- Email: [email protected]
Relative contributions of apocrine and eccrine secretion to digestive enzyme release from midgut cells of Stomoxys calcitrans (Insecta: Diptera)
0022-1910/91$3.00+ 0.00 Copyright 0 1991Pergamon Press plc
Vol. 37, No. 2, pp. 161-166,1991 Printed in Great Britain. All rights reserved
J. Imecr Physiol.
RELATIVE CONTRIBUTIONS OF APOCRINE AND ECCRINE SECRETION TO DIGESTIVE ENZYME RELEASE FROM MIDGUT CELLS OF STOMOXYS CALCIZ-‘RANS (INSECTA: DIPTERA) A. R. Woon and M. J. LEHANE* School of Biological Sciences, University of Wales, Bangor, Gwynedd LL57 2UW, U.K. (Received 2 October 1990; revised 27 November 1990)
Abstract-A morphometric analysis of the secretory process in the midgut opaque zone cells of the stablefly, Stomoxys calcitrans, shows that over half the stored secretory granules are released from the cell in the first 5 min after the blood meal. The analysis suggests that the majority of digestive enzyme secretion is achieved by eccrine means. Apocrine secretion may play a substantial role in the early burst of secretory activity which immediately follows the blood meal. Assuming that membrane recycling follows eccrine secretion from these cells the membrane recovery rates calculated (1.99 times the apical surface area of the cell in 5 min) are remarkably fast. Key Word Index: Digestive enzyme; secretion; tnorphometrics;
INTRODUCTION Secretion of digestive enzymes by the regulated route has been demonstrated in the opaque zone cells of the midgut of the stable fly Stomoxys calcitrans (Lehane,
1976; 1987; 1988a; 1989). It is clear that these enzymes can be released by the classical route of eccrine fusion of the secretory granule with the apical plasma membrane (Lehane, 1976). On histological grounds Lehane (1976) proposed that secretory granules could also be released from the cell contained in cytoplasmic extrusions (i.e. by apocrine secretion). Many other authors have also presented evidence that insects release cellular extrusions into the lumen of the midgut (for example van Gehuchten, 1890; Deegener, 1909; Cragg, 1920; Buchmann, 1928; Weber, 1928; Wigglesworth, 1929; Graham-Smith, 1934; Pradhan, 1934; Fain-Maurel et al., 1973; Andries, 1976; Bayon, 1981; Baker et al., 1984) and some have claimed that these represent the apocrine secretion of digestive enzymes. Other authors have produced evidence that apical extrusions seen in insect midgut cells are not related to the secretion of digestive enzymes (Day and Powning, 1949; Khan and Ford, 1962; Brunings and de Priester, 1971). Because apical extrusions can be produced as fixation artifacts (Brunings and de Priester, 1971; Bell, 1974; Heath, 1974; Hasty and Hay, 1978; Chandler, 1979; Chandler and Heuser, 1980) it was clear that the debate on the role of apical extrusions in digestive enzyme secretion could not be settled on direct *To whom all correspondence
should be addressed.
Stomoxys
histological evidence and so Lehane (1987; 1988a) took a different approach. He argued that if apocrine secretion was occurring in the opaque zone cells of S. calcitrans then there would be a considerable drop in the volume of the cytoplasm and mitochondria as well as a considerable drop in the associated membrane area of the cells following the burst of enzyme secretion immediately following the blood meal. By quantitatively describing the opaque zone cells before and after the period when apocrine secretion was presumed to occur (thus avoiding any possibility that apical extrusions could be produced as fixation artifacts) Lehane (1987, 1988) established that there was indeed a 34% fall in the volume of the cytoplasm, a 41% fall in the volume of mitochondria and concomitant falls in the area of cell membranes. This suggested that apocrine secretion may play a part in the release of digestive enzymes from the opaque zone cells of S. culcitrans. In this study the relative contributions that apocrine and eccrine secretion make during enzyme release are investigated. MATERIALS AND METHODS S. calcitrans were cultured in the laboratory at 26-28°C in a 12 h light-12 h dark cycle. Larvae were reared in a mixture of 0.5 litre measure each of bran, hardwood chips and Molichop (TM) (a mixture of hay, straw and molasses used as horse feed) to which is added one tablespoon of malt extract, 10 yeast tablets and 1 litre of water. Adults in the colony were fed daily on cotton wool swabs soaked with 161
162
A. R. WOOD and M. J. LEHANE
heparinized pig blood. The adult to adult cycle is approx. 16 days. Adult male flies, 24 h after eclosion, were used in the experiments. Flies were sacrificed unfed or at 5, 12, 18 or 25 min after a blood meal. A systematic, stratified sampling procedure was employed (Williams, 1977). Six flies were used in each treatment. They were anaesthetized with carbon dioxide gas and dissected under cold fixative. Portions of midgut containing the opaque zone were excised and transferred to fresh fixative at 4°C. Guts from unfed flies were fixed for 1 h at 4°C in 3% glutaraldehyde in 0.1 M sodium cacodylate/HCl buffer pH 7.1. After rinsing in 0.1 M sodium cacodylate buffer pH 7.1 containing 5% sucrose, unfed guts were post-fixed in 1% osmium tetroxide for 1 h. This is an unsuccessful fixation procedure for guts from blood fed flies (Lehane, 1976) which were fixed for 1 h at 4°C in a combination fixative (Hirsch and Fedorko, 1968; Brunings and de Priester, 1971) consisting of 0.5 % OsO,, 1.25% glutaraldehyde and 2% sucrose in 0.1 M sodium cacodylate buffer, pH 7.1. Tissues were dehydrated in a graded series of ethanol and embedded in Spurr resin. Two ribbons of gold/silver sections were cut every 150 ,um through the length of the opaque zone and stained in lead citrate and alcoholic uranyl acetate before being viewed and photographed in an AEI Corinth 275 electron microscope at a nominal magnification of x 4,000. Actual magnifications were calculated after photographing a diffraction grating inscribed with 2160 lines mm-‘. Fields to be photographed were randomly selected by choosing only those areas of tissue which lay closest to the upper right hand corner of the mesh square of the copper grid. Because the tissue is anisotropic the photographic sample taken from each selected field was a column of photographs extending vertically from the basal lamina of the cells to the lumenal surface with care being taken to avoid overlap