Beaudoin and Grondin, 1991). Their secretory products are segregated from the cytosol from the beginning and are transported through the cell and discharged, as a result of a series of fission-fusion reactions between adjacent endomembranous compartments (e.g., endoplasmic reticulum, Golgi complex, vesicles, secretion granules, and plasmalemma). As all of these compartments can be identified by the electron microscope, these tissues and the exocrine secretory process lend themselves to morphological quantification. Changes in compartments should reflect the passage of macromolecules through the cells. Thus, information concerning the formation, packaging, and discharge of secretory products can be obtained stereologically. Furthermore, analysis of the membrane envelopes themselves can provide information about membrane dynamics during the secretory cycle (Cope, 1983). Estimates of the size of parotid acinar cells vary between 550 and 1100 pm3, and those of the pancreas vary between 700 and 1700 pm3. Variations in cell size for a given species are probably attributable to differences in tissue-processing and analytical techniques, rather than to biological differences among strains. Although they look similar, significant morphological differences exist between pancreas and parotid exocrine acinar cells. These differences include the relative amounts of

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rough endoplasmic reticulum (RER) and the ratio of RER to secretion granules (Bolender, 1974; Amsterdam and Jamieson, 1974; Ferraz de Carvalho et al., 1978; Nevalainen, 1970; Bloom et ul., 1979). Indeed, pancreatic acinar cells have about twice the volume of the RER and about one-half the volume of secretory granules of parotid acinar cells. These values may reflect the greater synthetic capability of the pancreatic acinar cell and suggest that either its products are more highly condensed than those of the parotid gland or that the pancreas cells store less of their output in granular form than do parotid cells (Cope, 1983). In addition to secretory granules, one finds in the Golgi area various types of vesicles, including some minigranules (Beaudoin and Grondin, 1987). There are also small vesicles in transit between compartments; that is, vesicles shuttling back and forth between the RER and the Golgi apparatus, between the Golgi apparatus and the granules, and between the Golgi saccules and the plasmalemma. As the diameter of many of these vesicles is sometimes about the same order as section thickness, their numbers are difficult to quantify. One of the major factors that affect the number and size of secretory granules of these exocrine cells is their physiological state. In the rat pancreas the ZGs normally occupy 5-20% of the cell volume. Their number is generally higher after a short period of fasting. The mouse pancreas does not follow this pattern. Indeed, a 50% reduction in granule content after a 24-hr fast has been reported (Carlsoo et a / . , 1974). This is accompanied by a reduction in protein synthesis and a collapse of RER cisternae. Starvation causes crinophagy, or autolytic digestion of granules (Nevalainen and Janigan, 1974). In the rabbit values as high as 40% of the cell volume have been reported (Cope and Williams, 1973a; Bedi et af., 1974). In contrast, feeding causes a marked reduction (50%) in the granule compartment in rat pancreas as compared to fasted controls (Ermak and Rothman, 1981). Similar values were reported for the frog pancreas after 4 hr of feeding (Slot and Geuze, 1979). A comparable reduction in the granule compartment (40%) in rabbit parotid gland was observed within 1 hr of feeding (Carlsoo ef ul., 1974). The average size of the ZGs is also greatly influenced by stimulation (Fig. 2a-c). In the rat pancreas, in a resting state, the granule population shows approximately a gaussian size distribution with respect to diameter (0.7 and 1.0 pm) (Nadelhaft, 1973; Liebow and Rothman, 1973; Beaudoin et al., 1984; Aughsteen and Cope, 1987). Some studies reported a bimodal distribution of granule diameters with a minor peak in granule diameter around 0.5 and 0.6 pm and a major peak between 0.8 and 1.0 pm (Sato and Take, 1975). Yoshimura (1977) noticed that, among Wistar rats, the pancreata of some had two populations of granules and others had only one.

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Intriguingly, this bimodal distribution was also observed in stimulated pancreas, The average size of the small-granule population corresponds roughly to that of the minigranules (0.2-0.3 pm) described by Beaudoin and Grondin (1987). We have found that these minigranules are not present in all cells on a given section of pancreas. Secondly, their occurrence is variable from one species to another, being, for example, more frequent in pigs than in rats. In the rat azaserine-induced pancreatic tumor, some cells are filled with minigranules, whereas others contain a normal population of granules (Beaudoin et al., 1986b). A shift in the size-frequency distribution of granules has been reported in the rat pancreas after either feeding (Ermak and Rothman, 1981) or hormonal stimulation in vivo (Beaudoin et al., 1984; Aughsteen and Cope, 1987). Ermak and Rothman (1981) proposed that the number of granules discharged is not always in proportion to the amount of secretory material released by the gland. They suggested that granules shrink as their contents are released by diffusion-like processes. Beaudoin et al. (1984) proposed that this shift is, rather, due to a selective discharge of large granules and the concomitant formation of smaller new granules, especially after hyperstimulation. This view is shared by Aughsteen and Cope (1987). One must be cautious when interpreting data on granule size, since it could be influenced by many physiological and methodological factors. For example, cells in the vicinity of Langerhans islets are larger and contain more granules than are usually encountered in the rest of the tissue (Bendayan, 1984). After stimulation the so-called “periinsular cells” appear to retain more granules than cells more distant from the islets or the “teleinsular cells.” In addition some heterqgeneity has been observed among the teleinsular acini. The accuracy of granule size determinations is greatly reduced in stimulated glands, because the acini do not respond in synchrony to secretagogues. Indeed, differences in the number of granules was observed after feeding or following stimulation of the pancreas by pharmacological doses of secretagogues, some acini being more rapidly depleted of their granules than others (Phaneuf et al., 1985; Roberge et al., 198 1). Intriguingly, autoradiographic studies on the incorporation of [3H]leucineinto proteins of acini isolated form the pancreas of a fasting rat demonstrate great variations in labeling among acini (see Fig. 1). Another important parameter which may have been overlooked in the morphometrical studies mentioned above is circadian variation of both granule size and number (see Uchiyama and Saito, 1982; Uchiyama and Watanabe, 1984), which occur even under fasting conditions (A. R. Beaudoin, G. Grondin, and P. St-Jean, unpublished observations). The size distribution of the ZGs in the parotid gland cells shows some points of similarity with that observed in the pancreas. A unimodal size distribution of granules has been found in the starved rabbit (Cope and

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FIG. 1 Autoradiography illustrating the variations in the rate of protein synthesis among the acini isolated from the pancreas of a fasting rat. Proteins were pulse-labeled for 5 min with [3H]leucine and chased with cold leucine for 90 min in v i m . Note the differences between acini in both the density and localization of silver grains, indicating different rates of processing. Compare the density in l a (solid and open arrows) and localization in l b (arrowheads and arrow). Acini were prepared according to Roberge er ul. (1981). and autoradiography was carried out according to Kopriwa and Leblond (1962).

Williams, 1973b, 1981; Bedi et al., 1974), the mean granule size being close to 1 pm. As was reported for the pancreas, bimodal distribution in granule size has been observed in the rat parotid gland (Itoh, 1977). After feeding the cells of the rabbit parotid gland showed a greater reduction in the number of granules than in the total volume of granules per cell (Bedi ef al., 1974), indicating a preferential discharge of older granules that were smaller and more condensed than usual. Simson et al. (1974) observed the same phenomenon in the rat parotid gland following repeated stimulations with isoproterenol. Following stimulation in uiuo both the parotid gland and the pancreas demonstrate rapid granule depletion, whereas the pattern of regranulation is much slower (Amsterdam et al., 1969; Nevalainen, 1970; Geuze and Kramer, 1974; Cope and Williams, 1980, 1981). New granules usually begin to appear about 3-4 hr after the onset of secretion, then their volume rises sharply for the next 4-6 hr, during which time the rate of accumulation falls off, with complete restitution of granule stores after 12-16 hr (Cope and Williams, 1980, 1981). The size distribution of granules during regranulation has been analyzed for the rabbit parotid gland (Cope and Williams, 1981). Initially, the gland produces small granules relatively quickly, so that by 8 hr the cells con-

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tained nearly twice as many granules as the unstimulated controls. These, however, were only about two-thirds the volume of granules. Thereafter, the total volume of secretory material per cell continued to rise, but the number of granules per cell dropped to a number similar to that in the control group. A similar pattern of granule maturation was reported in the rat parotid gland, except that the transformation to larger granules, which occurs between 8 and 12 hr in the rabbit, was delayed in the rat (Itoh, 1977). Cope (1982) suggested that the gland first produces small granules with a mean diameter of about 0.5-0.6 pm, but as the gland fills up and the granules come into close contact, they fuse to form larger ones. The studies reported above show that, during fasting, there is generally an accumulation of large granules in both the pancreas and the parotid gland. A study of the pancreas suggests that the larger granules would concentrate in close vicinity to the apical plasma membrane. When the pancreas is stimulated either by feeding or by administered hormones, a reduction of mean granule diameter is generally observed, which reflects a selective exocytosis of large granules. If stimulation is maintained, newly formed granules are smaller, indicating that secretagogues exert an influence on the packaging process (see Fig. 2).

111. Fate of the ZG Membrane after Exocytosis: A Recycling Process

During exocytosis the ZG membrane fuses with the luminal plasmalemma, allowing the discharge of granule content. Occasionally, one could observe some membranous material in the gland lumen, suggesting that pieces of membrane are also expelled during secretion. In this respect Battistini et al. (1990) recently showed that, in response to stimulation, some particulate y-glutamyl transpeptidase is found in pancreatic juice. However, it cannot be excluded that this enzyme, which is believed to be present in ZG membrane of the rat pancreas, can also be released from ductal cells, as proposed by Yasuda et al. (1986). Despite this observation, which may involve minute amounts of membrane, it is believed that the granule membrane is almost entirely withdrawn into the cell by endocytosis (Kalina and Robinovitch, 1975; Herzog and Farquhar, 1977; Oliver and FIG. 2 Influence of stimulation on the ZG and the Golgi apparatus (Go) of the rat pancreas. (a) Pancreas after an overnight fast. (b) Pancreas after two injections of urecholine. (c) Pancreas after infusion of a cocktail of secretagogues. Note the swelling of the Golgi apparatus and the reduction in both number and size of the residual ZGs (arrowhead). Lu, Acinar lumen. Experimental conditions are as described by Beaudoin et al. (1984).

