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Calcium containing lysosomes in the normal chick duodenum: A histochemical and analytical electron microscopic study
TISSUE &CELL 1979 11 (1) 127-138 Published hr Longman Group Ltd. Printed in Gwat Britain
WALTER
L. DAVIS,
RUTH G. JONES and HERBERT K. HAGLER
CALCIUM CONTAINING LYSOSOMES NORMAL CHICK DUODENUM: A HISTOCHEMICAL AND ANALYTICAL ELECTRON MICROSCOPIC STUDY
IN THE
ABSTRACT. Absorptive cells of the normal chick duodenum contain numerous supranuclear vesicular/vacuolar structures. By routine transmission electron microscopy, such structures are membrane bound and demonstrate a granular content. These vesicles appear to move laterally and eventually coalesce with the lateral plasma membrane (exocytosis). The granular contents are resistant to high temperature microincineration, thus revealing their mineral-containing nature. The granular vesicular matrix also stains intensely with osmium pyroantimonate. EGTA chelation of pyroantimonate-stained vesicles selectively extracts the granules indicating a high concentration of calcium. X-ray microanalysis also demonstrates a significant intravesicular calcium localization. When tissues were incubated for the presence of acid phosphatase, the supranuclear vesicles were markedly positive for this lysosomal enzyme. A possible role for these calcium-containing lysosomes in the transcellular flux of calcium ions across the intestinal absorptive cell is discussed.
Introduction
and chick intestine (Warner and Coleman, 197.5). However, due to the limitations of microprobe resolution utilized in the latter study, no ultrastructural description of these sites was reported. We describe here the identification of intracellular calcium vesicles in absorptive cells of normal chick duodenum using routine electron microscopy, electron microscopic histochemistry, high temperature microincineration and analytical electron microscopy. Such vesicles also demonstrate acid phosphatase activity, indicative of their lysosomal nature.
PREVIOUSinvestigations, using a wide variety of morphologic techniques, have attempted to localize intra- and extracellular calcium binding sites in the intestinal epithelia of various species (Kashiwa and Atkinson, 1963; Sampson, et al., 1970; Oschman and Wall, 1972, I973 ; Schafer, 1973 ; Davis and Jones, 1976; Warner and Coleman, 1975; Halloran and Coleman, 1977; Davis et al., 1978). These studies demonstrated significant calcium accumulations at the level of the microvillar and lateral plasma membranes, the mitochondria, within membrane bound vesicles, and in association with intra- and extracellular goblet cell mucus. Additionsupranuclear and lateral calcium ally, localizations were identified by electron probe analysis in the absorptive cells of rat
Materials and Methods Four to six week old male White Leghorn chicks (Hornung Hatchery, Sherman, Texas), weighing between 300 and 600 g, were used for this investigation. Animals were maintained from I day post-hatching in temperature-regulated brooders and fed Startena (Ralston Purina Co.) and water ad libitum.
Departments of Microscopic Anatomy and Pathology, Baylor College of Dentistry and the University of Texas Health Science Center, Southwestern Medical School, Dallas, Texas. Received 3 July 1978. Revised 2 November 1978. 127
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At sacrifice, chicks were decapitated and exsanguinated. Blood was collected, allowed to clot, and the serum frozen for subsequent calcium analysis by atomic absorption spectrophotometry. The duodenal loop was surgically exposed. Both the proximal and distal segments were tied off (distally) and infused by syringe with the appropriate fixative (see below). Prior to removing the syringe, the intestinal segments were tied off (proximally) to form 2-4 cm duodenal ‘sausages’ containing the infused fixative. The tied segments were removed and placed in their respective fixative. The ‘sausages’ were subsequently diced under fixative into 1 mm thick closed loops. The fixatives used were: (1) phosphate buffered formalin, 4% (Carson et al., 1973), followed by osmication in phosphate buffered 1% 0~04; (2) 3 % glutaraldehyde buffered with 0.08 M s-collidine and containing 5 mM CaCls and 5 % sucrose (Oschman and Wall, 1972); post-osmication was in s-collidine buffered osmium tetroxide; (3) unbuffered 1% osmium tetroxide containing 2% potassium pyroantimonate (Carson et al., 1978). All tissues were dehydrated in graded ethanols and flat embedded in Spurr medium (Spurt-, 1969). For orientation purposes, 1 pm sections of Epoxy embedded tissues were cut with a glass knife on a Porter-Blum MT-2 ultramicrotome (Du Pont Instruments, Sorvall Operations, Newtown, Conn.). These were stained with Paragon (Martin et al., 1967). Following light microscopic evaluation, specimens were trimmed so that only the outer one-third of a single villus was thin sectioned for electron microscopy. Ultrathin sections, cut with a diamond knife and showing silver to gold interference colors (600-700 A), were picked up on 200 mesh uncoated copper grids and examined either unstained or doubly stained with uranyl acetate and lead citrate (Reynolds, 1963) in a Philips 300 electron microscope (Philips Electronics Instruments, Inc., Mt Vernon, N.Y.) operated at 40-300 kV. EGTA chelation
Unviewed, unstained sections of osmium pyroantimonate-fixed tissues mounted on copper grids were floated on solutions of 20 mM ethylene glycol bis-(B-amino ethyl ether)-N,N’-tetraacetic acid (EGTA), pH 7.6 7.8, for 30-60 min at 60°C. Grids were then
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thoroughly washed in deionized water, air dried, and viewed. High temperature microincineration
Ultrathin sections of either formalin or glutaraldehyde fixed, osmicated or nonosmicated tissues, were mounted on Formvar -silicon monoxide coated stainless steel grids (Thomas and Greenawalt, 1968). The unstained grids were then placed in a 500°C muffle furnace for 10-l 5 min. Energy dispersive X-ray spectroscopic analysis
For analytical electron microscopy, dark gold sections (approximately 1000-1200 8, thick) of glutaraldehyde fixed non-osmicated tissues were mounted on 200 mesh copper grids, left unstained, and subsequently carbon coated. Sections were viewed with a JEOL 100 C transmission electron microscope equipped with a JEOL high resolution scanning attachment STEM (JEOL, U.S.A., Medford, Mass.) (ASID), and a Kevex 30 mm2, 158 eV resolution, lithium-drifted silicon, energy dispersive X-ray detector (Kevex Corp., Burlingame, Calif.) placed within 20 mm of the sample through the objective pole piece (Hagler et al., 1977). Analyses were performed for 20 sec. X-ray counts were processed via a Tracer Northern NS 880 multichannel analyzer (Tracer Northern, Middleton, Wis.) operated at 20 eV/channel (Hagler et al., 1977). Ten vesicles were analyzed for 20 set each and their spectra were summed. The summed spectra were then subjected to a multipleleast squares fitting routine using reference spectra obtained from single crystal specimens (Shuman et al., 1976). The analyses of spectra were performed with the DEC PDP 1l/O5 computer-based Tracer Northern NS 880 unit. Acid phosphatase histochemistry