in the photographs comprising each columnar sample (Weibel, 1969). A positive transparency was made of each electron micrograph. These were analysed by projection onto a 45 x 45 cm screen on which was drawn the analysis grid. This consisted of 137 points arranged as,equally spaced equilateral triangles. Point counts were used to determine volume fractions (Vu) of the nucleus, mitochondria, microvilli, secretory granules and cytoplasm including the rough endoplasmic reticulum and Golgi. Each micrograph was analysed for a second time after rotation through 90”. The final magnification of the electron micrograph on the screen was calculated to be x 33,700 using the calibration micrograph of the diffraction grating and knowing the projection magnification. To calculate the concentration of secretory granules at the apical border of the cells, a separate analysis was performed by using only the final apical electron micrograph from each column. From this sub-sample
the volume fraction of secretory granules and cytoplasm were calculated. To determinate areas of apical plasma membrane the micrographs were projected onto a second analysis screen consisting of 18 7 cm lines drawn at an angle of 19” to the vertical. Final projection magnification was calculated to be x 29,900. To reduce bias due to the anisotropic nature of the cells, counts were also made after rotation of the negative through 90”. Surface density of membranes (Sv) was then calculated from the line cut data using the formula Sv = 2P, (Smith and Guttman, 1953). Data from the six experimental animals used at each experimental time are grouped for each feeding regime. Statistical calculations have been carried out on a DEC Vax computer using the statistical package Minitab. RESULTS
To test for quantitative differences between the flies which made up each Vu sample an analysis of variance was carried out. The data was non-normally distributed as shown by a normal scores analysis. Consequently we performed a Kruskal-Wallace analysis of the Vu estimates for the six flies measured at each sampling time. This revealed no significant variation in secretory granules or microvilli among individuals. Significant variation among individuals was observed only in cytoplasm/rer at 5 min (P = 0.021) and 18 min after a blood meal (P = 0.035), mitochondria at 5 min (P = 0.028) and 25 min after a blood meal (P = 0.04) and nucleus at 25 min after a blood meal (P = 0.02). Mitochondrial and cytoplasmic components of the cell are undergoing extreme changes in volume during this period (Lehane, 1987; 1988a) and relatively small differences in the timings of these events are likely to lead to the differences observed between flies. The volume fractions for each cellular compartment have been converted to real volumes by using the opaque cell nuclear volumes measured by Lehane (1988a). These are presented in Table 1. The time dependent changes after a blood meal in the volumes of the key organelles (cytoplasm and secretory granules) are presented in Table 2. Clearly there is a very large early loss of secretory granules whereas the largest losses in cytoplasmic volume occur later in the secretory period. The density of secretory granules in the cytoplasm of the apex of the cell are presented in Table 3. If we assume that the density of secretory granules in apical extrusions is the same as in the cell apex, and the histological evidence suggests this is so, then knowing the cytoplasmic loss over each time interval (Table 2) we can calculate the expected loss of secretory granules with this cytoplasm during apocrine secretion (Table 3). The conclusion is that the remainder of the secretory granules lost in that period are released by eccrine secretion (Table 3).
Digestive enzyme secretion in
163
Stomoxys
Table 1 Time @iin) 0 5 12 18 25
Point counts
Secretory granules
12,656 16,720 18,008 9,009 10,779
2,154 981 892 617 585
Cytoplasm/rer
Nucleus
Mitochondria
Microvilli
Mean cell volume
9,311 8,527 8,347 6,440 6,386
565 599 634 654 666
524 571 586 568 450
423 517 580 420 475
12,977 11,195 11,039 8,699 8,562
The real volumes of the cellular organelles at the different sampling times are presented in pm3 per cell There is a complication with the analysis as presented because it assumes that the volume of secretory granules remains static throughout the period investigated and this is not so. Although there is a considerable build up in secretory granules in the unfed fly there is in fact a continuous secretion of these granules at a low level (Moffatt and Lehane, 1990) as is the case in vertebrate cells undertaking regulated secretion (Case, 1978). This low level production of new secretory granules will mean that there is a small underestimate (probably less than 10%) in the overall prediction of secretory granules secreted over the experimental period. The consequences of this underestimate for the analysis are that for each experimental period (except 12-18 min when no eccrine release is calculated to take place) the amount of eccrine secretion taking place will be underestimated. There is no underestimate in the amount of apocrine secretion calculated to occur because this is fixed by the decrease in volume ;of cytoplasm and will be insensitive to the error outlined above. This underlying low level of production of new secretory granules is greatly accelerated once the blood meal is taken but this should not interfere with the analysis as new secretory granules induced in this way do not begin to appear until about 24-26min after a blood meal (Lehane, 1989). The area of apical plasma membrane in the opaque zone cells at the various times in the feeding cycle are presented in Table 4. Kruskal-Wallace analysis shows there were no significant differences between the area of the apical plasma membrane at any of the time intervals whether this is calculated as prn2/pm3 (P = 0.208) or as pm2 per cell (P = 0.557). The cell clearly painstakingly conserves the area of the apical plasma membrane despite the considerable changes in the cell during this period of intense activity. This agrees with the study of Lehane (1988a) who Table 2 Time @in) o-5 5-12 12-18 18-25
Secretory granules
Cytoplasm/rer
1,173 89 275 32
784 180 1,907 54
Losses over time in secretory granule volume km3 per cell) and cytoplasm/rer volume have been calculated from the information given in Table 1.