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Hand, 1978; Farquhar and Palade, 1981). Amsterdam et al. (1969) reported an increase in the number of smooth vesicles within the cytoplasm of the rat parotid gland during secretion. Geuze and Kramer (1974) made counts of vesicles following pilocarpine-stimulated secretion on the rat pancreas. They also reported increases in the number of coated vesicles per square micron of apical cytoplasm, which reached a peak 3 hr after the onset of secretion. However, it is unlikely that this peak marks the period of maximum membrane withdrawal. Indeed, exocytosis and endocytosis are usually considered to occur more or less simultaneously (Kalina and Robinovitch, 1975; Tamarin and Walker, 1976), with a peak in the number of vesicles about 2 hr after most of the granules have discharged their content. This peak in the numerical density of vesicles appears to coincide with the time that cytoplasmic volume is at its lowest level (Nevalainen, 1970). Cope (1982) suggested that this peak does not reflect a real increase in the number of vesicles per cell, but simply the concentration of vesicles into a smaller cytoplasmic volume. As reviewed by Farquhar and Palade (1981), results of early morphological studies with electron-dense tracers had established that, after exocytosis, membrane is recovered intact (i.e., exocytosis is coupled to endocytosis). However, because the tracers were found to be subsequently transported primarily or exclusively to lysosomes, it was concluded that the recovered surface membrane was degraded. The idea that the secretory granule membrane was recovered and degraded in lysosomes, rather than being reutilized or recycled, prevailed for some time until the studies by Holtzman et a / . (1977). In retrospect it is clear that these early studies were limited by the fact that the tracers used (usually horseradish peroxidase and native ferritin) were charged protein molecules that acted primarily as content markers. Hence, they were useful for following the fate of the vesicle contents, but not that of the vesicle membrane. Several groups (Farquhar, 1978; Herzog and Farquhar, 1977; Ottosen et al., 1980; Wilson et al., 1981; Herzog and Miller, 1979; Herzog and Reggio, 1980) later showed that, after exocytosis, the retrieved membrane is funneled through the Golgi complex. Evidence was obtained by using several tracers that had not been utilized before, that is, dextrans (uncharged relatively inert polysaccharide molecules) and cationized ferritin (which is known to bind electrostatically to membranes and, therefore, act as a membrane marker). As mentioned by Farquhar and Palade (1981), the most likely explanation for the bulk of this traffic in secretory cells is that it represents the recovery of granule membranes to be reutilized in the packaging of secretory granules; that is, it represents a recycling of granule membrane. Fate of the ZG membrane has also been studied during pancreas development. Indeed Carneiro and Sesso (1987) performed a morphometric

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evaluation of ZG membrane transfer to Golgi saccules following exocytosis in pancreatic acinar cells from newborn rats. More specifically, acinar cells from unfed newborn rats and suckling rats for 4,8, and 16 hr were examined morphometrically in semi- and ultrathin sections. In the cells of the unfed newborn rats numerical and volume densities of the ZG and the volume of the Golgi apparatus are, respectively, the highest and the lowest observed during peri- and postnatal life. Cisternae of the RER appear irregularly disposed among the ZG. Once feeding starts, cytoplasmic volume becomes progressively reduced until the 16th hour, owing to sustained exocytosis of ZG contents. The decline in the numerical density of ZGs between 0 and 4 hr revealed the minimum number of ZGs exocytosed in the first 240 min. The sum of the membrane surfaces measured in the various subcellular compartments [RER, condensing vacuoles (CVs), Golgi saccules, Golgi apparatus-associated microvesicles, “other structures,” apical and basolateral plasmalemmae, and mitochondria] did not vary significantly in the various groups of rats. After 4 and 8 hr the net amount of cellular ZGs is sufficient to account for the expressive increase in membrane surface occumng at these times in CVs, Golgi saccules, and microvesicles. The curves showing the membrane surface decrease in ZGs and the increase in the Golgi saccules appear to express a precursorproduct relationship. The results of topochemical reactions are consistent with the interpretation that part of the ZG membrane internalized after exocytosis, induced by alimentary stimulus, is reused to expand and/or form trans [thiamine pyrophosphatase (TPPase)-positive] and trans-most [acid phosphatase (AcPase)-positive] Golgi saccules. In the latter study there was no significant increase in apical plasma membrane in the first hours of suckling, despite a pronounced decrease in ZG content. The lack of increase in apical plasma membrane suggested an immediate retrieval of the ZG membrane (Carneiro and Sesso, 1987). However, some observations by Romagnoli (1988), on lobules of adult rats in uitro during a 10-min incubation period, showed a significant increase in apical surface upon stimulation. Indeed, the apical plasma membrane surface area and the number of ZGs less than 20 nm from the apical plasma membrane significantly increased and were directly correlated with increases in secretion. The diameter of ZGs decreased when lobules were stimulated by lop6M carbamylcholine, but increased at lo-’ and lop5 M concentrations as compared with controls (see Fig. 2c). In the parotid gland exocytosis and the ensuing membrane retrieval are restricted to a clearly delimited and easily recognizable portion of the plasmalemma and are not dispersed over the entire cell surface. Second, optimal stimulation of acinar cells with P-adrenergic agonists results not only in the rapid discharge of most secretion granules, but also in a clear

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enlargement of the acinar lumen (Amsterdam er al., 1969; Batzri et al., 1971). Such a situation, which is at variance with that observed in the exocrine pancreas, indicates that, at the luminal surface, membrane input (exocytosis) and output (retrieval) are not kept in balance during sustained stimulation. The obvious discrepancy between the large membrane surface disappearing from the granule pool and the considerable, though not massive, increase of the luminal plasmalemma suggests that the two processes are not completely dissociated (Koike and Meldolesi, 1981). In the experiments by Amsterdam et al. (1969), the acinar lumen remained clearly enlarged as long as 2 hr after isoprenaline injection, whereas in freeze-fracture studies membrane patches of the granule type were detected at the luminal surface until the fourth hour (Koike and Meldolesi, 1981). Moreover, a slow time course of membrane retrieval can be deduced from the careful stereological data in the rabbit parotid gland (Cope, 1983). Amsterdam et al. (1969) showed that the lumen perimeter increased markedly in the first 30 min of isoprenaline stimulation in the rat parotid gland. This increase corresponded well with the membrane added by exocytosis. By 2 hr the acinar lumen perimeter had already returned to normal values, with a massive increase in the number of apical vesicles. From the experiments described above it is clear that time after stimulation is a critical parameter when one wants to interpret the changes in acinar lumen in relation to ZG exocytosis. It appears that, at short term after stimulation in both the parotid gland and the pancreas, there is an enlargement of the lumen then the process of membrane retrieval is initiated and gradually catches up with the process of membrane addition by exocytosis. However, an equilibrium between the two processes is apparently reached more rapidly in pancreas than in parotid acinar cells. The intensity and duration of stimulation are major parameters that could shift this equilibrium one way or the other. The alterations of cell compartments associated with pancreas secretory activity are summarized in Table I.

IV. ZG Cytochemistry: Relationship between the Golgi Apparatus and ZGs

Cytochemistry has proved to be an invaluable tool to define the biochemical characteristics of the Golgi apparatus, and the relationship of this organelle to secretory granules, in a variety of cells. The central role played by the Golgi apparatus in the formation of secretory vesicles was recognized long ago by light microscopists. As mentioned by Farquhar and Palade (1981), early electron-microscopic studies (Sjostrand and Hanzon, 1954; Haguenau and Bernhard, 1955; Farquhar and Rinehart, 1954) noted

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ZYMOGEN GRANULES TABLE I Alterations of Cell Compartments Associated with Secretory Activity in the Pancreas"

Experimental conditions ~

Fasting

Cellular compartments cis-Golgi saccules trans-Golgi saccules and the trans-Golgi network trans-Golgi area microvesicles Condensing vacuoles ZGs Endocytic vesicles Acinar lumen

Normal Normal Few Few (large) Many (large and dark) Few Small (few microvilli)

Stimulated Normal Increased (volume and membrane surface) Many Many (small) Few (small, pale) Many < I hr enlarged, with few microvilli 1-3 hr, small, with many microvilli >3 hr, normal

" Unless otherwise stated, the fasting condition corresponds to a deprivation of food for 16-36 hr. The stimulated condition corresponds to intense stimulation for about 3 hr, by either intravenous infusion of a secretagogue or repeated stimulation. As demonstrated by Phaneuf er a / . (1985). some acini appear to be insensitive to secretagogues following short-term stimulation.

the close association between secretory granules and Golgi elements. Shortly thereafter, several investigators (Farquhar and Wellings, 1957; Palay, 1958) published electron micrographs in which material resembling the contents of secretory granules was clearly recognized within Golgi elements. Subsequent morphological and autoradiographic studies (reviewed by Whaley, 1975; Beams and Kessel, 1968; Farquhar, 1971; Bainton et al., 1976) established that, in most cell types, the concentration and packaging of secretory products usually occur in the dilated rims of the trans-most cisternae. However, in a few cell types (e.g., the exocrine pancreas and the parotid gland), concentration takes place in specialized condensing vacuoles, which are separate from the stacked cisternae. In either case concentration results in the production of a storage granule with a condensed content and a membrane acquired in the Golgi complex. The fact that concentration commonly takes place in the dilated ends of the Golgi cisternae raised the intriguing question of how concentration is brought about in the dilated ends of a continuous compartment. As re-