To demonstrate the presence of lysosomes, segments of duodenum were fixed in 0.1 M cacodylate buffered cold (4°C) glutaraldehyde (2.5%) for 4 hr. Specimens were washed overnight in cold buffer, then washed several times in 7.5 % sucrose prior to freezing on an IEC model CT1 cryostat (International Equipment Co., Needham. Mass.). Frozen sections, cut at 40 pm, were collected in the chilled sucrose wash and subsequently incubated in Novikoff’s CMP medium for
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the demonstration of acid phosphatase (Novikoff, 1963). Samples were incubated at room temperature for 1 hr with two changes of medium. Following incubation, tissues were washed with 7.5 o/o sucrose and osmicated in cacodylate buffered osmium tetroxide prior to embedding in Spurr medium. Results Serum calcium analyses All animals utilized in these studies had serum calcium values between 9 and 11 mg%. General description Numerous ovoid vesicles/vacuoles containing significant accumulations of electron dense granules were clearly identified clustered in the supranuclear absorptive cell cytoplasm of normal chick intestinal epithelium (Figs. l-4). Similar structures were not seen within the terminal web, or below the level of the cell nucleus. Additionally, such entities were not identified in goblet cells. Such vesicles were membrane bound (Fig. 2), generally located in close proximity to both mitochondria and endoplasmic reticulum cisternae (Figs. l-3), and frequently appeared to fuse or coalesce with the lateral plasma membrane at the supranuclear level of the lateral intercellular space (Fig. 3). These structures ranged in size from less than 0.1 pm to greater than 1.0 pm in diameter. Internally, in addition to the electron-dense granules, lipoidal and membranous elements were often identifiable within the vacuoles. [The lipid-containing nature of these aforementioned elements was ascertained by their removal (washing out) when tissues were not osmicated prior to their rapid ethanolic dehydration (specimens not shown)]. The larger vesicles demonstrated a compartmentalized or compound architecture, appearing to consist of fused smaller vesicles (Figs. 3, 6, 7). Microincineration Following the high temperature (500°C) ashing of unstained sections from either formalin or glutaraldehyde fixed tissues, the granular contents of the vesicular elements pErsisted (Fig. 5). The ability of the deposits to survive such high temperatures is indicative of their inorganic (mineral) content.
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Osmium pyroantimonate fixed tissues In the presence of the pyroantimonate anion, which complexes with various cellular cations (Tandler et al., 1970; Simson and Spicer, 1975), most specifically calcium (Davis et a/., 1974; Klein et al., 1972), the internal substructure of the described vesicles was markedly labelled (Figs. 6, 7). The glycocalyx, the microvilli, and the mitochondria were also characterized by the presence of a localized heavy precipitate (Fig. 6). Goblet cells (not shown) were also intensely reactive. Evidence of endocytosis was seen at the base of the microvilli. These vesicles were heavily labelled with reaction product (Fig. 8). When sections from osmium pyroantimonate fixed tissues were floated on an EGTAcontaining calcium chelating solution, the intravesicular granules were drastically depleted (Fig. 9), indicative of their calcium content. Microvillar deposits were also markedly reduced. X-ray microanalysis of’ vesicles The results of the energy dispersive X-ray analyses of the supranuclear vesicles clearly showed the presence of significant amounts of calcium (Kcr 3.69 keV) and phosphorus (Kcr 2.01 keV) within these organelles (Fig. 10). Fig. 1 I demonstrates the resulting ‘probe spot’ produced during electron probe analysis of the vesicles. Such spots clearly indicate that we were indeed probing the elemental content of the so-called granular vesicles. Acid phosphatase localization Because of similarities in the ultrastructure of the granular vesicles reported here and the structure of lysosomes in a wide variety of other tissues (Novikoff, 1963), including intestine (Barka, 1964), we utilized ultrastructural histochemistry to demonstrate the presence of acid phosphatase in chick intestinal epithelium. The calcium containing vesicles described above were positive for acid phosphatase indicative of their lysosomal nature (Fig. 12). Discussion Summarizing the results, we have identified membrane bound vesicular or vacuolar structures in the supranuclear cytoplasm of chick intestinal absorptive cells. Internally, these structures are characterized by the
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presence of electron-dense granules which resist high temperature microincineration and are thus concluded to be inorganic (mineral) in nature. Ion selective EGTA chelation, staining with osmium pyroantimonate, and X-ray microanalysis confirmed the presence of calcium within these vesicles. Additionally, positive acid phosphatase histochemistry of these structures indicates their apparent lysosomal character. Thus, we have apparently identified a population of lysosomes containing substantial quantities of calcium ions. What is the possible function or functions of these organelles? Could they conceivably be instrumental in the uptake (absorption) and transcellular flux of calcium ions in the intestinal absorptive cell? Lysosomes are especially numerous in
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epithelial cells, including the absorptive cells of the intestinal epithelium (Barka, 1964; Hsu and Tappel, 1964). One of the many proposed functions of these organelles is that of absorption and digestion (De Duve and Wattiaux, 1966). Some evidence has demonstrated that lysosomes may functon in the sequestration of ions and ion translocation, including calcium. For example, in insect (Musca domestica) Malpighian tubules, various cations (calcium included) were identified by X-ray microanalysis within lysosomes prior to the formation of mineralized concretions (Sohal et al., 1976). Similar mineral-containing lysosomes were also seen during concretion formation in Musca midgut epithelial cells (Sohal et al., 1977). In both instances, a link between lysosomes,
Fig. 1. Supranuclear cytoplasm from two adjacent duodenal absorptive cells. Numerous granular vesicles (arrows) are seen scattered amongst the mitochondria (M) and endoplasmic reticulum (ER). These are often in close proximity to the lateral intercellular space (L). Phosphate buffered formalin fixation, doubly stained. x 33,000. Fig. 2. High magnification electron micrograph showing the single tri-laminar unit membrane (arrows) which surrounds a granular vesicle. Mitochondria (M) and endoplasmic reticulum (ER) are shown. Phosphate buffered formalin fixation, doubly stained. x 130,000. Fig. 3. In many instances, the granular vesicles (V) appear to fuse (arrows) with the plasma membrane of the lateral intercellular spaces (L) in a process similar to exocytosis. Mitochondria (M) and endoplasmic reticulum (ER) are shown. Calciumglutaraldehyde fixation, doubly stained. x 86,000. Fig. 4. Granular vesicles from a tissue sample fixed in calcium-glutaraldehyde, not osmicated, and not stained. The granular nature of these organelles is clearly demonstrable. x 128,000. Fig. 5. High temperature microincineration of calcium-glutaraldehyde The granules resist incineration, indicative of their inorganic content. Fig. 6. precipitate reacted as (M) show Unstained.