found that the quantity of apical plasma membrane appeared to be carefully conserved. Eccrine secretion will lead to an increase in the area of the apical plasma membrane. In contrast apocrine secretion will lead to the loss of apical plasma membrane. Knowing the size of the secretory granules (Lehane, 1988b) and having calculated the total volume of secretory granule lost during each sampling interval the area of secretory granule membrane added to the apical plasma membrane can be calculated for each sampling interval (Table 4). Calculations of the loss of apical plasma membrane due to apocrine secretion will be less accurate. This is because although the total volume of cytoplasm/rer lost is known the shape and diameter of the extruded vesicle is not known. In Table 4 we have estimated the area of apical plasma membrane lost for the simplest possible case where all the cytoplasm/rer lost in that sample interval is assumed to bud off as one vesicle which is completely covered in microvilli. This is likely to underestimate losses because the increased volume/area associated with smaller vesicles will lead to increased losses of apical plasma membrane. DISCUSSION
It is a reasonable assumption that apocrine secretion, involving as it does the loss of considerable quantities of cellular material in addition to the secretory product, is energetically less favourable to the cell than eccrine secretion with its built in mechanisms for the retrieval and reuse of vesicle membranes. The cell must have a powerful reason to sanction apocrine rather than eccrine secretion. The most obvious advantage of apocrine secretion is that it may enable a very rapid release of large quantities of material from cells. We estimate that over half of the accumulated secretory granules in opaque zone cells are released in the first 5 min after a blood meal, and so it might reasonably be predicted that apocrine secretion would be used at this time. In fact, although we calculate that about 30% of the secretory granules are lost by apocrine secretion in this period, the bulk of this early release (about 70%) is by the eccrine secretion. If the measured decrease in cytoplasmic volume throughout the experimental period is exclusively related to apocrine secretion there should be a close correlation between cytoplasmic volume and the loss of secretory granules-this is not the case (Table 3).
A. R. WOODand M. J. LEHANE
164
Table 3
Time (min)
Secretory granule density in cell apex
0
0.45
5
0.22
12
0.22
18
0.24
Calculated loss of secretory granule volume with cytoplasm
Remainder of secretory granule volume lost
784
353
820
180
40
49
1907
“419” (275)
0
13
19
681
888
Cytoplasm/rer loss
54 25 Totals
Having measured the secretory granule density in the apex of the opaque zone cell, assuming that secretory granule density is the same in apical extrusions and knowing the cytoplasm loss over each time period, the volume of secretory granules potentially lost with that cytoplasm has been calculated. The remaining secretory granule volume lost over that time period (presumably by eccrine means) is also presented. All volumes in pm” per cell.
Nearly 75% of the secretory granules released into the gut lumen during the experimental period are secreted in the first 5 min after a blood meal, a time when only about 27% of the total decrease in cytoplasm occurs. In contrast the bulk (65%) of the decrease in cytoplasmic volume occurs during the period 12-18 min after a blood meal when there is a relatively minor (< 18%) loss of secretory granule volume. This calls into question whether apocrine secretion is the main cause of the decrease in cytoplasmic volume observed in these cells. It can reasonably be argued that the early losses in cytoplasm are due to apocrine secretion. However, given the minor levels of enzyme released between 12 and 18 min after a blood meal, it is unlikely that the major decreases in cytoplasm volume seen at this time could be due to apocrine secretion. This is particularly so as the cell has shown how effectively it can use the more energetically efficient eccrine secretory mechanisms.
Lehane (1976) has reported apical extrusions occurring up to 15 min after a blood meal. This is in accord with results of this study that show the majority (> 65%) of the decrease in cytoplasmic volume occurs between 12 and 18 min after a blood meal. As argued above this later decrease in cytoplasmic volume is unlikely to be associated with apocrine secretion as comparatively few secretory granules are now available for release. However, most of the cellular extrusions reported from insect midgut cells are from cells with few secretory granules in their apices. The purpose of such cellular extrusions is unknown. Of course, as well as producing apical extrusions, the cell may also be changing its volume by physiological means. After feeding there is a sharp increase in the synthetic activity of opaque zone cells. Morphologically this is mirrored in the considerable rearrangement of rough endoplasmic reticulum
Table 4 Time (min)
n
P
pm2/pm3
~m*/cell
0
6
0.863
0.591
7,656
5
5
0.072
0.68
7,612
12
4
0.572
0.75
8,279
18
6
0.529
0.95
8,264
25
4
0.093
0.80
6,849
Membrane added
Membrane lost
17,186
1,986
1,027
744
0 (3,693) 398
3,594 333
The area of the apical plasma membranes of the opaque zone cells is presented both before and at various times after feeding. Each estimate is a median from several flies (n). Kruskal-Wallace tests showed there were no significant differences between flies at the same time after feeding (P). Nor were there significant differences between flies at the different time intervals whether this is calculated as ~m2/~m3 (P = 0.208) or as pm2 per cell (P = 0.557). The calculated area of membrane added to the apical plasma membrane due to eccrine secretion and the loss due to apocrine secretion is also given for each time interval. The figure in parentheses is the membrane added if secretion occurs by eccrine means during this time interval.