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viewed by Farquhar and Palade (1981), the first information on this problem came from the experiments by Jamieson and Palade (1971), who showed that concentration in both condensing vacuoles and ZGs was maintained in situ in the absence of ATP synthesis. The findings led to the conclusion that concentration is not dependent on a continuous expenditure of energy. AcPase and TPPase cytochemistries have provided some insights into the details of the secretory pathway involving the CVs of the pancreas and the parotid gland (Novikoff et al., 1977; Hand and Oliver, 1984; Fujita and Okamoto, 1979; Beaudoin et al., 1983; Rambourg et al., 1988). These studies showed that CVs were formed from the trans-Golgi apparatus and the Golgi-associated endoplasmic reticulum-lysosome system (GERL). This region of the cell is included in what is now known as the trans-Golgi network. Novikoff et al. (1977) defined the GERL concept as a lysosome formation system. According to them, the GERL consisted of smooth endoplasrnic reticulum, multivesicular bodies, small vesicles, coated vesicles, and lysosomes. From their observations they proposed that in the pancreatic cell the nascent secretory vesicles or CVs are expanded cisternal portions of the GERL, a structure originating from the endoplasmic reticulum. The studies by Hand and Oliver (1977) confirmed the role of the GERL in granule formation in exocrine cells. Their observations suggested that the GERL might be derived from the inner Golgi saccule and indicated a close relationship between the GERL and the endoplasrnic reticulum. They also mentioned that endoplasmic reticulum-GERL continuities were encountered only rarely, and the marked differences in enzyme content suggested that the transfer of proteins is probably minimal. Later, Fujita and Okamoto (1979) reported that AcPase and TPPase were both present in the trans-Golgi saccule, in rigid lamellae, on CVs, and on coated vesicles in the trans area of the Golgi apparatus. They proposed to consider the GERL of Novikoff et al. (1977) as part of the Golgi apparatus. Beaudoin et al. (1983) reported cytochemical distributions of AcPase, TPPase, and ATP-diphosphohydrolase activities on thin sections of rat pancreas and on isolated ZG membranes. AcPase was found in the rigid lamellae separated from the Golgi stacked saccules, CVs, and the transGolgi saccules, but it was not detected in purified ZG membranes. TPPase was detected in trans-Golgi saccules, purified ZG membrane and occasionally ZG membrane in situ, and the plasmalemma of the acinar cell, but it was not seen in CVs. The ATP-diphosphohydrolase activity had a distribution similar to that of TPPase. These observations illustrated the similarity between the trans-Golgi saccules and the membrane of mature ZGs and further corroborated the view that the trans-Golgi saccules are involved in the formation of secretory granules. The interpretation of the cytochemical observations is complex, as illustrated in Fig. 3. For example,

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acid phosphatase is highly reactive in CVs, but is undetectable in mature ZGs; Trimetaphosphatase is not detectable in the trans-Golgi saccules or the GERL (the trans-Golgi network), but is present in mature granules and acinar lumen. Another example is the NADPH-phosphohydrolase which is detected in the medial saccules of the Golgi stack. It is undetectable in CVs and ZGs, but is present in the acinar lumen. It is noteworthy that some vesicles that appear to be “minigranules” do not seem to bear any type of phosphatase activities. In the parotid gland the cytochemical localizations of AcPase and TPPase in the Golgi apparatus, the GERL, and forming granules are essentially comparable to those in the pancreas. As reported by Hand and Oliver (1984), the Golgi apparatus consist of several stacks of four to six saccules, numerous vesicles, and immature secretory granules of variable sizes and densities located near the trans face. The saccules at the cis face tend to be dilated and irregular, while those at the trans face are narrower and more regular. Reaction product is usually present in one or two trans-Golgi saccules after incubation for TPPase activity. No difference was noted in the localization of reaction products when TPPase, uridine diphosphate, or inosine diphosphate was used as substrate. The GERL was identifiable as short narrow saccule adjacent to, or, more frequently, separated from, the trans-Golgi saccule. Occasionally, continuities between the GERL and an immature granule were observed. The GERL and some of the immature secretory granules contained reaction product after incubation for AcPase activity. However, the reactivity of the GERL in control acinar cells was variable, and in many instances little reaction product was present under their fixation and incubation conditions. The GERL and the immature granules were usually unreactive for TPPase. Modified cisternae of RER, lacking ribosomes on the surface adjacent to the GERL, were frequently observed paralleling the GERL and the immature granules. Perhaps one of the most significant cytochemical observations were made when these cells were stimulated. Discharge of mature secretory granules was complete within 1 hr after isoproterenol injection, but immature granules in the Golgi region or near the lumen were not released. At early times (1-5 hr) after isoproterenol administration, AcPase activity was markedly increased in the GERL and the immature secretory granules compared to uninjected controls. The GERL increased, and numerous continuities with immature granules were observed. Reaccumulation of mature secretory granules was first evident at 5 hr and was almost complete by 16 hr after isoproterenol injection. TPPase activity, normally restricted to the trans-Golgi saccules, was frequently present in immature granules during this period. Narrow saccules resembling the GERL, occasionally in continuity with immature granules, also contained TPPase

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reaction product. By 16-24 hr after stimulation, the activity and distribution of AcPase and TPPase were similar to those of control cells. These results demonstrate the dynamic nature of the Golgi apparatus and the GERL in parotid acinar cells and emphasize the close structural and functional relationship between these organelles. Along the same line Novikoff et al. (1977) also reported the effect of stimulation on AcPase in both pancreas and parotid acinar cells. In resting cells no phosphatase activity was present in mature ZGs, but high levels of activity were seen in smaller granules, located close to the Golgi apparatus, corresponding to early secretory granules. When the discharge was induced by pilocarpine, small AcPase-rich granules were seen at the apical pole of the cell. Their interpretation was that newly formed granules were lysosomes. Some recent observations by Sesso et af. (1990) have shown that there is a microvesicle budding process from the surface of CVs during conversion to mature ZGs. Along this process the cortex of the CVs, as well as vesicles that are budding, show AcPase reaction products. These observations indicate that, under resting conditions, there is a maturation process that removes AcPase and perhaps other lysosomal enzymes from the immature secretory granules. Under intense and prolonged stimulation this maturation process would be impaired, as evidenced by the presence of smaller granules containing AcPase, but the identity of these secretory vesicles must be confirmed by combined immunocytochemistry and cytochemistry.

V. Freete-Fracture Observations: Evidence That the ZG Membrane Undergoes Some Major Topographical Alterations of Protein and Lipids Fusion of the ZG membrane with the plasma membrane is normally restricted to the portion of the cell surface facing the secretory lumen. By the use of freeze-fracture techniques, De Camilli et al. (1974) compared the secretory portion of the plasma membrane to the nonsecretory portion and found some important differences. In general, after cleavage, most of the FIG. 3 Cytochemistry of secretory vesicles in the apical cytoplasm of pancreatic acinar cells. (a) Acid phosphatase. Condensing vacuoles (CVs) are positive; ZGs and minigranules (arrow) are negative. (b) Trimetaphosphatase. Note the patchy distribution of the stain and its concentration at budding sites (arrowheads) on the ZG surface. (c) P-NADPHase. Note that CVs and ZGs are negative, whereas the acinar lumen (Lu) is highly reactive. (d) P-NADPHase precipitates (arrowhead) in the acinar lumen. Go. Golgi apparatus. Experimental conditions are as described by Beaudoin el ul. (1985).

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membrane particles are associated with the half of the membrane left frozen to the cytoplasm (the P face). The face of the half-membrane left frozen to the extracellular space (the E face) bears fewer particles per unit of area. In pancreatic acinar cells the typical P face pattern can be found only in the basal and lateral portions of the plasma membrane (including the regions close to the tight junctions and within the chambers delimited by their network). However, over the entire luminal area there are far fewer particles per unit of area of the P face. The transition is sharp across the inner continuous line of the tight junction delimiting the lumen. On the E face the number of particles is always small, with no clear difference between the luminal and lateral portions of the plasma membrane. The limiting membranes of the ZGs stored within the cells bear much fewer particles per unit of area than the membranes of other cytoplasmic organelles. The distribution is, again, asymmetrical, with more particles in the half-membrane left frozen to the surrounding cytoplasm (P face). Hence, if we look at the particle patterns, the E face of the ZG membrane resembles the E face of the whole plasma membrane, while the P face of the ZG membrane looks like the luminal plasma membrane P face. This observation is expected, since during exocytosis the P leaflet of the ZG membrane becomes continuous with the P leaflet of the luminal plasma membrane and the same occurs with the two E leaflets. Jamieson (1975) and De Camilli et al. (1976) later confirmed the polarity of the external fracture leaflet with less than half the number of particles as compared to the plasmic fracture leaflet. Cabana et al. (1981) observed a comparable distribution on purified ZGs in uitro. Indeed, ultrastructural examination of the ZG membrane by rapid-freezing and freeze-fracture techniques revealed that the E leaflet has a highly textured subparticle background with a significantly lower frequency of intramembrane particles (IMPs) (44per square micron), showing diameters of 9-18 nm and a shift to larger IMPs (12.3 nm). Two hitherto undescribed types of IMPs were found on both membrane leaflets. First, a population of 13-nm particles with an electron-lucent center, or “pore,” the most frequent type on the E face (26%),and a second population of large IMPs (15 nm) characterized by a large pore (5 nm in diameter), subdivided by a delicate crossshaped structure. Under alkaline conditions, at pH 8.2, ZG lysis occurs rapidly. Membrane ghosts thus obtained were rapidly frozen or suspended in dextran and filtered immediately. Transmission electron microscopy showed many opened ghosts with adhering amorphous material and numerous small vesicles near or still attached to openings in the ghosts. Freeze-fracture preparations also showed that granule lysis is accompanied by major alterations, suggesting a system that controls the topological distribution of membrane particles. More recently, Beaudoin et at. (1988) demonstrated that the isolated ZG

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membranes of the rat pancreas can be subfractionated on a sucrose gradient. Four discrete types of membranes corresponding to densities of I . 105, 1.085, 1.075, and 1.020 were obtained, designated types A, B, C, and D, respectively, and characterized by both morphological and biochemical criteria. Electrophoretic profiles showed that they contain the same protein bands, but in different proportions. Type A membranes comprise four major bands corresponding to 80,69,54, and 20 kDa, being in higher concentrations. Types B and C contain three major bands at 80, 54, and 20 kDa, whereas type D comprises only two major bands at 69 and 54 kDa; the latter polypeptide, corresponding to ATP-diphosphohydrolase activity, is present in all four types of membrane. Freeze-fracture of rapidly frozen membranes, followed by transmission electron microscopy (TEM), showed that type A membranes are large superimposed sheets of membranes with amorphous material between them. The surface area of these sheets corresponds roughly to the surface of intact ZGs, with a few IMPs (distributed at random or in small aggregates on large smooth fracture planes). Types B and C exhibited a totally different aspect, forming closed vesicles about the size of small ZGs, with few IMPs (distributed at random or in small aggregates on smooth fracture planes). Type D membranes were small vesicles, with no detectable IMPs on relatively smooth fracture planes. Various explanations can be suggested for the existence of different types of ZG membranes. There could be a maturation process involving changes in the ratio of lipid to protein, as well as the proportions of the various proteins; there could also be different populations of ZGs with different proportions of the same membrane proteins; the isolation procedure could have separated various domains of the in situ ZG membrane; or finally, it could have induced an artificial phase separation in the ZG membrane, followed by fragmentation. Some information regarding the alteration of architecture of the newly formed granules with time was obtained by Sesso et al. (1980), who conducted a freeze-fracture and thin-section study of CVs in rat pancreatic acinar cells in the suckling rat pancreas. The CVs showed what appeared to be a biphasic evolution. During the first stage the CVs enlarge, accumulating contents of rather low electron density. Fracture faces with irregular patterns, possibly the result of fusion (pinching off) of microvesicles with (from) the CVs, were occasionally encountered. The infrequency of such images indicates that fusion-fission during the growth stage must be a rapid event. One common type of surface irregularity is gibbosities (or convexities) in the P fracture face, with complementary images on the E fracture face. The significance of these irregularities, which are in apparent discordance with the theory of microvesicular transport, is unclear. By the end of the growing period, the CVs are large and smooth surfaced (referred to as CVd, with contents of intermediate elec-