fixed vesicles. x 96,000.
Micronrauh A heavv - . from suecimen fixed in osmium-ovroantimonate. is seen over the microvilli (MV). The granular ves’lcles (arrows) are markedl; well. Note the compound globular structure of these entities. Mitochondria a finely granular internal precipitate pattern. L, lateral intercellular space. x 23,000.
Fig. 7. High magnification of granular compound vesicles from osmium-pyroantimonate fixed tissue. Again, note the globular or compound vesicular nature of these structures. M, mitochondria; L, lateral intercellular space. Unstained. x 47,000. Fig. 8. Osmium-pyroantimonate pyroantimonate-stained material Unstained. x 40,000.
fixed tissue demonstrating lumenal endocytosis of (arrows). Microvilli (MV) are again heavily labelled.
Fig. 9. Micrograph from osmium-pyroantimonate fixed tissue, sectioned and subsequently floated on a calcium chelating EGTA solution. Note that the granular contents of the vesicles (V) have been markedly depleted. Unstained. x 54,000.
.
,.:I.
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4.000
6.000
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.3?
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Energy (KEV) Fig. IO. Typical X-ray spectrum obtained from granular vesicles. Prominent calcium (Ca) and phosphorus (P) peaks are apparent. Chloride (Cl) is from the embedding medium; silicon (Si) and Copper (01) are related to contamination during analysis. Peaks represent summed data obtained from the sequential analysis of ten vesicles, each analyzed for 20 set, i.e. a total of 200 sec. Tissue fixed in calcium-glutaraldehyde, not osmicated. unstained.
cations,
and
excretion
was
hypothesized
(Sohal CTal., 1976, 1977). In higher animals, lysosomes in renal proximal tubules of rats reportedly take up experimentally administered metallic ions (Fe”-, Cu2+) (Koenig, 1963; Galle, 1974). Iron has also been identified in lysosomes of intestinal absorptive cells (Deutschlander et al., 1975; Davis, Jones, Hagler, manuscript in preparation). In resorbing osteoclasts, phagocytosed mineral crystals can be observed in vacuolar structures not unlike secondary lysosomes (Bonucci, 1974). Despite the recent evidences regarding lysosomes and the accumulation of minerals, little is known about the significance of these findings. It is generally agreed that cells maintain a constant resting cytosolic calcium ion con-
centration at IO-” M (Rasmussen, 1970). Transient cytoplasmic elevations in calcium ion concentration for extended periods of time can have a deleterious effect on normal cellular metabolism (Kimmich and Rasmussen, 1969). Because of this, excess cytosolic calcium must be sequestered (either protein or organelle bound). Such constraints place special demands on the intestinal epithelial cell which is charged with the task of absorbing large quantities of calcium while acting in concert with other tissues (bone, kidney) to maintain normal plasma calcium levels. Three cell structures are known to function in regulating intra- and extra-cellular calcium homeostasis: (I) the plasma membrane; (2) the mitochondrion; and (3) the endoplasmic reticulum (Rasmussen, 1970; Rasmussen
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Fig. 11. ‘Probe spot’ or carbon residue produced as a result of the electron beam during the X-ray analvsis of a single calcium containing vesicle. Such contamination spotsmdicate the specific locus ofanalysis so that the spectra obtained in Fig. 10 can be precisely localized to the so-called granular vesicles. x 95,000. Fig. 12. Intestinal tissue incubated in CMP medium for the demonstration of acid phosphatase. The granular vesicles (arrows) are markedly positive for this enzyme, indicative of their lysosomal nature. The microvillar border is at the upper left; L, lateral intercellular space. x 20,500.
et al., 1972; Rasmussen and Bordier, 1974). However, in the calcium transporting intestinal absorptive cell, as well as in other ion transporting epithelia, an additional mechanism, perhaps involving lysosomes, may have evolved. Morphological studies on intestinal epithelia have identified calcium in association with
apical and lateral plasma membranes (Oschman and Wall, 1972, 1973 ; Sampson et al., 1970). Calcium containing mineral granules in mitochondria have also been observed, and apparently their number reflects changes in both the normal and pathological transcellular flux of this cation (Sampson et al., 1970; Krawitt et al., 1974).