Digestive enzyme secretion in Stomoxys which takes place (Lehane, 1976). These changes may well be accompanied by volumetric adjustments in the cell. There is also a change in the gut lumenal contents after feeding which may place osmotic stresses on the cells of the opaque zone causing volumetric changes. With the possibility of physiological changes in the volume of cellular compartments in mind it is interesting to note the lack of synchrony between the decreases in the volumes of mitochondria and cytoplasm (Table 1). It might be expected that the ratio between these two cellular compartments would remain more or less constant if decrease in cellular volume were due exclusively to the loss of cellular extrusions. This may suggest that some physiological adjustment of the volume of these organelles is taking place rather than their loss from the cell. In the case of mitochondria, which decrease in volume by > 40% by 40 min after a blood meal (Lehane, 1988a), any physiological change in overall volume is more likely to be caused by a change in the volume of individual mitochondria rather than a reduction in their number as there is no dramatic rise in lysosome numbers. It is estimated that twice the original area of the apical plasma membrane is added and removed from the apical plasma membrane in the first 5 min of secretion following the blood meal. Presumably this .is conserved in the cell by membrane recycling which is a common occurrence in secretory cells (Douglas and Nagasawa, 1971). The recovery rates calculated to occur in this system are remarkably fast. Frog neuromuscular junctions can recycle five times the surface area of the nerve terminus in the muscle over a 4 h period (Ceccarelli et al., 1973) or about half the plasma membrane area of end plates within 15 min of the secretory burst (Heuser and Reese, 1973). Mouse macrophages and L-cells can internalize an area equivalent to their surface area in 32 and 111 min, respectively (Steinman et al., 1973). In conclusion, this study suggests that opaque zone cells make efficient use of eccrine secretory mechanisms in the release of secretory material. Apocrine secretion is most likely to occur during the rapid secretory burst immediately following feeding when it may account for as much as 30% of the release taking place. Later decreases in cytoplasmic volume are less likely to be associated with secretory activity. REFERENCES
Andries J. C. (1976) Variations ultrastructurales au sein des cellules epitheliales mesenteriques d’deshna cyanea (Insecte, Odonate) en fonction de la prise de nourriture. Cytophiologie 13, 451-468. Baker J. E., Woo S. M. and Byrd R. V. (1984) Ultrastructural features of the gut of Sitophilus granarius (L) (Coleoptera: Curculionidae) with notes on distribution of proteinases and amylases in crop and midgut. Can. J. 2001. 62, 1251-1259. Bayon C. (1981) Ultrastructure d’epithelium intestinal et flore parientale chez le larve xylophage d’&yctes
165
nasicornis L. (Coleoptera : Scarbaeidae). J. Insect Morph. Embryol. 10, 359-371. Bell M. (1974) The ultrastructure of human axilary apocrine glands after epinephrine injection. J. invest. Derm. 63, 147-159. Brunings E. A. and de Priester W. (1971) Effects of mode of fixation on formation of extrusions in the midgut epithelium of Calliphora erythrocephalu (Diptera : Calliphoridea). Cytobiologie 4, 487492. Buchmann W. W. (1928) Zellveranderungen im mitteldarm wahrend der sekretion. Zool. Anz. 79, 223-243. Case R. M. (1978) Synthesis, intracellular transport and discharge in the pancreatic acinar cell and other cells. Biol. Rev. 53, 211-354. Ceccarelli B., Hurlbut W. P. and Mauro A. (1973) Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction. J. Ck Biol. 57, 499-524. Chandler D. E. (1979) Quick-freezing avoids specimen preparation artefacts in membrane fusions studies. In Freeze Fraclure. Methods, Artefacts and Interpretations (Eds Rash J. C. and Hudson C. S.). Raven Press, New York. Chandler D. E. and Heuser J. E. (1980) Arrest of membrane fusion events in mast cells by quick freezing. J. Cell Biol. 86, 666-674. Cragg F. W. (1920) Secretion and epithelial regeneration in the mid intestine of Tabanus. Indian J. med. Res. 7. 648-663. Day M. F. and Powning F. G. (1949) A study of the process of digestion in certain insects. Aust. J. scient. Res. 149B, 175-215. Deegener P. (1909) Beitrage zur kenntnis der darmsekretion (Deilephila euphorbia). Arch. Naturgesch. 75, 71-110. Douglas W. W. and Nagasawa J. (1971) Membrane vesiculation at sites of exocytosis in neurohyposphysis, adenohypophysis and adrenal medulla; a device for membrane conservation. J. Physiol. 218, l-94. Fain-Maurel M. A., Cassier P. and Alibert J. (1973) Etude infrastructurale et cytochimique de l’intestine moyen de Petrobius maritimus-Leach en rapport avec ses fonctions excretrices et digestives. Tissue Cell 5. 603-631. van Gehuchten A. (1890) Recherches’ histologiques sur l’appareil digestif de Ptychoptera contaminata. Cellule 6, 185-290. Graham-Smith G. S. (1934) The alimentary canal of Culliphora erythrocephnla L. with special references to its musculature and the proventriculus, rectal valve and rectal papillae. Parasitology 26, 176248. Hastv D. L. and Hav E. D. 11978) \ , Freeze-fracture studies of the developing cell surface--II. Particle-free membrane blisters on glutaraldehyde fixed cornea1 fibroblasts are artefacts. J. Cell Biol. 78, 756-768. Heath E. (1974) Cytological observations on the secretory cells of the chin (submandibular) gland and brown inguinal gland in the rabbit. Cell Tissue Res. 154, 399408. Heuser J. E. and Reese T. S. (1973) Evidence of recycling of synaptic vesicle membrane during transmitter release at frog neuromuscular junctions. J. Cell Biol. 57, 315-344. Hirsch J. G. and Fedorko M. E. (1968) Ultrastructure of human leucocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and ‘post-fixation’ in uranyl acetate. J. Cell Biol. 38, 615-627. Khan M. R. and Ford J. B. (1962) Studies of digestive enzyme production and its relationship to the cytology of the mid-gut epithelium in Dysdercus fasciatus Sign. (Hemiptera, Pyrrhocordiae). J. Insect Physiol. 8, 597-608. Lehane M. J. (1976) Digestive enzyme secretion in Stomoxys calcitrans (Diptera: Muscidae). Cell Tissue Res. 170, 275-287. Lehane M. J. (1987) Quantitative evidence for merocine secretion in insect midgut cells. Tissue Cell 19, 451461.
166
A. R.
WQOD
and M. J.
Lehane M. J. (1988a) Evidence for secretion by the production of cytoplasmic extrusions in the midgut cells of the stablefly, Stomoxys calcitrans J. Insect Physiol. 34, 949-953. Lehane M. J. (1988b) Size of secretory granules from midgut cells of the stablefly, Stomoxys calcitrans. Tissue Cell 20, 763-770. Lehane M. J. (1989) The intracellular pathway and kinetics of digestive enzyme secretion in an insect midgut cell. Tissue Cell 21, 101-111. Moffatt M. A. and Lehane M. J. (1990) Trypsin is stored as an inactive zymogen in the midgut of Stomoxys calcitrans. Insect Biochem. 20, 719-723. Pradhan S. (1934) The alimentary canal and pro-epithelial regeneration in Coccinella septempunctata with a comparison of carnivorous coccinellids. Q. JI microsc. Sci. 81, 451-478.
bHANE
Smith C. S. and Guttman L. (1953) Measurement of internal boundaries in three dimensional structures by random sectioning. Trans. Am. Inst. Min. metall. Engrs 197, 81. Steinman R. M., Brodie S. E. and Cohn Z. A. (1976) Membrane flow during pinocytosis. A stereological analysis. J. Cell biol. 68, 665-687. Weber H. (1928) Skelett, musculatur und darm der schwarzen blattlaus. Aphis fabae, Zoologica 28, l-20. Weibel E. R. (1969) Stereological principles for morphometry in electron microscopical cytology. Reu. Cytol. 26, 235-302. Wigglesworth V. B. (1929) Digestion in the tsetse fly: a study of structure and function. Parasitology 21, 288-321. Williams M. A. (1977) Quantitative methods in biology. In Practical Methods in Electron Microscopy (Ed. Glauert A. M.).