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tron density (between that of the initial growing stage and that of the mature ZGs). The number of intercalated particles on both the large irregularly surface (CV,) and large smooth-surfaced CV (CV,) membranes is high and comparable to that of the Golgi saccule and the endoplasmic reticulum membranes. During the second stage the smooth-surfaced CVs undergo a volume reduction associated with a progressive increase in the electron density of their contents, thus becoming ZGs. Concomitant with size reduction, the number of intercalated particles in the membranes with CVt diminishes markedly. The process of membrane retrieval appears to be accomplished selectively by pinching off coated microvesicles, heavily studded with intercalated particles. More recently, Sesso er al. (19%) reported that, as the CVs mature into ZGs, they lose about 80% of their IMPS. Some preliminary results led them to suggest that lysosomal enzymes would be sorted out of CVs via mannose 6-phosphate receptors by microvesicular budding, as the CVs are converted to ZGs (Sesso et af., 1990). Using the lectin-gold technique, Kan and Bendayan (1989) studied the distribution binding sites of Helix pomatia lectin on thin sections and freeze-fractured preparations of rat pancreas submitted to fracture label. On thin sections of acinar cells, whereas the content of the ZG was negative or weakly labeled, the limiting membrane displayed a high degree of labeling. In the Golgi complex labeling by the lectin was localized on the trans saccules and the limiting membrane of the CVs. The latter appeared to be more intensely labeled than the membrane of the ZGs. Intense labeling by the lectin was also observed along the microvilli and the plasma membrane. In contrast to the weak labeling of the ZG content, labeling of the acinar lumen was intense. Fracture-label preparations revealed preferential partition of lectin binding sites to the exoplasmic half of the ZG and plasma membranes. The population of ZGs was, however, heterogeneous with respect to the degree of labeling; the exoplasmic fracture face of the plasma membrane was intensely and uniformly labeled, while the protoplasmic membrane halves were only weakly labeled. These observations were further confirmed and extended by the thinsection fracture-label approach. In addition favorable profiles of thin sections of freeze-fractured ZGs showed that the labeling was not associated with the external surface of the limiting membrane, but, rather, localized over the exoplasmic fracture face. Kan and Bendayan (1989) concluded that ( 1 ) ZGs contain little lectin-binding glycoconjugates; (2) lectin binding sites are preferentially associated with the exoplasmic half of the ZG and plasma membranes, and (3) the limiting membrane of the immature CVs carries a greater number of lectin binding sites than that of the mature ZGs. The latter, in turn, constitute a heterogeneous population with respect to labeling density. These results support the current view

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that glycoconjugates are directed toward the lumen in secretory granules, but become external to the cell surface after fusion of the secretory granule membrane with the plasma membrane. Also, the results reflect membrane modifications during the maturation process of secretory granules in the exocrine pancreas, in which glycoproteins are removed from the limiting membrane of the granule to become soluble and secreted with their content. Cholesterol distribution is another aspect of the architecture of the ZG membrane. Indeed, Orci et al. (1980) carried out freeze-fracture studies using filipin, an antibiotic that binds to membrane cholesterol and 3phydroxysteroid and produces deformations of the membrane. They demonstrated an asymmetrical distribution of these sterols in the plane of the membrane of both ZGs and CVs. Intriguingly, they showed a change in the polarity of filipin-induced deformations associated with the conversion of CVs into ZGs. If one assumes that the observed protuberances correspond to cholesterol molecules, then the latter would be concentrated on the P face in CVs, whereas in the mature ZGs the E leaflet would be enriched in cholesterol. When exocytosis occurs, it would end up on the E face of the luminal plasma membrane. There is no reason to believe that these observations were artifactual; therefore, this rapid transfer of cholesterol from one layer to the other raises some questions. First, at what precise moment does this transfer of cholesterol occur? Second, why does it occur? Freeze-fracture observations performed in our laboratory on isolated ZGs could provide a possible answer to some of these questions. Indeed, when we exposed ZGs to filipin, most of the ZGs showed a clear concentration of protuberances on the E leaflet, as reported by Orci et al. (1980). However, we noticed some small buddings at the surface of the isolated ZGs. These buddings were apparently filled with cholesterol, as evidenced by the protuberances and pits produced by the filipin treatment. At these sites the planar structure of the membrane was totally disrupted. From a thermodynamical viewpoint such an arrangement could greatly facilitate the transfer of cholesterol from the P layer to the E layer during the conversion of CVs to ZGs. Observations of buddings at the surface of the CVs and immature granules by scanning electron microscopy and on thin sections suggest that the same phenomenon also exists in uiuo (see Naguro and Lino, 1989). The reason that this would occur remains speculative. However, such a transfer of cholesterol from one layer to the other results in an increase in the fluidity and fusogenic properties of the P layers from both ZG and luminal plasma membranes. Information regarding the fusion process itself has been obtained by freeze-fracture studies. Indeed, De Camilli et af. (1974) examined the influence of stimulation on the distribution of IMPS in three exocrine

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glands: pancreas, parotid, and lacrimal. They found that the distribution of IMPs on the fracture faces of the luminal plasmalemma appeared random before stimulation. However, after injection of secretagogues, IMPs were rapidly cleared from the areas of granule-plasmalemma apposition in the parotid cells and especially the lacrimocytes. In the latter, the cleared areas appeared as large bulges toward the lumen, whereas in the parotid cells they were less pronounced. Exocytotic openings were usually large, and the fracture faces of their rims were covered with IMPs. In contrast, in stimulated pancreatic acinar cells the IMP distribution remained apparently random after stimulation. Exocytoses were established through the formation of narrow necks, and no image that might correspond to early stages of membrane fusion was revealed. Within the cytoplasm of parotid and lacrimal cells (but not in the pancreas), both at rest and after stimulation, secretion granules were often closely apposed by means of flat circular areas, also devoid of IMPS. In thin sections the images corresponding to IMP-free areas were close granule-granule and granule-plasmalemma appositions, sometimes with focal merging of the membrane outer layers to yield pentalaminar structures (Tanaka et al., 1980). Finally, an internal reticulation associated with the isolated ZG membranes has been put in evidence by the freeze-fracture studies by Cabana et al. (1981). Moreover, this reticulation was seen after washing the membrane with a carbonate buffer at pH 11.O. Such a procedure removes all the IMPs and major proteins, except one from the membrane. Electrophoretic analysis led these authors to conclude that this protein was GP2. More recent results give strong support to their conclusion, as discussed in Section VII.

VI. ZG lmmunocytochemistry and the Concept of Nonparallel Secretion

Immunocytochemistry has brought some major information about the distribution of zymogens in the acinar cell of the pancreas which is related to the concept of nonparallelism, well known to pancreatologists. This concept has been the subject of debates and confrontations for many years. It is not our intention to reanimate these discussions here, but, rather, to simply point out the contribution of some immunological techniques to the explanation of the secretory process. In simple words nonparallel secretion of digestive enzymes corresponds to changes in the relative proportions of the different secretory proteins (e.g., a-amylase, chymotrypsinogen A, and lipase) which occur in response to different physiological or pharmacological stimuli.

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According to this definition, nonparallelism presupposes the existence of different pools of secretory digestive enzymes in the gland. These pools of zymogens could potentially be localized at different levels of the gland organization; thus, they could correspond to different populations of granules within cells, acini, or different areas of the gland. In addition to these possibilities, Rothman (1975) proposed the existence of a cytoplasmic pool of enzyme in the pancreas. As early as 1954, Marshall (1954) studied the localization of four enzymes in the bovine pancreas by immunofluorescence. He then reported that the distributions of chymotrypsinogen and procarboxypeptidase could be determined to a resolution of less than 0.5 pm. His results indicated that, at this level of organization, the acinar cells of the resting pancreas are alike with respect to zymogen synthesis and storage. He did not find any specialization among the cells, nor among the ZGs within each cell. Two decades later Kraehenbuhl et al. (1977) performed the first immunocytochemical localization of secretory proteins in bovine pancreatic exocrine cells and isolated ZGs. Their goal was to determine whether regional differences exist in the bovine gland with regard to the production of individual secretory proteins, and whether specialization of product handling occurs at the subcellular level. A double-antibody technique was used in which the first step consisted of rabbit F(ab')z antibovine secretory protein, and the detection step consisted of sheep F(ab')z anti-rabbit F(ab')* conjugated to ferritin. The results showed that all exocrine cells in the gland, and all ZGs and Golgi cisternae in each cell, were qualitatively alike with regard to the content of secretory proteins examined (i.e., trypsinogen, chymotrypsinogen A, carboxypeptidase A, RNase, and DNase). The data suggested that these secretory proteins are transported through the cisternae of the Golgi complex, where they are intermixed before copackaging in ZGs; passage through the Golgi complex is apparently obligatory for these (and likely all) secretory proteins and is independent of the extent of glycosylation (e.g., trypsinogen, a nonglycoprotein, versus DNase, a glycoprotein). It has long been known, on the basis of morphological and biochemical differences, that the exocrine pancreatic gland can be divided into periand teleinsular regions (Jarotsky, 1899; Sergeyeva, 1938; Hellman et af., 1962; Kramer and Tan, 1968; Malaise-Lagae et al., 1975). More recently, the enzyme profile of these regions has been investigated by Bendayan and Ito (1979) by the immunofluorescence technique, using antibodies against nine pancreatic enzymes (i.e., a-amylase, lipase, chymotrypsinogen A, trypsinogen, elastase, carboxypeptidases A and B, DNase, and RNase A). These antibodies were specific to their antigens without cross-reaction. By immunofluorescence most acinar cells of the normal rat pancreas were