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There is no doubt that mitochondria function in buffering the cytosolic calcium ion concentration; however, a precise role for these organelles in transcellular calcium flux is more difficult to assume. For example, how does calcium get from the intestinal lumen to the mitochondrial matrix space? Secondly, how does calcium pass from the mitochondria to the lateral intercellular space and subsequently into the vascular system? So far, these questions remain unanswemd. Thus, increased intramitochondrial mineral densities may indicate elevated mitochondrial calcium buffering activity due to an increased cytosolic calcium load. They may not necessarily identify a component of a transcellular calcium flux pathway. Using electron probe analysis, discrete calcium localizations were concentrated in absorptive cells along lateral cell borders and in the supranuclear cytoplasm (Warner and Coleman, 1975). The latter calcium foci probably reflect the presence of calcium containing lysosomes reported here. The localizations at the lateral intercellular spaces may represent lysosome extrusion or exocytosis, a process known to occur in other cells (De Duve and Wattiaux, 1966). In addition, finite calcium localizations have also been reported in intra- and extracellular (brush border) goblet cell mucin (Kashiwa and Atkinson, 1963 ; Warner and Coleman, 1976; Halloran and Coleman, 1977) and reflect the distribution of the socalled intestinal calcium binding protein (CaBP) (Taylor and Wasserman, 1970), the precise function of which, other than its propensity to bind calcium, has really never been demonstrated. Assimilating the above information and coupling it to our concept of calcium containing lysosomes, we propose the following model as one possibility (pathway) for explaining the transcellular movement of calcium ions across the normal intestinal absorptive cell (Fig. 13). First of all, it appears evident, based on both immunofluorescent (Taylor and Wasserman, 1970) and microprobe analysis (Warner and Coleman, 1975 ; Halloran and Coleman, 1977), that CaBP is secreted from the intestinal goblet cell, binds calcium, and is subsequently distributed across the intestinal brush border as a component of the glycocalyx. Here, CaBP probably binds additional
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lumenal calcium ions. Calcium, still attached to CaBP, is then taken up into the absorptive cell by endocytosis (Fig. 8). The endocytotic uptake of macromolecules (proteins) and ions (iron), and their subsequent confinement in membrane bound vesicles, is known to occur during their intestinal absorption (Cornell et al., 1971; Lev and Orlic, 1972: Rodewald, 1971; Deutschlander et al., 1975). Next, the vesicular structures, delimited by a unit membrane and containing CaBP and its bound calcium load, fuse with supranuclear primary lysosomes to form secondary lysosomes. Many vesicles may fuse with each other; similarly, several CaBP-Ca vesicles may fuse with a single primary lysosome resulting in the larger compound secondary vacuolar lysosomes we have described (Fig. 7). The process of endocytosis-lysosome fusion is not unique. It has been clearly demonstrated in many systems (De Duve and Wattiaux, 1966), including intestinal epithelia (Cornell et al., 1971). Because of their cytoplasmic location, these calcium containing secondary lysosomes may have been responsible for the discrete supranuclear calcium foci detected by microprobe analysis (Warner and Coleman, 1975). After fusion of the CaBP-Ca vesicles with lysosomes, the acid hydrolases (proteases) in the secondary lysosome come in contact with, and eventually denature CaBP. As a result of the denaturation, calcium is released within the membrane constraints of the secondary lysosome. While this is occurring, the secondary lysosome is migrating laterally toward the lateral plasma membrane; its supranuclear location being maintained at all times. Here, the lysosome membrane coalesces with the lateral plasma membrane. The contents of the vacuole, i.e. calcium, denatured protein (amino acids) lipid, are later liberated into the lateral intercellular space from which they gain easy access to the blood. Such an event would explain the numerous calcium foci identified at the lateral supranuclear cell borders of intestinal absorptive cells observed by microprobe analysis (Warner and Coleman, 1975), as well as the frequent association (fusation) of calcium containing secondary lysosomes with the lateral plasma membrane reported here (Fig. 3). The extracellular unloading (defecation) of lysosomes is known to occur by exocytosis (De Duve and Wattiaux, 1966).
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Fig. 13. Schematic depicting the possible pathways involved in the transcellular flux of calcium across the intestinal absorptive cell. (1) The mitochondrial pathway whereby calcium ions (Ca) enter the cell, gain access to the mitochondrial (Mi) matrix space, and are subsequently passed back into the cytosol from which they are pumped across the lateral plasma membrane into the extracellular fluid compartment (Ecf). With such a mechanism, several points are difficult to explain: First, the precise intracellular role for the glycocalyx-associated intestinal calcium binding protein (CaBP); secondly, how does calcium gain access to the mitochondrial matrix from either the intestinal lumen or from the absorptive cell cytoplasm; thirdly, how does calcium leave the mitochondrial matrix and eventually enter the extracellular fluid? We are postulating an alternate route (2) involving pinocytotic vesicles and lysosomes, the latter shown here to demonstrate a marked ability to accumulate calcium. In this proposed pathway, calcium (Ca) bound to the calcium binding protein component of the glycocalyx (CaBP-Ca), is taken up into the cell as a CaBP-Ca complex in pinocytotic (endocytotic) vesicles (PV). Similarly formed vesicles (V) may fuse with each other; all such vesicles (V) eventually fuse with primary lysosomes (LY-I) to form secondary lysosomes (LY-2), the so-called granular vesicles described in this report. In the secondary lysosomes (LY-2) specific acid hydrolytic enzymes (E) denature CaBP to cleave calcium from its binding agent. During the process of vesicle fusion or exocytosis (EX), the free calcium (Ca) is liberated into the extracellular space (Ecf). Some calcium may also become associated with lipids (L) within the secondary lysosomes (LY-a). A certain amount of interplay may exist between the vesicles (V) and/or the lysosomes (LY-2) and the mitochondrial calcium stores and vice versa. Thus, any vesicular calcium ‘spillover’ may be taken up by the mitochondrial calcium buffer system (Mi); alternatively, any mitochondrial calcium ‘spillover’ may be sequestered by either the vesicles (V) or lysosomes (LY-2), or both. N, nucleus; M, microvilli.
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While this proposed intestinal calcium transport pathway is intriguing and explains some heretofore isolated pieces of information such as the intracellular role of CaBP and the known inhibitory effects of steroids such as glucocorticoids (lysosome stabilizing agents) on calcium transport (Kimberg, 1969), such a mechanism must await further scrutiny. What is the effect of rachitogenic diets of intestinal lysosomes? What effect does vitamin D and its metabolites have on intestinal lysosomes? These and other experiments are currently in progress. At this time, however, it is tempting to speculate that intestinal lysosomes might function in calcium homeostatic mechanisms like their counterparts in bone resorbing osteoclasts.
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However, less provocative functions for these calcium containing lysosomes cannot at present be ruled out. These include: intracellular calcium storage: calcium accumulation via the intracellular autophagy of calcium containing organelles (mitochondria); and calcium secretion (lumenal) via the desquamation of absorptive cells rich in calcium containing lysosomes.