Vol. 37, No. 2, pp. 161-166,1991 Printed in Great Britain. All rights reserved
J. Imecr Physiol.
RELATIVE CONTRIBUTIONS OF APOCRINE AND ECCRINE SECRETION TO DIGESTIVE ENZYME RELEASE FROM MIDGUT CELLS OF STOMOXYS CALCIZ-‘RANS (INSECTA: DIPTERA) A. R. Woon and M. J. LEHANE* School of Biological Sciences, University of Wales, Bangor, Gwynedd LL57 2UW, U.K. (Received 2 October 1990; revised 27 November 1990)
Abstract-A morphometric analysis of the secretory process in the midgut opaque zone cells of the stablefly, Stomoxys calcitrans, shows that over half the stored secretory granules are released from the cell in the first 5 min after the blood meal. The analysis suggests that the majority of digestive enzyme secretion is achieved by eccrine means. Apocrine secretion may play a substantial role in the early burst of secretory activity which immediately follows the blood meal. Assuming that membrane recycling follows eccrine secretion from these cells the membrane recovery rates calculated (1.99 times the apical surface area of the cell in 5 min) are remarkably fast. Key Word Index: Digestive enzyme; secretion; tnorphometrics;
INTRODUCTION Secretion of digestive enzymes by the regulated route has been demonstrated in the opaque zone cells of the midgut of the stable fly Stomoxys calcitrans (Lehane,
1976; 1987; 1988a; 1989). It is clear that these enzymes can be released by the classical route of eccrine fusion of the secretory granule with the apical plasma membrane (Lehane, 1976). On histological grounds Lehane (1976) proposed that secretory granules could also be released from the cell contained in cytoplasmic extrusions (i.e. by apocrine secretion). Many other authors have also presented evidence that insects release cellular extrusions into the lumen of the midgut (for example van Gehuchten, 1890; Deegener, 1909; Cragg, 1920; Buchmann, 1928; Weber, 1928; Wigglesworth, 1929; Graham-Smith, 1934; Pradhan, 1934; Fain-Maurel et al., 1973; Andries, 1976; Bayon, 1981; Baker et al., 1984) and some have claimed that these represent the apocrine secretion of digestive enzymes. Other authors have produced evidence that apical extrusions seen in insect midgut cells are not related to the secretion of digestive enzymes (Day and Powning, 1949; Khan and Ford, 1962; Brunings and de Priester, 1971). Because apical extrusions can be produced as fixation artifacts (Brunings and de Priester, 1971; Bell, 1974; Heath, 1974; Hasty and Hay, 1978; Chandler, 1979; Chandler and Heuser, 1980) it was clear that the debate on the role of apical extrusions in digestive enzyme secretion could not be settled on direct *To whom all correspondence
should be addressed.
Stomoxys
histological evidence and so Lehane (1987; 1988a) took a different approach. He argued that if apocrine secretion was occurring in the opaque zone cells of S. calcitrans then there would be a considerable drop in the volume of the cytoplasm and mitochondria as well as a considerable drop in the associated membrane area of the cells following the burst of enzyme secretion immediately following the blood meal. By quantitatively describing the opaque zone cells before and after the period when apocrine secretion was presumed to occur (thus avoiding any possibility that apical extrusions could be produced as fixation artifacts) Lehane (1987, 1988) established that there was indeed a 34% fall in the volume of the cytoplasm, a 41% fall in the volume of mitochondria and concomitant falls in the area of cell membranes. This suggested that apocrine secretion may play a part in the release of digestive enzymes from the opaque zone cells of S. culcitrans. In this study the relative contributions that apocrine and eccrine secretion make during enzyme release are investigated. MATERIALS AND METHODS S. calcitrans were cultured in the laboratory at 26-28°C in a 12 h light-12 h dark cycle. Larvae were reared in a mixture of 0.5 litre measure each of bran, hardwood chips and Molichop (TM) (a mixture of hay, straw and molasses used as horse feed) to which is added one tablespoon of malt extract, 10 yeast tablets and 1 litre of water. Adults in the colony were fed daily on cotton wool swabs soaked with 161
162
A. R. WOOD and M. J. LEHANE
heparinized pig blood. The adult to adult cycle is approx. 16 days. Adult male flies, 24 h after eclosion, were used in the experiments. Flies were sacrificed unfed or at 5, 12, 18 or 25 min after a blood meal. A systematic, stratified sampling procedure was employed (Williams, 1977). Six flies were used in each treatment. They were anaesthetized with carbon dioxide gas and dissected under cold fixative. Portions of midgut containing the opaque zone were excised and transferred to fresh fixative at 4°C. Guts from unfed flies were fixed for 1 h at 4°C in 3% glutaraldehyde in 0.1 M sodium cacodylate/HCl buffer pH 7.1. After rinsing in 0.1 M sodium cacodylate buffer pH 7.1 containing 5% sucrose, unfed guts were post-fixed in 1% osmium tetroxide for 1 h. This is an unsuccessful fixation procedure for guts from blood fed flies (Lehane, 1976) which were fixed for 1 h at 4°C in a combination fixative (Hirsch and Fedorko, 1968; Brunings and de Priester, 1971) consisting of 0.5 % OsO,, 1.25% glutaraldehyde and 2% sucrose in 0.1 M sodium cacodylate buffer, pH 7.1. Tissues were dehydrated in a graded series of ethanol and embedded in Spurr resin. Two ribbons of gold/silver sections were cut every 150 ,um through the length of the opaque zone and stained in lead citrate and alcoholic uranyl acetate before being viewed and photographed in an AEI Corinth 275 electron microscope at a nominal magnification of x 4,000. Actual magnifications were calculated after photographing a diffraction grating inscribed with 2160 lines mm-‘. Fields to be photographed were randomly selected by choosing only those areas of tissue which lay closest to the upper right hand corner of the mesh square of the copper grid. Because the tissue is anisotropic the photographic sample taken from each selected field was a column of photographs extending vertically from the basal lamina of the cells to the lumenal surface with care being taken to avoid overlap in the photographs comprising each columnar sample (Weibel, 1969). A positive transparency was made of each electron micrograph. These were analysed by projection onto a 45 x 45 cm screen on which was drawn the analysis grid. This consisted of 137 points arranged as,equally spaced equilateral triangles. Point counts were used to determine volume fractions (Vu) of the nucleus, mitochondria, microvilli, secretory granules and cytoplasm including the rough endoplasmic reticulum and Golgi. Each micrograph was analysed for a second time after rotation through 90”. The final magnification of the electron micrograph on the screen was calculated to be x 33,700 using the calibration micrograph of the diffraction grating and knowing the projection magnification. To calculate the concentration of secretory granules at the apical border of the cells, a separate analysis was performed by using only the final apical electron micrograph from each column. From this sub-sample