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positive to the nine enzymes tested. However, an inhomogeneity in the staining pattern was found; specifically, the cells located in the periinsular region of many islets showed a brighter fluorescence than did acinar cells in the teleinsular tissue. The interesting observation emerging from this study was that not all of the acini showed the same fluorescence intensity, suggesting that the enzymatic content of the acinar cells varies quantitatively from one acinus to another. The variation in fluorescence intensity was present not only between periinsular and teleinsular acini, but also between different acini in the same region. In addition conventional light and electron microscopy have shown that the periinsular acinar cells are bigger, contain a higher concentration of ZGs, and have a larger nucleus and nucleolar volume (Jarotsky, 1899; Ferner, 1958; Hellman et al., 1962; Kramer and Tan, 1968). Moreover, in the first hours following an intravenous injection, the distribution pattern of radioactively labeled amino acid uptake showed markedly more radioactivity in the periinsular than in the teleinsular tissue (Hansson, 1959). A quantitative immunocytochemical localization of these proteins was later carried out by Bendayan et af. (1980), this time using the protein A-gold technique applied on thin sections. An increasing gradient of the labeling from the RER to the Golgi apparatus and to the ZG was found for a-amylase, carboxypeptidases A and B, chymotrypsinogen A, trypsinogen, and RNase A, while a comparable low degree of labeling in the Golgi apparatus and in the ZG was observed for DNase, lipase, and elastase. These results suggested that the nine enzymes are processed through the same intracellular compartments, but that they are concentrated to different degrees in the ZG before being released in the acinar lumen. The distributions of a-amylase and chymotrypsinogen in peri- and teleinsular cells of the rat pancreas were reinvestigated by Posthuma et al. (1986). They used ultrathin cryosections from tissue blocks consisting of tele- and periinsular tissue elements. Consecutive sections of these blocks were alternatively immunolabeled for a-amylase and chymotrypsinogen, using protein A-gold as the marker. The density of gold particles over ZGs of both peri- and teleinsular cells was measured. It appeared that the a-amylase/chymotrypsinogen labeling density ratio was significantly lower in periinsular than in teleinsular cells. This difference resulted from a lower a-amylase labeling, as well as a higher chymotrypsinogen labeling density over ZGs in periinsular cells. Their results were in agreement with those of Malaisse-Lagae et al. (1975). This immunocytochemical observation reported above provided a strong argument against the concept of the “equilibrium hypothesis” (Rothman, 1975) as a model of secretion. Immunocytochemistry has also brought some important clues regarding the packaging process. Indeed,

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Bendayan (1984) reported the concentration of a-amylase along its secretory pathway in the rat pancreatic acinar cell by high-resolution immunocytochemistry. Quantitative evaluations of the degree of labeling demonstrated an increasing intensity which progresses from the RER, through the Golgi apparatus, to the ZG and have identified the sites where protein concentration occurs. The results obtained have thus demonstrated that a-amylase is processed through the conventional RER-Golgi-granule secretory pathway in the pancreatic acinar cells. In addition a concomitance has been found between some sites where protein concentration occurs: the trans-most Golgi cisternae, the CVs, and the premature and mature ZGs. It is noteworthy that minigranules also transport a-amylase to the cell surface, as illustrated in Fig. 4. If immunocytochemistry has not indicated any major differences in granule composition among the granules of a given cell (see Fig. 5 ) , there would, however, be some important variations among granules isolated from the whole pancreas, as proposed by Mroz and LechCne (1986). Indeed, these authors measured the activities of both chymotrypsinogen and a-amylase in individual ZGs of the rat pancreas by micromanipulation and microfluorometric methods. The enzyme content and the ratio of a-amylase to chymotrypsinogen vaned widely among granules from the same animal. These results are compatible with short-term nonparallel bulk secretion of the two enzymes through exocytosis. The distribution of each enzyme activity in a population of granules suggests quanta1 packaging of a-amylase and chymotrypsinogen into the granules. From the immunocytochemical studies described above it is clear that ZGs all contain a-amylase and other secretory proteins, but these studies do not provide information about the turnover rate of the secretory proteins.

VII. Pancreas ZG Membrane Proteins

Meldolesi et af. (1971) and MacDonald and Ronzio (1972) were among the first to isolate the ZG fraction from pancreas acinar cells and to analyze the protein composition of their membrane. Meldolesi et al. (1971) noticed that ZG membranes were enriched in phospholipids, and Meldolesi and Cova (1972) later analyzed the profile of protein composition obtained after polyacrylamide gel electrophoresis (PAGE). They noticed a certain degree of similarity between isolated ZG membranes and a plasma membrane fraction. In the same period MacDonald and Ronzio (1974) made a comparative analysis of ZG membrane polypeptides from several species by sodium dodecyl sulfate (SDS)-PAGE. They found that these membranes contained very few proteins, among which was a common major

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glycoprotein component identified as GP2. Paquet et al. (1982) later corroborated this analysis of ZG membrane protein components of the rat pancreas by two-dimensional isoelectric focusing and SDS-PAGE. Following the identification of GP2, several groups determined its localization by immunological methods. Immunocytochemical observations by Geuze et al. (1981) showed that GP2, on the one hand, exhibited the characteristics of a membrane protein by its occurrence in the cell membrane and the Golgi membranes and its association with ZG membranes. On the other hand, GP2 was present in the contents of the ZG and the acinar and ductal lumena. A GP2-like glycoprotein was also found in the cannulated pancreatic secretion (Scheffer et al., 1980). The presence of GP2 in the pancreatic juice was confirmed later in both the rat and the pig pancreas (Havinga et al., 1984; Beaudoin et al., 1986a). Scheffer et al. (1980) and later Beaudoin et al. (1986a) found, by immunological techniques, that GP2 was in a sedimentable form in the pancreatic juice of the rat. Moreover, Beaudoin et al. (1986a) proposed that GP2 was associated with some microvesicles recovered in a pellet obtained by ultracentrifugation of the pancreatic juice collected under resting conditions. Recently, Rindler and Hoops (1990) reinvestigated the localization and some of the biochemical properties of GP2 in the rat pancreas and pancreatic juice. Using affinity-purified antibodies, they found it to be concentrated in the ZG and the acinar lumen. Label was also present on the apical and basolateral plasma membranes, but prior treatment of the sections with periodate to eliminate the contribution of highly antigenic oligosaccharide moieties substantially reduced the staining of the basolateral surface, suggesting that their antibodies were reactive to oligosaccharides. Approximately 45% of the GP2 in the granules was not membrane associated, but appeared, instead, in the granule lumen. Parallel biochemical characterization of GP2 in isolated secretory granules demonstrated that 60% of the total GP2 fractionated with the membranes after granule lysis, while the remaining 40% was found in the content. Unlike the membrane-associated form of the protein, which is linked to the membrane via glycosyl phosphatidylinositol (GPI), GP2 in the content did not enter the detergent phase upon Triton X-I14 extraction; nor was it sedimentable at 200,000 g , as is characteristic of the form collected in the pancreatic juice. In addition, GP2 in the pancreatic juice was recovered in FIG. 4 Minigranules in the rat pancreas under “resting” conditions. Immunocytochemical localization shows that minigranules are filled with a-amylase. The fact that the content of these granules is more concentrated than in the lumen rules out the possibility that these are endocytic vesicles. (a) Minigranules (arrows) dispersed in the trans-Golgi area. (b) Minigranules (arrows) close to the luminal plasmalemma. Experimental methods are as described by Beaudoin ef al. (1991).

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the aqueous phase during Triton X-114 extraction and yet remained sedimentable after detergent extraction, demonstrating that its ability to remain in large aggregates was independent of lipid. In a more recent study Beaudoin et al. (1991) reinvestigated the immunocytochemical localization of GP2 with polyclonal antibodies reactive to the protein moiety of the glycoprotein. Their immunocytochemical studies have demonstrated GP2 to be present on the membrane and in the matrix of ZGs, over the Golgi saccules, on the apical and basolateral surfaces of the plasma membrane, and in the acinar lumen. In addition this protein was observed in small vacuoles and tubular structures previously identified as “basal lysosomes” or “snakelike tubules,” and in lysosomes. Its presence in the latter group of structures involved in endocytosis suggests a possible role of GP2 in this cellular process. GP2 was readily detectable in the pancreatic juice and was totally sedimentable by ultracentrifugation, as assessed by Western blot analysis. Induced lysis of isolated ZGs also caused the release of GP2 in a sedimentable form, which, by electron microscopy, appeared as some fibrillar material. This probably corresponds to the internal reticulation found on carbonate buffer-washed ZG membrane, as observed by freeze-fracture techniques (Cabana et al., 1981). Various biochemical labeling techniques have been used to determine the localization of GP2 in the plane of the ZG membrane. No labeling of GP2 could be obtained on intact ZGs from the rat (Ronzio et al., 1978) or the pig pancreas (LeBel, 1988), indicating that it was associated with the inner layer of the ZG membrane; It was later shown by Paquette et al. ( 1986) that GP2 was associated with the membrane via a GPI linkage. Although subjected to contradictory views (see Scheffer et al., 1980; Beaudoin et al., 1991), the presence of GP2 in ZG granule lysates (40%) would suggest that a fraction of the GP2 molecules is released from the ZG membranes, as demonstrated by aqueous-phase Triton X- 1 14 partition techniques (Paquette et al., 1986; Rindler and Hoops, 1990). The absence of GP2 after high-speed centrifugation of the pancreatic juice is still puzzling, and it leads one to believe that the hydrophilic GP2 molecules would form aggregates during secretion. In this respect the immunocytochemical observations by Beaudoin e f al. FIG. 5 ZGs (asterisks) of the rat pancreas in siru and in uitro. Immunocytochemical localization shows that all of the granules contain a-amylase in about the same proportions. High magnification does not reveal any concentration of a-amylase at the membrane surface. Isolated granules provide the same labeling and size distribution patterns as in situ. (a) a-Amylase distribution among ZGs in sitrr. (b) a-Amylase distribution in a population of isolated ZGs. (c) High magnification showing the distribution of a-amylase in a ZG matrix. Notice the ZG membrane in 5c (arrowheads).