Acknowledgements The authors gratefully acknowledge the continued support of the Research Committee, Baylor College of Dentistry. Excellent manuscript preparation and secretarial assistance provided by Jane Coleman.
References
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KIMBERG, D. 1969. The effects of vitamin D and steroid hormones on the active transport of calcium by the intestine. New Eng. J. Med., 280, 1396-1405. KIMMICH, C. A. and RASMUSSEN,H. 1969. Regulation of pyruvate carboxylase activity by calcium in intact rat liver mitochondria. J. biol. Chem., 244, 190-199. KLEIN, R. L., YEN, S.-S. and THURESON-KLEIN, A. 1972. Critique on the K-pyroantimonate method for semiquantitative estimation of cations in conjunction with electron microscopy. J. Histochem. Cytochem., 20, 65-78. KOENIG, H. 1963. lntravital staining of lysosomes by basic dyes and metallic ions. J. Histochem. Cytochem., 11, 120-121.
KRAWITT, E. L., SAMPSON,H. W., KUNIN, A. S. and MATTHEWS,J. L. 1974. Effect of ethane-l-hydroxyl-l,ldiphosphonate (EHDP) administration on duodenal calcium transport. C&if. Tiss. Res., 15, 21-29. NOVIKOFF, A. B. 1963. Lysosomes in the physiology and pathology of cells: contributions of staining methods. In Ciba Foundation Symposium Lysosomcs (ed. A. V. S. de Reuck and M. P. Cameron), pp. 36-73. Little, Brown and Co., Boston. LEV, R. and ORLIC, D. 1972. Protein absorption by the intestine of the fetal rat in utero. Science, 177, 522-524. MARTIN, I. H., LYNN, J. A. and NICKEY, W. M. 1967. A rapid polychrome stain for epoxy-embedded tissues. Am. J. clin. Path., 46, 250-251. OSCHMAN, J. L. and WALL, B. J. 1972. Calcium binding to intestinal membranes. J. Cell &of., 55, 58-73. OSCHMAN, J. L. and WALL, B. J. 1973. Binding of calcium to cell membranes. In Transport Mechanisms In Epithelia (ed. H. H. Ussing and N. A. Thorn), pp. 392403. Academic Press, New York. RASMUSSEN,H. 1970. Cell communication, calcium ion, and cyclic adenosine monophosphate. Science, 170, 4044412.
RASMUSSEN,H., GOODMAN, D. P. B. and TENENHOUSE,A. 1972. The role of cyclic AMP and calcium in cell activation. CRC Critical Rev. Biochem., 1, 95-148. RASMUSSEN,H. and BORDIER, P. 1974. The Physiological and CeNular Basis of Metabolic Bone Disease. Williams and Wilkins Co., Baltimore. REYNOLDS,E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cc/l Biol., 17, 208-212. RODEWALD, R. 1971. Selective antibody transport in the proximal small intestine of the neonatal rat. J. Cell Biol., 45, 635-640. SAMPSON,H. W., MATTHEWS, J. L., MARTIN, J. H. and KUNIN, A. S. 1970. An electron microscopic localization of calcium in the small intestine of normal, rachitic, and vitamin-D-treated rats. C&if. Tiss. Res., 5, 305-316. SCHAFER, H.-J. 1973. Ultrastructural and ion distribution of the intestinal cell during experimental vitamin-D deficiency rickets in rats. Virchows Arch. Abt. A, Path. Anat., 359, 11 l-123. SHUMAN, H., SOMLYO, A. Y. and SOMLYO, A. P. 1976. Quantitative electron probe microanalysis of biological thin sections: methods and validity. Ultramicroscopy, 1, 317-339. SIMSON, J. A. V. and SPICER, S. S. 1975. Selective subcellular localization of cations with variants of the potassium (pyre) antimonate technique. J. Histochem. Cytochem., 23, 575-598. SOHAL, R., PETERS, P. D. and HALL, T. A. 1976. Fine structure and X-ray microanalysis of mineralized concretions in the Malpighian tubules of the housefly, Musca domestica. Tissue & Cell, 8, 447-458. SOHAL, R. S., PETERS, P. D. and HALL, T. A. 1977. Origin, structure, composition and age-dependence of mineralized dense bodies (concretions) in the midgut epithelium of the adult housefly, Musca domestica. Tissue & Cell, 9, 87-102. SPURR, A. R. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultmstruct. Res., 26, 31-43. TANDLER, C. J., LIBANATI, C. and SANCHIS, C. A. 1970. The intracellular localization of inorganic cations with potassium pyroantimonate: electron microscopic and microprobe analysis. J. Cell Biol., 45, 355366. TAYLOR, A. N. and WASSERMAN,R. H. 1970. Immunofluorescent localization ofvitamin D-dependent calciumbinding protein. J. Histochem. Cytochem., 18, 107-l 15. THOMAS, R. S. and GREENAWALT,J. W. 1968. Microincineration, electron microscopy, and electron diffraction of calcium phosphate loaded mitochondria. J. CeN Biol., 39, 55-76. WARNER, R. R. and COLEMAN,J. R. 1975. Electron probe analysis of calcium transport by small intestine. J. Cell Biol., 64, 54-74.
WALTER
L. DAVIS,
RUTH G. JONES and HERBERT K. HAGLER
CALCIUM CONTAINING LYSOSOMES NORMAL CHICK DUODENUM: A HISTOCHEMICAL AND ANALYTICAL ELECTRON MICROSCOPIC STUDY
IN THE
ABSTRACT. Absorptive cells of the normal chick duodenum contain numerous supranuclear vesicular/vacuolar structures. By routine transmission electron microscopy, such structures are membrane bound and demonstrate a granular content. These vesicles appear to move laterally and eventually coalesce with the lateral plasma membrane (exocytosis). The granular contents are resistant to high temperature microincineration, thus revealing their mineral-containing nature. The granular vesicular matrix also stains intensely with osmium pyroantimonate. EGTA chelation of pyroantimonate-stained vesicles selectively extracts the granules indicating a high concentration of calcium. X-ray microanalysis also demonstrates a significant intravesicular calcium localization. When tissues were incubated for the presence of acid phosphatase, the supranuclear vesicles were markedly positive for this lysosomal enzyme. A possible role for these calcium-containing lysosomes in the transcellular flux of calcium ions across the intestinal absorptive cell is discussed.