the volume fraction of secretory granules and cytoplasm were calculated. To determinate areas of apical plasma membrane the micrographs were projected onto a second analysis screen consisting of 18 7 cm lines drawn at an angle of 19” to the vertical. Final projection magnification was calculated to be x 29,900. To reduce bias due to the anisotropic nature of the cells, counts were also made after rotation of the negative through 90”. Surface density of membranes (Sv) was then calculated from the line cut data using the formula Sv = 2P, (Smith and Guttman, 1953). Data from the six experimental animals used at each experimental time are grouped for each feeding regime. Statistical calculations have been carried out on a DEC Vax computer using the statistical package Minitab. RESULTS
To test for quantitative differences between the flies which made up each Vu sample an analysis of variance was carried out. The data was non-normally distributed as shown by a normal scores analysis. Consequently we performed a Kruskal-Wallace analysis of the Vu estimates for the six flies measured at each sampling time. This revealed no significant variation in secretory granules or microvilli among individuals. Significant variation among individuals was observed only in cytoplasm/rer at 5 min (P = 0.021) and 18 min after a blood meal (P = 0.035), mitochondria at 5 min (P = 0.028) and 25 min after a blood meal (P = 0.04) and nucleus at 25 min after a blood meal (P = 0.02). Mitochondrial and cytoplasmic components of the cell are undergoing extreme changes in volume during this period (Lehane, 1987; 1988a) and relatively small differences in the timings of these events are likely to lead to the differences observed between flies. The volume fractions for each cellular compartment have been converted to real volumes by using the opaque cell nuclear volumes measured by Lehane (1988a). These are presented in Table 1. The time dependent changes after a blood meal in the volumes of the key organelles (cytoplasm and secretory granules) are presented in Table 2. Clearly there is a very large early loss of secretory granules whereas the largest losses in cytoplasmic volume occur later in the secretory period. The density of secretory granules in the cytoplasm of the apex of the cell are presented in Table 3. If we assume that the density of secretory granules in apical extrusions is the same as in the cell apex, and the histological evidence suggests this is so, then knowing the cytoplasmic loss over each time interval (Table 2) we can calculate the expected loss of secretory granules with this cytoplasm during apocrine secretion (Table 3). The conclusion is that the remainder of the secretory granules lost in that period are released by eccrine secretion (Table 3).
Digestive enzyme secretion in
163
Stomoxys
Table 1 Time @iin) 0 5 12 18 25
Point counts
Secretory granules
12,656 16,720 18,008 9,009 10,779
2,154 981 892 617 585
Cytoplasm/rer
Nucleus
Mitochondria
Microvilli
Mean cell volume
9,311 8,527 8,347 6,440 6,386
565 599 634 654 666
524 571 586 568 450
423 517 580 420 475
12,977 11,195 11,039 8,699 8,562
The real volumes of the cellular organelles at the different sampling times are presented in pm3 per cell There is a complication with the analysis as presented because it assumes that the volume of secretory granules remains static throughout the period investigated and this is not so. Although there is a considerable build up in secretory granules in the unfed fly there is in fact a continuous secretion of these granules at a low level (Moffatt and Lehane, 1990) as is the case in vertebrate cells undertaking regulated secretion (Case, 1978). This low level production of new secretory granules will mean that there is a small underestimate (probably less than 10%) in the overall prediction of secretory granules secreted over the experimental period. The consequences of this underestimate for the analysis are that for each experimental period (except 12-18 min when no eccrine release is calculated to take place) the amount of eccrine secretion taking place will be underestimated. There is no underestimate in the amount of apocrine secretion calculated to occur because this is fixed by the decrease in volume ;of cytoplasm and will be insensitive to the error outlined above. This underlying low level of production of new secretory granules is greatly accelerated once the blood meal is taken but this should not interfere with the analysis as new secretory granules induced in this way do not begin to appear until about 24-26min after a blood meal (Lehane, 1989). The area of apical plasma membrane in the opaque zone cells at the various times in the feeding cycle are presented in Table 4. Kruskal-Wallace analysis shows there were no significant differences between the area of the apical plasma membrane at any of the time intervals whether this is calculated as prn2/pm3 (P = 0.208) or as pm2 per cell (P = 0.557). The cell clearly painstakingly conserves the area of the apical plasma membrane despite the considerable changes in the cell during this period of intense activity. This agrees with the study of Lehane (1988a) who Table 2 Time @in) o-5 5-12 12-18 18-25
Secretory granules
Cytoplasm/rer
1,173 89 275 32
784 180 1,907 54
Losses over time in secretory granule volume km3 per cell) and cytoplasm/rer volume have been calculated from the information given in Table 1.