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(1991) raise some questions regarding the origin of GP2 found in the lumen. Indeed, these authors compared the immunoreactivity of GP2 found in the acinar lumen with that in some ZG at the outset of exocytosis, and the images leave no doubt that GP2 is much more reactive in the lumen than in the corresponding ZG content. It is our opinion that this behavior is not a question of antibody accessibility, due to protein aggregation, since immunocytochemical localization of a-amylase does not show such behavior. These observations strongly suggest that some of the GP2 molecules in the lumen do not derive from ZG, but perhaps, as we previously proposed, via a paragranular pathway or a special class of ZGs (see Beaudoin and Grondin, 1991). The biochemical studies by Havinga et al. (1984) bring strong support to the latter hypothesis. Indeed, their pulse-chase experiments on isolated acinar cells showed that the incorporation of the first newly synthesized GP2 molecules into ZG membranes occurred about 60 min after the beginning of the pulse. They also demonstrated that newly made GP2 molecules reach the cell surface within the same time span. After a 6- to 8-hr chase considerably more newly synthesized GP2 molecules have reached the cell surface than would be expected from the secretion of newly synthesized zymogens. These observations strongly suggest that at least part of the GP2 molecules bypass the mature ZG compartment on their way to the plasma membrane. GP2 is the only protein that appears in discernible quantity in the plasma membrane 1-4 hr after a pulse label. Nevertheless, GP2 comprises only a small percentage of externally '251-iodinatedplasma membrane proteins. Havinga et al. concluded that GP2 has a high turnover rate at the plasma membrane level. Finally, treatment of the acinar cells with the N-glycosylation inhibitor tunicamycin does not block the intracellular transport of GP2. In these in vitro experiments GP2 is not released into the medium. We and others (Phaneuf et al., 1985; Arvan and Castle, 1987) have noticed the same phenomenon with rat pancreas acini and lobule preparations. Some molecular biology information about this protein has been reported recently by Fukuoka et al. (1990). In a carbohydrate-shift strategy amino-terminal and internal peptide sequences were obtained on glycosylated and deglycosylated forms of GP2, respectively, by gas phase sequencing. Sets of mixed oligonucleotides and the polymerase chain reaction were used to obtain a double-stranded cDNA probe, which was used to isolate overlapping cDNA clones from a dog pancreatic cDNA library constructed in XZAP-11. The sequence of these clones revealed an open reading frame which encodes a protein of 509 amino acids, containing eight N-linked oligosaccharide attachment sites. The carboxy terminus shows a 20-residue hydrophobic transmembrane domain preceded by a potential GPI attachment site. GP2, completely released from the ZG membranes with phospholipase C, showed similar immunochemical properties and

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electrophoretic mobilities compared to the form associated with ZG membranes. A similar form of GP2 was released from ZGs permeabilized with saponin. Kinetic analysis indicated two distinct pools of GP2 released from permeabilized granules. According to Fukuoka et al., these findings indicate that GP2 is a GPI-linked membrane protein, which is released from ZG membranes by GP1-anchor cleavage activity present in ZGs. In parallel to these molecular biology studies, Freedman et al. (1990) demonstrated homology between GP2 and the Tamm-Horsfall protein (THP) from the kidney. Extensive conservation of structure is demonstrated between the two proteins. Over the carboxy-terminal sequence observed between Asp-54 and Phe-530 in the rat sequence, there is 63% identity and 91% homology between rat GP2 and human THP. According to these authors, the similarities observed in molecular structure and cellular localization between GP2 and THP in the pancreas and the kidney, respectively, and the complementary information obtained from studies of GP2 (GPI-tail nature) and THP (self-aggregating property) suggest that (1) both proteins are synthesized as GPI-tailed proteins, (2) aggregation of GP2 and THP on the cisternal leaflet of trans-Golgi elements plays important roles in the assembly of ZGs and apical vesicles in the pancreas and the kidney, respectively, and (3) the release of GP2 and THP from granules and apical vesicle membranes, respectively, is required for retrieval of these membranes from the plasmalemma after exocytosis. A family of genes appears to be responsible for the expression of granule membrane assembly proteins from diverse epithelia (Freedman et al., 1990). y-Glutamyltransferase (GGT) is another glycoprotein found in the ZG membrane of the rat pancreas (Castle et al., 1985). Antibodies were produced in rabbits, using purified GGT from the rat kidney. trans-Blot experiments showed some immunoreactivity with a 58- and 30-kDa polypeptide when ZG membrane was used as the source of antigens. Battistini et al. (1990) reported the release of this protein in the rat pancreatic juice under resting and stimulated conditions. Under resting conditions in uiuo, high levels of GGT were found in the pancreatic juice, and these levels were not related to protein concentration. Under secretin infusion a relatively constant level of GGT was released, and again, there was no correlation between GGT activity and protein secretion. However, following a bolus injection of cerulein, an analog of cholecystokinin, marked and concomitant rises in protein and GGT levels were observed. Ultracentrifugation, as well as gel filtration on Sepharose 4B, demonstrated that the enzyme was not released in a soluble form. It cannot be excluded that some of the GGT in the pancreatic juice could originate from ductal cells (Yasuda et al., 1986). In addition to GGT, several other enzymes have been put into evidence in ZG membranes by cytochemical immunological and biochemical methods, including TPPase (Paquet et al., 1982), ATP-diphosphohydro-

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lase (LeBal et al., 1980; Laliberte et al., 1982), and protein disulfide isomerase (Akagi et at., 1988). Like GP2 and GGT, the latter protein is apparently secreted by the acinar cell. The hypothesis according to which phosphorylation of specific proteins could change granule membrane properties and facilitate the fusion with the plasma membrane during exocytosis led some authors (Lambert et al., 1974; Wren, 1984; MacDonald and Ronzio, 1974) to examine protein kinase activity in these membranes. An endogenous protein kinase activity was demonstrated, but these observations fail to establish any direct relationship between phorphorylation and exocytosis. A renewed interest for this hypothesis has risen recently by the detection of GTP-binding proteins (G proteins) associated with ZG membranes of the rat pancreas (Padfield et al., 1990).A 28-kDa G protein and a 25-kDa ADP-ribosylated protein were also described by Lambert et al. (1990). These authors suggested that a 28-kDa protein could be involved in protein transport between intracellular compartments along the secretory pathway andlor exocytosis. Some previous observations, using an in vitro system, support this hypothesis. Indeed, Nadin et al. (1989) developed a cell-free assay for the interaction between pancreatic ZG and plasma membranes. They showed that plasma membranes are able to trigger the release of the granule contents and that this effect is specific to pancreatic membranes. It involves membrane fusion, it requires membrane proteins, and it is stimulated by activators of G proteins, but not by Ca”.

VIII. Parotid ZG Membrane Proteins

Early attempts to characterize the proteins of the membranes of parotid secretory granules have been hampered by difficulties in obtaining membrane preparations free of secretory proteins (Castle and Palade, 1978; Wallach et al., 1975a,b). Wallach et al. (1975a) measured the a-amylase activity in a granule membrane preparation from the rat parotid gland and estimated that 10% of the protein was contributed by secretory proteins. Castle and Palade (1978) compared the proteins of secretory granules and granule membranes of rabbit parotid glands by SDS-PAGE after labeling unfractionated glands with ‘‘C-labeled amino acids. They estimated that 25-30% of the total protein in the membrane preparation are secretory proteins. Putative secretory proteins were removed by treating the membranes with saponin and sodium sulfate, conditions which caused disruption of the typical bilayer structure of the membrane. After treatment SDS-PAGE of the parotid granule membrane revealed 70 polypeptide bands. Wallach et al. (1975a) reported a simple composition of parotid

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granule membranes of the rat, whereas Casieri and Somberg (1983) found that the granule membrane contains 166 polypeptides, of which only 26 are also present in the granule content. The membrane proteins have isoelectric points between 4.75 and 6.45 and apparent molecular masses varying from 17 to 190 kDa. In a more recent study Cameron et ul. (1986) made a comparative analysis of two-dimensional isoelectric focusing: SDS-PAGE of radioiodinated granule membranes from parotid, pancreatic, lacrimal, and submandibular glands. Each profile comprises about 25 species with electrophoretic mobilities corresponding to apparent molecular masses ranging from 18 to 150 kDa. In the case of the parotid gland, the number and distribution by apparent molecular mass are consistent with the onedimensional profile shown previously using either tyrosine- or amino group-directed labeling (Cameron and Castle, 1984). Also, polypeptides of low apparent molecular mass are more prevalent than larger species in all cases. As well, the majority of granule membrane polypeptides are acidic, focusing between pH 5.0 and 7.0. This observation is consistent with the previous reports for the general nature of polypeptides from adrenal chromaffin granules (Bader and Aunis, 1983), pancreatic ZGs (Paquet et al., 1982), and parotid granules (Casieri and Somberg, 1983), although in the latter case, in which residual mitochondria1 contamination appears to be significant, the pattern is considerably more complex than the one Cameron er al. have observed. As mentioned by Cameron et ul. (1986), the most sriking observation made by comparing the profiles of the four types of membranes is the presence of apparent extensive polypeptide homology. Not only identities are suggested by overlapping mobilities for individual species, but the two-dimensional patterns of spots are also similar, if not identical, in a number of regions. More specifically, major radiolabeled species (- 10 distinct polypeptides) that coincide in isoelectric point and molecular mass are located in the range of 24-30 kDa and, in part, 2 8 5 kDa. Evidently, these common polypeptides do not correspond to incompletely removed secretory proteins, since neither radiolabeling nor protein staining of parotid granule content reveals a pattern having comparable isoelectric points and apparent molecular masses. In addition, at least four to six species in the range of 40-70 kDa exhibit common mobilities, but show much wider quantitative variations in intensity among the different types of membranes. Some of these species are observed in more than one, but not all, preparations. Finally, each sample contains unique polypeptides for which there is no counterpart in the other patterns. Peptide mapping after chymotrypsin and trypsin digestion show very clearly that membrane and content proteins are structurally unrelated and that one of the principal overlapping species (-29 kDa) is essentially

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identical in parotid and pancreatic two-dimensional polypeptide profiles (Cameron et al., 1986). In addition different enzyme activities have been associated with the ZGs of the parotid gland, using cytochemical, biochemical, and immunological approaches, including AcPase, TPPase (Hand and Oliver, 1977), GGT (Castle et al., 1985) and Ca2+ ATPase (Watson et af., 1974).