Introduction
and chick intestine (Warner and Coleman, 197.5). However, due to the limitations of microprobe resolution utilized in the latter study, no ultrastructural description of these sites was reported. We describe here the identification of intracellular calcium vesicles in absorptive cells of normal chick duodenum using routine electron microscopy, electron microscopic histochemistry, high temperature microincineration and analytical electron microscopy. Such vesicles also demonstrate acid phosphatase activity, indicative of their lysosomal nature.
PREVIOUSinvestigations, using a wide variety of morphologic techniques, have attempted to localize intra- and extracellular calcium binding sites in the intestinal epithelia of various species (Kashiwa and Atkinson, 1963; Sampson, et al., 1970; Oschman and Wall, 1972, I973 ; Schafer, 1973 ; Davis and Jones, 1976; Warner and Coleman, 1975; Halloran and Coleman, 1977; Davis et al., 1978). These studies demonstrated significant calcium accumulations at the level of the microvillar and lateral plasma membranes, the mitochondria, within membrane bound vesicles, and in association with intra- and extracellular goblet cell mucus. Additionsupranuclear and lateral calcium ally, localizations were identified by electron probe analysis in the absorptive cells of rat
Materials and Methods Four to six week old male White Leghorn chicks (Hornung Hatchery, Sherman, Texas), weighing between 300 and 600 g, were used for this investigation. Animals were maintained from I day post-hatching in temperature-regulated brooders and fed Startena (Ralston Purina Co.) and water ad libitum.
Departments of Microscopic Anatomy and Pathology, Baylor College of Dentistry and the University of Texas Health Science Center, Southwestern Medical School, Dallas, Texas. Received 3 July 1978. Revised 2 November 1978. 127
128
At sacrifice, chicks were decapitated and exsanguinated. Blood was collected, allowed to clot, and the serum frozen for subsequent calcium analysis by atomic absorption spectrophotometry. The duodenal loop was surgically exposed. Both the proximal and distal segments were tied off (distally) and infused by syringe with the appropriate fixative (see below). Prior to removing the syringe, the intestinal segments were tied off (proximally) to form 2-4 cm duodenal ‘sausages’ containing the infused fixative. The tied segments were removed and placed in their respective fixative. The ‘sausages’ were subsequently diced under fixative into 1 mm thick closed loops. The fixatives used were: (1) phosphate buffered formalin, 4% (Carson et al., 1973), followed by osmication in phosphate buffered 1% 0~04; (2) 3 % glutaraldehyde buffered with 0.08 M s-collidine and containing 5 mM CaCls and 5 % sucrose (Oschman and Wall, 1972); post-osmication was in s-collidine buffered osmium tetroxide; (3) unbuffered 1% osmium tetroxide containing 2% potassium pyroantimonate (Carson et al., 1978). All tissues were dehydrated in graded ethanols and flat embedded in Spurr medium (Spurt-, 1969). For orientation purposes, 1 pm sections of Epoxy embedded tissues were cut with a glass knife on a Porter-Blum MT-2 ultramicrotome (Du Pont Instruments, Sorvall Operations, Newtown, Conn.). These were stained with Paragon (Martin et al., 1967). Following light microscopic evaluation, specimens were trimmed so that only the outer one-third of a single villus was thin sectioned for electron microscopy. Ultrathin sections, cut with a diamond knife and showing silver to gold interference colors (600-700 A), were picked up on 200 mesh uncoated copper grids and examined either unstained or doubly stained with uranyl acetate and lead citrate (Reynolds, 1963) in a Philips 300 electron microscope (Philips Electronics Instruments, Inc., Mt Vernon, N.Y.) operated at 40-300 kV. EGTA chelation
Unviewed, unstained sections of osmium pyroantimonate-fixed tissues mounted on copper grids were floated on solutions of 20 mM ethylene glycol bis-(B-amino ethyl ether)-N,N’-tetraacetic acid (EGTA), pH 7.6 7.8, for 30-60 min at 60°C. Grids were then
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thoroughly washed in deionized water, air dried, and viewed. High temperature microincineration
Ultrathin sections of either formalin or glutaraldehyde fixed, osmicated or nonosmicated tissues, were mounted on Formvar -silicon monoxide coated stainless steel grids (Thomas and Greenawalt, 1968). The unstained grids were then placed in a 500°C muffle furnace for 10-l 5 min. Energy dispersive X-ray spectroscopic analysis
For analytical electron microscopy, dark gold sections (approximately 1000-1200 8, thick) of glutaraldehyde fixed non-osmicated tissues were mounted on 200 mesh copper grids, left unstained, and subsequently carbon coated. Sections were viewed with a JEOL 100 C transmission electron microscope equipped with a JEOL high resolution scanning attachment STEM (JEOL, U.S.A., Medford, Mass.) (ASID), and a Kevex 30 mm2, 158 eV resolution, lithium-drifted silicon, energy dispersive X-ray detector (Kevex Corp., Burlingame, Calif.) placed within 20 mm of the sample through the objective pole piece (Hagler et al., 1977). Analyses were performed for 20 sec. X-ray counts were processed via a Tracer Northern NS 880 multichannel analyzer (Tracer Northern, Middleton, Wis.) operated at 20 eV/channel (Hagler et al., 1977). Ten vesicles were analyzed for 20 set each and their spectra were summed. The summed spectra were then subjected to a multipleleast squares fitting routine using reference spectra obtained from single crystal specimens (Shuman et al., 1976). The analyses of spectra were performed with the DEC PDP 1l/O5 computer-based Tracer Northern NS 880 unit. Acid phosphatase histochemistry
To demonstrate the presence of lysosomes, segments of duodenum were fixed in 0.1 M cacodylate buffered cold (4°C) glutaraldehyde (2.5%) for 4 hr. Specimens were washed overnight in cold buffer, then washed several times in 7.5 % sucrose prior to freezing on an IEC model CT1 cryostat (International Equipment Co., Needham. Mass.). Frozen sections, cut at 40 pm, were collected in the chilled sucrose wash and subsequently incubated in Novikoff’s CMP medium for
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the demonstration of acid phosphatase (Novikoff, 1963). Samples were incubated at room temperature for 1 hr with two changes of medium. Following incubation, tissues were washed with 7.5 o/o sucrose and osmicated in cacodylate buffered osmium tetroxide prior to embedding in Spurr medium. Results Serum calcium analyses All animals utilized in these studies had serum calcium values between 9 and 11 mg%. General description Numerous ovoid vesicles/vacuoles containing significant accumulations of electron dense granules were clearly identified clustered in the supranuclear absorptive cell cytoplasm of normal chick intestinal epithelium (Figs. l-4). Similar structures were not seen within the terminal web, or below the level of the cell nucleus. Additionally, such entities were not identified in goblet cells. Such vesicles were membrane bound (Fig. 2), generally located in close proximity to both mitochondria and endoplasmic reticulum cisternae (Figs. l-3), and frequently appeared to fuse or coalesce with the lateral plasma membrane at the supranuclear level of the lateral intercellular space (Fig. 3). These structures ranged in size from less than 0.1 pm to greater than 1.0 pm in diameter. Internally, in addition to the electron-dense granules, lipoidal and membranous elements were often identifiable within the vacuoles. [The lipid-containing nature of these aforementioned elements was ascertained by their removal (washing out) when tissues were not osmicated prior to their rapid ethanolic dehydration (specimens not shown)]. The larger vesicles demonstrated a compartmentalized or compound architecture, appearing to consist of fused smaller vesicles (Figs. 3, 6, 7). Microincineration Following the high temperature (500°C) ashing of unstained sections from either formalin or glutaraldehyde fixed tissues, the granular contents of the vesicular elements pErsisted (Fig. 5). The ability of the deposits to survive such high temperatures is indicative of their inorganic (mineral) content.