found that the quantity of apical plasma membrane appeared to be carefully conserved. Eccrine secretion will lead to an increase in the area of the apical plasma membrane. In contrast apocrine secretion will lead to the loss of apical plasma membrane. Knowing the size of the secretory granules (Lehane, 1988b) and having calculated the total volume of secretory granule lost during each sampling interval the area of secretory granule membrane added to the apical plasma membrane can be calculated for each sampling interval (Table 4). Calculations of the loss of apical plasma membrane due to apocrine secretion will be less accurate. This is because although the total volume of cytoplasm/rer lost is known the shape and diameter of the extruded vesicle is not known. In Table 4 we have estimated the area of apical plasma membrane lost for the simplest possible case where all the cytoplasm/rer lost in that sample interval is assumed to bud off as one vesicle which is completely covered in microvilli. This is likely to underestimate losses because the increased volume/area associated with smaller vesicles will lead to increased losses of apical plasma membrane. DISCUSSION
It is a reasonable assumption that apocrine secretion, involving as it does the loss of considerable quantities of cellular material in addition to the secretory product, is energetically less favourable to the cell than eccrine secretion with its built in mechanisms for the retrieval and reuse of vesicle membranes. The cell must have a powerful reason to sanction apocrine rather than eccrine secretion. The most obvious advantage of apocrine secretion is that it may enable a very rapid release of large quantities of material from cells. We estimate that over half of the accumulated secretory granules in opaque zone cells are released in the first 5 min after a blood meal, and so it might reasonably be predicted that apocrine secretion would be used at this time. In fact, although we calculate that about 30% of the secretory granules are lost by apocrine secretion in this period, the bulk of this early release (about 70%) is by the eccrine secretion. If the measured decrease in cytoplasmic volume throughout the experimental period is exclusively related to apocrine secretion there should be a close correlation between cytoplasmic volume and the loss of secretory granules-this is not the case (Table 3).
A. R. WOODand M. J. LEHANE
164
Table 3
Time (min)
Secretory granule density in cell apex
0
0.45
5
0.22
12
0.22
18
0.24
Calculated loss of secretory granule volume with cytoplasm
Remainder of secretory granule volume lost
784
353
820
180
40
49
1907
“419” (275)
0
13
19
681
888
Cytoplasm/rer loss
54 25 Totals
Having measured the secretory granule density in the apex of the opaque zone cell, assuming that secretory granule density is the same in apical extrusions and knowing the cytoplasm loss over each time period, the volume of secretory granules potentially lost with that cytoplasm has been calculated. The remaining secretory granule volume lost over that time period (presumably by eccrine means) is also presented. All volumes in pm” per cell.
Nearly 75% of the secretory granules released into the gut lumen during the experimental period are secreted in the first 5 min after a blood meal, a time when only about 27% of the total decrease in cytoplasm occurs. In contrast the bulk (65%) of the decrease in cytoplasmic volume occurs during the period 12-18 min after a blood meal when there is a relatively minor (< 18%) loss of secretory granule volume. This calls into question whether apocrine secretion is the main cause of the decrease in cytoplasmic volume observed in these cells. It can reasonably be argued that the early losses in cytoplasm are due to apocrine secretion. However, given the minor levels of enzyme released between 12 and 18 min after a blood meal, it is unlikely that the major decreases in cytoplasm volume seen at this time could be due to apocrine secretion. This is particularly so as the cell has shown how effectively it can use the more energetically efficient eccrine secretory mechanisms.
Lehane (1976) has reported apical extrusions occurring up to 15 min after a blood meal. This is in accord with results of this study that show the majority (> 65%) of the decrease in cytoplasmic volume occurs between 12 and 18 min after a blood meal. As argued above this later decrease in cytoplasmic volume is unlikely to be associated with apocrine secretion as comparatively few secretory granules are now available for release. However, most of the cellular extrusions reported from insect midgut cells are from cells with few secretory granules in their apices. The purpose of such cellular extrusions is unknown. Of course, as well as producing apical extrusions, the cell may also be changing its volume by physiological means. After feeding there is a sharp increase in the synthetic activity of opaque zone cells. Morphologically this is mirrored in the considerable rearrangement of rough endoplasmic reticulum
Table 4 Time (min)
n
P
pm2/pm3
~m*/cell
0
6
0.863
0.591
7,656
5
5
0.072
0.68
7,612
12
4
0.572
0.75
8,279
18
6
0.529
0.95
8,264
25
4
0.093
0.80
6,849
Membrane added
Membrane lost
17,186
1,986
1,027
744
0 (3,693) 398
3,594 333
The area of the apical plasma membranes of the opaque zone cells is presented both before and at various times after feeding. Each estimate is a median from several flies (n). Kruskal-Wallace tests showed there were no significant differences between flies at the same time after feeding (P). Nor were there significant differences between flies at the different time intervals whether this is calculated as ~m2/~m3 (P = 0.208) or as pm2 per cell (P = 0.557). The calculated area of membrane added to the apical plasma membrane due to eccrine secretion and the loss due to apocrine secretion is also given for each time interval. The figure in parentheses is the membrane added if secretion occurs by eccrine means during this time interval.