IX. ZG Membrane Lipid Composition

During exocytosis the ZG membrane fuses with the plasma membrane. This cellular process, which can be mimicked by isolated preparations in vitro (Rand and Parsegian, 1986), is in great part determined by the physicochemical properties of the lipid components of the interacting membranes. In this respect several years ago Blashcko et at. (1967) reported that chromaffin granules of the adrenal medulla, which perform a function analogous to that of the ZGs in the pancreas, contain a high proportion of lysophosphatidylcholine (lyso-PC) [ 17% of the granule lipid phosphorus (P)]. These authors suggested that, during secretion, the lyso-PC might be involved in the fusion of the granule membrane with the plasma membrane of the cell. White and Hawthorne (1970) analyzed the lipid composition of ZGs from the ox pancreas. The pancreatic ZG fraction had slightly less PC than either the mitochondria1 or microsomal fractions (41% compared with 50%), but had significantly more phosphatidylethanolamine (PE) (35% compared with 23% and 29%,respectively). No abnormally high values for Iyso-PC were obtained. The ZG membrane resembles the surface membrane in its relatively high cholesterol/phospholipid molar ratio (0.56). A corresponding value for rat liver plasma membrane is 0.53 (Coleman et al., 1967). It is noteworthy that White and Hawthorne (1970) observed hydrolysis of phospholipids during fractionation, sometimes leading to high levels of lyso-PC and lyso-PE. Therefore, they retained only those experiments in which lyso-PE was inferior to 2% of lipid P. The hypothesis that cAMP would activate phospholipase activity, which would lead to locally increased lysophospholipid formation, resulting in a fusion between the ZG and apical plasma membranes was tested by Rutten et af. (1975). cAMP added to isolated pig pancreatic ZGs leads to an increased lysis of these granules. However, the slowness of this effect renders its physiological significance dubious. In pancreatic homogenates or ZGs no stimulating effect of cAMP on lipase or phospholipase activity could be demonstrated. Isolated ZGs have a high lysophospholipid content (27% of total phospholipids), consisting of the 1- and 2-acyl forms of lyso-PC and lyso-PE. Experiments with radioactive PC indicate that the

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lysophospholipids are due to the action of endogenous (phospho)lipases during the isolation procedure. These experiments did not lend support to the hypothesis mentioned above for the mechanism of action of cAMP in pancreatic enzyme secretion. Immunocytochemical evidence for an endogenous phospholipase A2 distinct from the secreted enzyme has been obtained recently in our laboratory. Properties of ZG membrane phospholipids and their fatty acyl compositions have also been measured in the rat parotid gland by Mizuno et al. (1987). A comparison of the ZG fraction with the microsomal fraction showed that the ZG membrane had higher levels of lysophospholipids (8%) and PE (31%) and lower levels of PC (40%) and phosphatidylserine (PS) (2.1%). However, fatty acid compositions of individual phospholipid classes from the two subfractions were found to be similar to each other. Electron spin resonance analysis demonstrated that extracted phospholipids from the secretory granular fraction were more fluid than those from microsomes. Castle et al. (1981) had previously reported that rabbit parotid secretory granules had a soluble phospholipase A, which was activated by Ca2+and inhibited by EDTA. However, they inferred that this high level of lysophospholipids in ZGs does not result from enzymatic degradation during cell fractionation, since they used EDTA-containing buffer during cell fractionation, and that such lysophospholipids showed a low level in other subfractions. Thus, the higher level of lysophospholipids, as well as PE, and the lower levels of PC and PS may be characteristic properties in secretory granular membrane lipids from rat parotid glands. Considering the ZG membrane composition from the pancreas and the parotid gland, one is struck by the high level of PE, a composition which would favor the transition from a bilayer to an hexagonal configuration of the membrane. Second, it cannot be excluded that the high level of lysophospholipids, which is thought to be derived from phospholipase A activity, might contribute to the increased fluidity in the fusion area.

X. ZG Ion Transport in the Pancreas

As mentioned by Gasser et al. (1988), investigations of stimulus-secretion coupling in exocrine glands were focused mainly on the intracellular messengers that play a role in this process. Both calcium- and CAMPdependent pathways have been identified. For example, changes in cytosolic calcium have been reported in pancreatic acinar cells in response to the secretagogues cholecystokinin, bombesin and acetylcholine and its analogs (Maruyama and Petersen, 1982; Merritt and Rubin, 1985), while vasoactive intestinal peptide and secretin elevate cAMP (Kimura et a / .,

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1986; Robberecht er al., 1976; Singh, 1979). Although calcium and CAMP probably contribute to the secretory process by activation of protein kinases and selective phosphorylation of plasma andlor granule membrane proteins (Burnham et al., 1985; Spearman et af., 1984), the physiological targets and function of secretagogue-dependent phosphorylation remain speculative. In an effort to understand the underlying physiology and biochemistry of macromolecule secretion, Gasser et af. (1988) concentrated their efforts on the characterization of transport properties of the granule membrane. Regulation of the ion permeabilities of this membrane could have significant consequences in several aspects of the secretory process, particularly the accumulation and storage of secretory proteins (Johnson et al., 1981; Njus et al., 1985), fluid secretion accompanying protein release (DeLisle and Hopfer, 1986), or osmotic swelling of granules, which has been postulated to play a role in membrane fusion or fission (Cohen et al., 1980; Kachadorian et al., 1981; Zimmerberg and Whitaker, 1985). Rat pancreatic ZGs have been shown to possess an anion exchange and an anion conductance pathway. However, they typically lack cation permeabilities when isolated in a low-calcium and detergent-free environment (DeLisle and Hopfer, 1986; Gasser et al., 1988). It has been postulated that the C1conductance of the granule membrane contributes significantly to the secretagogue-stimulated C1- conductance of the plasma membrane after fusion of the granules with the plasma membrane (DeLisle and Hopfer, 1986). Secretagogue-stimulated C1- conductance of the plasma membrane has been measured by electrophysiological techniques and is thought to play an essential role in fluid secretion (Petersen, 1986). Thus, the C1- conductance of the granule membrane may have a major role in the coupling of fluid and macromolecule secretion and the determination of fluidity in the primary secretion. Gasser et al. (1988) examined the membrane permeability of rat pancreatic ZGs in uirro with granules isolated from rats in different secretory states: (1) untreated, (2) pretreated with a muscarinic antagonist, (3) pretreated with a muscarinic and an adrenergic antagonist, (4) pretreated as in the previous state and then stimulated with the secretagogue cholecystokinin 4 min before death, and ( 5 ) pretreated as in state 3 and then stimulated with the secretagogue secretin 4 min before death. Granules isolated from untreated rats had variable ionic permeabilities. In general, however, they possessed both CI- conductance and electroneutral exchange pathways, with low permeabilities to alkali metal ions. In contrast, granules from animals pretreated with secretory antagonists had low ion permeabilities to both inorganic anions (e.g., chloride) and alkali metal ions. An injection of the peptide secretagogues cholecystokinin or secretin resulted in a relatively fast (i.e., within 4 min) activation or induction of high chloride permeabilities through both C1- conductance and chloride hydroxide (or

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chloride/bicarbonate) exchange pathways. In addition the secretagogues increased the cation permeability of the granule membrane, which exhibited a distinct potassium selectivity. These results demonstrate that granules may actively participate in the secretory process and suggest that some of the physiological targets in the cascade of events leading to secretion are anion and cation transporters in the ZG membrane. The influences of secretagogues and second messengers were also examined more recently by Fuller et al. (1989). They examined ion permeability pathways in ZGs isolated from control cells and cells pretreated with the acetylcholine analog carbachol, the peptide hormone cholecystokinin, and second messengers of hormone action such as CAMPand the diacylglycerol analog 12-0-tetradecanoyl phorbol- 13-acetate (TPA). Ion and water influx rates in ZGs and consequent swelling and lysis of granules were monitored by measuring changes in the optical densities of ZG suspensions at 540 nm following additions of the electrogenic or electroneutral ionophores valinomycin and nigericin, respectively. The data show that a C1- conductance and an anion exchange pathway are both present in the granule membrane. These two pathways are activated by pretreatment of isolated cells with cholecystokinin or isolated permeabilized cells with CAMP, whereas only the C1- conductance is increased by pretreatment with carbachol or TPA. The anion transport inhibitor diisothiocyanostilbene disulfonic acid abolishes CI- conductance, but it also has no effect on the anion exchanger. Granular lysis is also inhibited by buffers of high osmotic strength. The direct application of the catalytic subunit of protein kinase A and ATP to isolated ZGs inhibits both the CIconductance and the anion exchange pathway probably mediated by phosphorylation. This result does not seem to be consistent with the observation that in isolated ZGs from CAMP-prestimulated cells both C1- conductance and the Cl- anion exchanger are activated. Fuller et al. concluded that the stimulation of pancreatic acinar cells by secretagogues results in the activation of C1- permeability pathways in the ZG membrane mediated by cAMP-protein kinase A and by a diglyceride-protein kinase Cmediated phosphorylation. It is possible that, in addition to “on” reactions, “off” reactions also exist which close the C1- channel (e.g., by dephosphorylation) and that in activation of C1- permeability pathways different sites are involved for CAMP-protein kinase A action or different protein kinases A.