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Osmium pyroantimonate fixed tissues In the presence of the pyroantimonate anion, which complexes with various cellular cations (Tandler et al., 1970; Simson and Spicer, 1975), most specifically calcium (Davis et a/., 1974; Klein et al., 1972), the internal substructure of the described vesicles was markedly labelled (Figs. 6, 7). The glycocalyx, the microvilli, and the mitochondria were also characterized by the presence of a localized heavy precipitate (Fig. 6). Goblet cells (not shown) were also intensely reactive. Evidence of endocytosis was seen at the base of the microvilli. These vesicles were heavily labelled with reaction product (Fig. 8). When sections from osmium pyroantimonate fixed tissues were floated on an EGTAcontaining calcium chelating solution, the intravesicular granules were drastically depleted (Fig. 9), indicative of their calcium content. Microvillar deposits were also markedly reduced. X-ray microanalysis of’ vesicles The results of the energy dispersive X-ray analyses of the supranuclear vesicles clearly showed the presence of significant amounts of calcium (Kcr 3.69 keV) and phosphorus (Kcr 2.01 keV) within these organelles (Fig. 10). Fig. 1 I demonstrates the resulting ‘probe spot’ produced during electron probe analysis of the vesicles. Such spots clearly indicate that we were indeed probing the elemental content of the so-called granular vesicles. Acid phosphatase localization Because of similarities in the ultrastructure of the granular vesicles reported here and the structure of lysosomes in a wide variety of other tissues (Novikoff, 1963), including intestine (Barka, 1964), we utilized ultrastructural histochemistry to demonstrate the presence of acid phosphatase in chick intestinal epithelium. The calcium containing vesicles described above were positive for acid phosphatase indicative of their lysosomal nature (Fig. 12). Discussion Summarizing the results, we have identified membrane bound vesicular or vacuolar structures in the supranuclear cytoplasm of chick intestinal absorptive cells. Internally, these structures are characterized by the
130
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presence of electron-dense granules which resist high temperature microincineration and are thus concluded to be inorganic (mineral) in nature. Ion selective EGTA chelation, staining with osmium pyroantimonate, and X-ray microanalysis confirmed the presence of calcium within these vesicles. Additionally, positive acid phosphatase histochemistry of these structures indicates their apparent lysosomal character. Thus, we have apparently identified a population of lysosomes containing substantial quantities of calcium ions. What is the possible function or functions of these organelles? Could they conceivably be instrumental in the uptake (absorption) and transcellular flux of calcium ions in the intestinal absorptive cell? Lysosomes are especially numerous in
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epithelial cells, including the absorptive cells of the intestinal epithelium (Barka, 1964; Hsu and Tappel, 1964). One of the many proposed functions of these organelles is that of absorption and digestion (De Duve and Wattiaux, 1966). Some evidence has demonstrated that lysosomes may functon in the sequestration of ions and ion translocation, including calcium. For example, in insect (Musca domestica) Malpighian tubules, various cations (calcium included) were identified by X-ray microanalysis within lysosomes prior to the formation of mineralized concretions (Sohal et al., 1976). Similar mineral-containing lysosomes were also seen during concretion formation in Musca midgut epithelial cells (Sohal et al., 1977). In both instances, a link between lysosomes,
Fig. 1. Supranuclear cytoplasm from two adjacent duodenal absorptive cells. Numerous granular vesicles (arrows) are seen scattered amongst the mitochondria (M) and endoplasmic reticulum (ER). These are often in close proximity to the lateral intercellular space (L). Phosphate buffered formalin fixation, doubly stained. x 33,000. Fig. 2. High magnification electron micrograph showing the single tri-laminar unit membrane (arrows) which surrounds a granular vesicle. Mitochondria (M) and endoplasmic reticulum (ER) are shown. Phosphate buffered formalin fixation, doubly stained. x 130,000. Fig. 3. In many instances, the granular vesicles (V) appear to fuse (arrows) with the plasma membrane of the lateral intercellular spaces (L) in a process similar to exocytosis. Mitochondria (M) and endoplasmic reticulum (ER) are shown. Calciumglutaraldehyde fixation, doubly stained. x 86,000. Fig. 4. Granular vesicles from a tissue sample fixed in calcium-glutaraldehyde, not osmicated, and not stained. The granular nature of these organelles is clearly demonstrable. x 128,000. Fig. 5. High temperature microincineration of calcium-glutaraldehyde The granules resist incineration, indicative of their inorganic content. Fig. 6. precipitate reacted as (M) show Unstained.
fixed vesicles. x 96,000.