Digestive enzyme secretion in Stomoxys which takes place (Lehane, 1976). These changes may well be accompanied by volumetric adjustments in the cell. There is also a change in the gut lumenal contents after feeding which may place osmotic stresses on the cells of the opaque zone causing volumetric changes. With the possibility of physiological changes in the volume of cellular compartments in mind it is interesting to note the lack of synchrony between the decreases in the volumes of mitochondria and cytoplasm (Table 1). It might be expected that the ratio between these two cellular compartments would remain more or less constant if decrease in cellular volume were due exclusively to the loss of cellular extrusions. This may suggest that some physiological adjustment of the volume of these organelles is taking place rather than their loss from the cell. In the case of mitochondria, which decrease in volume by > 40% by 40 min after a blood meal (Lehane, 1988a), any physiological change in overall volume is more likely to be caused by a change in the volume of individual mitochondria rather than a reduction in their number as there is no dramatic rise in lysosome numbers. It is estimated that twice the original area of the apical plasma membrane is added and removed from the apical plasma membrane in the first 5 min of secretion following the blood meal. Presumably this .is conserved in the cell by membrane recycling which is a common occurrence in secretory cells (Douglas and Nagasawa, 1971). The recovery rates calculated to occur in this system are remarkably fast. Frog neuromuscular junctions can recycle five times the surface area of the nerve terminus in the muscle over a 4 h period (Ceccarelli et al., 1973) or about half the plasma membrane area of end plates within 15 min of the secretory burst (Heuser and Reese, 1973). Mouse macrophages and L-cells can internalize an area equivalent to their surface area in 32 and 111 min, respectively (Steinman et al., 1973). In conclusion, this study suggests that opaque zone cells make efficient use of eccrine secretory mechanisms in the release of secretory material. Apocrine secretion is most likely to occur during the rapid secretory burst immediately following feeding when it may account for as much as 30% of the release taking place. Later decreases in cytoplasmic volume are less likely to be associated with secretory activity. REFERENCES
Andries J. C. (1976) Variations ultrastructurales au sein des cellules epitheliales mesenteriques d’deshna cyanea (Insecte, Odonate) en fonction de la prise de nourriture. Cytophiologie 13, 451-468. Baker J. E., Woo S. M. and Byrd R. V. (1984) Ultrastructural features of the gut of Sitophilus granarius (L) (Coleoptera: Curculionidae) with notes on distribution of proteinases and amylases in crop and midgut. Can. J. 2001. 62, 1251-1259. Bayon C. (1981) Ultrastructure d’epithelium intestinal et flore parientale chez le larve xylophage d’&yctes
165
nasicornis L. (Coleoptera : Scarbaeidae). J. Insect Morph. Embryol. 10, 359-371. Bell M. (1974) The ultrastructure of human axilary apocrine glands after epinephrine injection. J. invest. Derm. 63, 147-159. Brunings E. A. and de Priester W. (1971) Effects of mode of fixation on formation of extrusions in the midgut epithelium of Calliphora erythrocephalu (Diptera : Calliphoridea). Cytobiologie 4, 487492. Buchmann W. W. (1928) Zellveranderungen im mitteldarm wahrend der sekretion. Zool. Anz. 79, 223-243. Case R. M. (1978) Synthesis, intracellular transport and discharge in the pancreatic acinar cell and other cells. Biol. Rev. 53, 211-354. Ceccarelli B., Hurlbut W. P. and Mauro A. (1973) Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction. J. Ck Biol. 57, 499-524. Chandler D. E. (1979) Quick-freezing avoids specimen preparation artefacts in membrane fusions studies. In Freeze Fraclure. Methods, Artefacts and Interpretations (Eds Rash J. C. and Hudson C. S.). Raven Press, New York. Chandler D. E. and Heuser J. E. (1980) Arrest of membrane fusion events in mast cells by quick freezing. J. Cell Biol. 86, 666-674. Cragg F. W. (1920) Secretion and epithelial regeneration in the mid intestine of Tabanus. Indian J. med. Res. 7. 648-663. Day M. F. and Powning F. G. (1949) A study of the process of digestion in certain insects. Aust. J. scient. Res. 149B, 175-215. Deegener P. (1909) Beitrage zur kenntnis der darmsekretion (Deilephila euphorbia). Arch. Naturgesch. 75, 71-110. Douglas W. W. and Nagasawa J. (1971) Membrane vesiculation at sites of exocytosis in neurohyposphysis, adenohypophysis and adrenal medulla; a device for membrane conservation. J. Physiol. 218, l-94. Fain-Maurel M. A., Cassier P. and Alibert J. (1973) Etude infrastructurale et cytochimique de l’intestine moyen de Petrobius maritimus-Leach en rapport avec ses fonctions excretrices et digestives. Tissue Cell 5. 603-631. van Gehuchten A. (1890) Recherches’ histologiques sur l’appareil digestif de Ptychoptera contaminata. Cellule 6, 185-290. Graham-Smith G. S. (1934) The alimentary canal of Culliphora erythrocephnla L. with special references to its musculature and the proventriculus, rectal valve and rectal papillae. Parasitology 26, 176248. Hastv D. L. and Hav E. D. 11978) \ , Freeze-fracture studies of the developing cell surface--II. Particle-free membrane blisters on glutaraldehyde fixed cornea1 fibroblasts are artefacts. J. Cell Biol. 78, 756-768. Heath E. (1974) Cytological observations on the secretory cells of the chin (submandibular) gland and brown inguinal gland in the rabbit. Cell Tissue Res. 154, 399408. Heuser J. E. and Reese T. S. (1973) Evidence of recycling of synaptic vesicle membrane during transmitter release at frog neuromuscular junctions. J. Cell Biol. 57, 315-344. Hirsch J. G. and Fedorko M. E. (1968) Ultrastructure of human leucocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and ‘post-fixation’ in uranyl acetate. J. Cell Biol. 38, 615-627. Khan M. R. and Ford J. B. (1962) Studies of digestive enzyme production and its relationship to the cytology of the mid-gut epithelium in Dysdercus fasciatus Sign. (Hemiptera, Pyrrhocordiae). J. Insect Physiol. 8, 597-608. Lehane M. J. (1976) Digestive enzyme secretion in Stomoxys calcitrans (Diptera: Muscidae). Cell Tissue Res. 170, 275-287. Lehane M. J. (1987) Quantitative evidence for merocine secretion in insect midgut cells. Tissue Cell 19, 451461.
166
A. R.
WQOD
and M. J.
Lehane M. J. (1988a) Evidence for secretion by the production of cytoplasmic extrusions in the midgut cells of the stablefly, Stomoxys calcitrans J. Insect Physiol. 34, 949-953. Lehane M. J. (1988b) Size of secretory granules from midgut cells of the stablefly, Stomoxys calcitrans. Tissue Cell 20, 763-770. Lehane M. J. (1989) The intracellular pathway and kinetics of digestive enzyme secretion in an insect midgut cell. Tissue Cell 21, 101-111. Moffatt M. A. and Lehane M. J. (1990) Trypsin is stored as an inactive zymogen in the midgut of Stomoxys calcitrans. Insect Biochem. 20, 719-723. Pradhan S. (1934) The alimentary canal and pro-epithelial regeneration in Coccinella septempunctata with a comparison of carnivorous coccinellids. Q. JI microsc. Sci. 81, 451-478.
bHANE
Smith C. S. and Guttman L. (1953) Measurement of internal boundaries in three dimensional structures by random sectioning. Trans. Am. Inst. Min. metall. Engrs 197, 81. Steinman R. M., Brodie S. E. and Cohn Z. A. (1976) Membrane flow during pinocytosis. A stereological analysis. J. Cell biol. 68, 665-687. Weber H. (1928) Skelett, musculatur und darm der schwarzen blattlaus. Aphis fabae, Zoologica 28, l-20. Weibel E. R. (1969) Stereological principles for morphometry in electron microscopical cytology. Reu. Cytol. 26, 235-302. Wigglesworth V. B. (1929) Digestion in the tsetse fly: a study of structure and function. Parasitology 21, 288-321. Williams M. A. (1977) Quantitative methods in biology. In Practical Methods in Electron Microscopy (Ed. Glauert A. M.).