XI. Cytoskeleton and ZG Movement It is well established that the cytoskeleton plays an important role in many cellular processes, including cytoplasmic transport and secretion. As re-

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ADRIEN R. BEAUDOIN AND GILLES GRONDIN

ported by Bonder and Mooseker (1986), cytochalasins B and D, both of which bind the barbed end of actin microfilaments in uitro, disturb the microfilament system. Several groups have shown that these cytochalasins inhibit the protein discharge in response to secretagogues in both the exocrine pancreas and the parotid gland (Butcher and Goldman, 1974; Morisset and Beaudoin, 1977; Williams, 1977; Burnham and Williams, 1982; Busson-Mabillot et d.,1985; Stock et d . , 1978). Indeed, maximal stimulation of enzyme release from in uitro preparations of the mouse and the rat pancreas was shown to be inhibited by cytochalasin B at concentrations high enough to disrupt the network of microfilaments that underlies the luminal membrane (Bauduin et al., 1975; Williams, 1977). Cytochalasin B at these concentrations did not affect either pancreatic 45Ca2f fluxes or ATP levels (Bauduin et al., 1975; Williams, 1977). In addition Williams (1977) noted that cytochalasin B caused the disappearance of apical microvilli and considerable luminal dilation in mouse pancreatic fragments and acini. It was suggested that the apical network of microfilaments acts as a contractile ring to stabilize the luminal membrane and that loss of this structure in cytochalasin B-treated tissue leads to luminal dilation as the result of incorporation of the microvillar membrane into the luminal membrane (Williams, 1977). The finding that cytochalasin inhibits protein discharge was taken to indicate that the microfilament, and more generally, the cytoskeleton, was involved in the movement of secretory granules to the site of exocytosis (Burridge and Phillipps, 1975). These observations with cytochalasins B and D are difficult to evaluate because of the multiple effects of cytochalasin on cellular metabolism and membranes (Lin et al., 1973). Among these effects some are directly related to the intracellular messengers. In the rat parotid gland the involvement of the microfilament system in the cellular signal transmission mechanism was tested by measuring the effect of cytochalasin D (which disturbs the microfilament system) on the production of intracellular second messengers. Cytochalasin D did not affect unstimulated calcium movements (measured by the 45Caefflux technique), inositol phosphate production, or cAMP accumulation. Neither did it modify the generation of intracellular second messengers induced by activation of the cholinergic muscarinic receptor (calcium and inositol phosphates). Cytochalasin D did not affect the cAMP accumulation induced by the activation of the P-adrenergic receptor, whereas it strongly inhibited the calcium movements induced by activation of the same receptor. These data suggest that, in the rat parotid gland, calcium movemements induced by fi-adrenergic receptor stimulation need an intact microfilament system to occur, whereas the rnuscarinic pathway (via inositol triphosphate) does not. Microtubules constitute another important element of the cytoskeleton

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of exocrine glands. Markham and Cope (1976) showed that pretreatment of the rabbit parotid gland with colchicine or vinblastine sulfate resulted in a 80-85% inhibition of isoproterenol-induced granule discharge. This effect was also seen when dibutyryl CAMPwas used as the secretagogue. Several groups (Nevalainen, 1975; Seybold et al., 1975; Stock et al., 1978; Williams and Lee, 1976) showed that agents that disturb microtubules (e.g., colchicine and vinblastine) cause cellular alterations and interfere with the pancreatic secretory process. In addition to inhibiting enzyme discharge, antimicrotubular agents such as vinblastine and colchicine reduce amino acid entry and intracellular transport (Seybold et al., 1975). The localization of some cytoskeleton proteins was studied by Drenckhahn and Mannherz (1983). Actrin, myosin, and the actinassociated proteins tropomyosin, a-actinin, vinculin, and vilin were localized in acinar cells of rat and bovine pancreas, parotid, and prostate glands by immunofluorescent staining of both frozen tissue sections and semithin sections of quick-frozen, freeze-dried, and plastic-embedded tissues. Antibodies to actin, myosin, tropomyosin, a-actinin, and villin reacted strongly with a narrow cytoplasmic band extending beneath the luminal border of acinar cells. Fluorescently labeled phalloidin, which reacts specifically with F-actin, gave staining, within the cell apex, similar to that obtained with antibodies to actin, myosin, tropomyosin, a-actinin, and villin. In contrast, immunostaining with antibodies to vinculin was restricted to the area of the junctional complex. Ultrastructurally, the apical immunoreactive band corresponded to a dense web composed of interwoven microfilaments, which could be decorated with heavy meromyosin. Outside this apical terminal web antibodies to myosin and tropomyosin gave only weak immunostaining (confined to the lateral cell borders), whereas antibodies to actin and a-actinin led to a rather strong beadlike staining along the lateral and basal cell membranes, probably marking microfilament-associated desmosomes. Antivillin immunofluorescence was confined to the apical terminal web. Drenckhahn and Mannherz suggested that the apical terminal web is important for the control of transport and access of secretory granules to the luminal plasma membrane and that villin, which is known to bundle or sever actin filaments in a Ca2+dependent manner, might participate in the regulation of actin polymerization within this strategically located network of contractile proteins. Bendayan et al. (1982)in the same period also reported the immunocytochemical localization of actin by the protein A-gold technique. The labeling was found at the level of the filamentous cell web and in close association with the Golgi cisternae, CVs, and ZG-delimiting membranes, as well as with the plasma membrane. Weak labeling was also present over the dense content of the ZGs. The association of actin with different membr.anes implicates that contractile proteins might constitute structural

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membrane proteins and, thus, might play an important role in protein secretion. Intermediate filaments are other important components of the cytoskeleton. These have been observed in pancreatic acinar cells and some keratins have been seen in close association with ZGs (Bendayan, 1985). Despite all of these biochemical and morphological observations, an active role of the cytoskeleton in the migration of ZGs toward the cell apex remains to be demonstrated. With the advent of the confocal microscope and techniques for cell permeabilization, this phenomenon can be better studied. Some freeze-substitution techniques, which better preserve the integrity of the microtubules, have been applied recently on rat pancreas acinar cells and as illustrated in Fig. 6 , there is definitely a close relationship between ZGs and the cytoskeleton.

FIG. 6 Some aspects of the terminal web in the rat pancreatic acinar cell. On tangential section one can observe (a) bundles of filaments (arrow) and (b) microtubules (arrow). Both appear to be apposed to the ZG surface. Lu, Acinar lumen; Go, Golgi apparatus.

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The nature of the interaction between microtubules and the protoplasmic face of the ZG membrane is undefined. In addition to contractile proteins such as actin, there could also be some interactions with phospholipids. On the basis of their results obtained with colchicine analogs on the rat lacrimal gland, Herman et al. (1989) proposed that it is during the maturation stage that microtubules would be required. According to these authors, during this stage the secretory granules would acquire an additional property that allows eventual exocytosis. Without microtubules granule maturation would be impossible because of granular inability to acquire this property, rendering these granules incapable of exocytosis.

XII. Concluding Remarks

Early autoradiographic studies by Warshawski et al. (1963) and by Kramer and Poort (1972) on the rat pancreas have shown that newly formed granules do not mix with older granules. These findings confirm some in uivo observations by Cove11 (1928) on the mouse pancreas showing that ZGs are relatively immobile in the apical cytoplasm. However, as reported by the same authors, some movement of ZGs can be observed after pilocarpine stimulation. The fact that ZG mobility is restricted in the apex cytoplasm, together with the fact that antimicrotubular agents interacting with the cytoskeleton interfere with the secretory response, led us to believe that the cytoskeleton controls the movement of ZGs toward the cell apex. The nature of these interactions may be much more complex than a simple ZG alignment in the apical cytoplasm. The life of a ZG starts in the trans-Golgi saccule, or the so-called GERL (which is part of the trans-Golgi network) in the form of a CV, as shown by electron microscopy and cytochemistry. Both size and electron opacity of the forming CV are highly variable. The intensity and duration of stimulation are among the most important parameters regulating the aspect of CV, and they probably determine the amount of membrane available for the packaging process. Indeed, ultrastructural observations clearly show that after 3-4 hr of sustained stimulation in the rat pancreas, the amount of membrane accumulated in the trans-Golgi area is at its maximum, presumably as a result of the endocytic process. Under these conditions the need for optimization of the membrane/volume ratio is not a limiting factor; hence, the cell responds by producing smaller ZGs. However, as the pancreas gradually recovers, the pool of membrane, available for packaging secretory protein, diminishes. Under the latter condition acinar cells react by producing large

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granules, thereby optimizing the amount of membrane used per amount of material packaged. The acinar cell has developed the capacity to increase the aggregation of the secretory products in the ZG matrix to refine the packaging process. As a result of this aggregation, if water is eliminated from the ZG matrix, there would be an excess of membrane and the latter is removed by a shedding process. Such a hypothesis is supported by different observations: Indeed, freeze-fracture studies (Sesso et al., 1980) and scanning electron microscopy (Naguro and Lino, 1989) have clearly shown the presence of small blebs and buds on the ZG surface. Sesso et al., (1980) reported that some of the microvesicles shed in the cytoplasm are covered with intercalated particles. This is also accompanied by a reduction in lectin binding sites that could be attributable to glycoproteins or other glycoconjugates. Since there is no detectable AcPase in the mature ZG membrane, as demonstrated by both cytochemistry and biochemical analysis, one is led to believe that this enzyme is one of the proteins eliminated from the ZG membrane during maturation. Some unpublished observations by F. Kan, A. R. Beaudoin, and G. Grondin support the view that GP2 would be shed in the cytoplasm during maturation. These microvesicles could migrate to the lysosomal system, as suggested in Fig. 7. One can predict that, during prolonged and intense stimulation, the maturation process would be impaired. As a result one could observe some

cv

AcPase

GP2

0

TMPase

Lysosornes and Snakellke Tubules

GP2

Apical Membrane

FIG. 7 Fate of the membrane components during maturation of the condensing vacuoles (CVs) in the pancreas. During maturation acid phosphatase (AcPase), trimetaphosphatase, (TMPase), and GP2 are removed from the immature granules. These proteins could be targeted to the lysosornes or snakelike tubules (basal lysosomes). MG,Mature granules.

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AcPase in the immature secretory granules. This is indeed the case, as demonstrated in both the parotid gland (Hand and Oliver, 1984) and the pancreas by cytochemistry (Novikoff et al., 1962). We have corroborated this observation in our laboratory. We even occasionally observed some AcPase activity in the acinar lumen. Such a shedding process could provide an explanation for the fact that a fraction of the newly synthesized GP2 and amylase molecules escape from the normal granule pathway and exit from the cell much faster than the bulk of newly synthesized secretory proteins. As early as 1982, Roberge and Beaudoin proposed the existence of a paragranular pathway to account for these observations (see Beaudoin and Grondin, 1991). Finally, minigranules are, in many respects, intriguing. They could well be responsible for another type of accelerated transport of some secretory proteins to the cell surface. Their small size would allow them to escape from the constraints of the cytoskeleton which would affect larger ZG. In support of this, we recently found that GP2 is much more concentrated in these minigranules than in regular-sized ZGs. Acknowledgments We thank Anne Rousseau. Marielle Martin, and Louise Hamel for their contribution in the preparation of the manuscript. This work was supported by Natural Sciences and Engineering Research Council of Canada and Formation de Chercheurs et Aide a la Recherche Quebec.

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