Micronrauh A heavv - . from suecimen fixed in osmium-ovroantimonate. is seen over the microvilli (MV). The granular ves’lcles (arrows) are markedl; well. Note the compound globular structure of these entities. Mitochondria a finely granular internal precipitate pattern. L, lateral intercellular space. x 23,000.
Fig. 7. High magnification of granular compound vesicles from osmium-pyroantimonate fixed tissue. Again, note the globular or compound vesicular nature of these structures. M, mitochondria; L, lateral intercellular space. Unstained. x 47,000. Fig. 8. Osmium-pyroantimonate pyroantimonate-stained material Unstained. x 40,000.
fixed tissue demonstrating lumenal endocytosis of (arrows). Microvilli (MV) are again heavily labelled.
Fig. 9. Micrograph from osmium-pyroantimonate fixed tissue, sectioned and subsequently floated on a calcium chelating EGTA solution. Note that the granular contents of the vesicles (V) have been markedly depleted. Unstained. x 54,000.
.
,.:I.
CALCIUM
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010,000
IN
2.000
DUODENAL
ABSORPTIVE
4.000
6.000
CELLS
I
.3?
8.000
Energy (KEV) Fig. IO. Typical X-ray spectrum obtained from granular vesicles. Prominent calcium (Ca) and phosphorus (P) peaks are apparent. Chloride (Cl) is from the embedding medium; silicon (Si) and Copper (01) are related to contamination during analysis. Peaks represent summed data obtained from the sequential analysis of ten vesicles, each analyzed for 20 set, i.e. a total of 200 sec. Tissue fixed in calcium-glutaraldehyde, not osmicated. unstained.
cations,
and
excretion
was
hypothesized
(Sohal CTal., 1976, 1977). In higher animals, lysosomes in renal proximal tubules of rats reportedly take up experimentally administered metallic ions (Fe”-, Cu2+) (Koenig, 1963; Galle, 1974). Iron has also been identified in lysosomes of intestinal absorptive cells (Deutschlander et al., 1975; Davis, Jones, Hagler, manuscript in preparation). In resorbing osteoclasts, phagocytosed mineral crystals can be observed in vacuolar structures not unlike secondary lysosomes (Bonucci, 1974). Despite the recent evidences regarding lysosomes and the accumulation of minerals, little is known about the significance of these findings. It is generally agreed that cells maintain a constant resting cytosolic calcium ion con-
centration at IO-” M (Rasmussen, 1970). Transient cytoplasmic elevations in calcium ion concentration for extended periods of time can have a deleterious effect on normal cellular metabolism (Kimmich and Rasmussen, 1969). Because of this, excess cytosolic calcium must be sequestered (either protein or organelle bound). Such constraints place special demands on the intestinal epithelial cell which is charged with the task of absorbing large quantities of calcium while acting in concert with other tissues (bone, kidney) to maintain normal plasma calcium levels. Three cell structures are known to function in regulating intra- and extra-cellular calcium homeostasis: (I) the plasma membrane; (2) the mitochondrion; and (3) the endoplasmic reticulum (Rasmussen, 1970; Rasmussen
DAVIS,
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Fig. 11. ‘Probe spot’ or carbon residue produced as a result of the electron beam during the X-ray analvsis of a single calcium containing vesicle. Such contamination spotsmdicate the specific locus ofanalysis so that the spectra obtained in Fig. 10 can be precisely localized to the so-called granular vesicles. x 95,000. Fig. 12. Intestinal tissue incubated in CMP medium for the demonstration of acid phosphatase. The granular vesicles (arrows) are markedly positive for this enzyme, indicative of their lysosomal nature. The microvillar border is at the upper left; L, lateral intercellular space. x 20,500.
et al., 1972; Rasmussen and Bordier, 1974). However, in the calcium transporting intestinal absorptive cell, as well as in other ion transporting epithelia, an additional mechanism, perhaps involving lysosomes, may have evolved. Morphological studies on intestinal epithelia have identified calcium in association with
apical and lateral plasma membranes (Oschman and Wall, 1972, 1973 ; Sampson et al., 1970). Calcium containing mineral granules in mitochondria have also been observed, and apparently their number reflects changes in both the normal and pathological transcellular flux of this cation (Sampson et al., 1970; Krawitt et al., 1974).
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There is no doubt that mitochondria function in buffering the cytosolic calcium ion concentration; however, a precise role for these organelles in transcellular calcium flux is more difficult to assume. For example, how does calcium get from the intestinal lumen to the mitochondrial matrix space? Secondly, how does calcium pass from the mitochondria to the lateral intercellular space and subsequently into the vascular system? So far, these questions remain unanswemd. Thus, increased intramitochondrial mineral densities may indicate elevated mitochondrial calcium buffering activity due to an increased cytosolic calcium load. They may not necessarily identify a component of a transcellular calcium flux pathway. Using electron probe analysis, discrete calcium localizations were concentrated in absorptive cells along lateral cell borders and in the supranuclear cytoplasm (Warner and Coleman, 1975). The latter calcium foci probably reflect the presence of calcium containing lysosomes reported here. The localizations at the lateral intercellular spaces may represent lysosome extrusion or exocytosis, a process known to occur in other cells (De Duve and Wattiaux, 1966). In addition, finite calcium localizations have also been reported in intra- and extracellular (brush border) goblet cell mucin (Kashiwa and Atkinson, 1963 ; Warner and Coleman, 1976; Halloran and Coleman, 1977) and reflect the distribution of the socalled intestinal calcium binding protein (CaBP) (Taylor and Wasserman, 1970), the precise function of which, other than its propensity to bind calcium, has really never been demonstrated. Assimilating the above information and coupling it to our concept of calcium containing lysosomes, we propose the following model as one possibility (pathway) for explaining the transcellular movement of calcium ions across the normal intestinal absorptive cell (Fig. 13). First of all, it appears evident, based on both immunofluorescent (Taylor and Wasserman, 1970) and microprobe analysis (Warner and Coleman, 1975 ; Halloran and Coleman, 1977), that CaBP is secreted from the intestinal goblet cell, binds calcium, and is subsequently distributed across the intestinal brush border as a component of the glycocalyx. Here, CaBP probably binds additional
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lumenal calcium ions. Calcium, still attached to CaBP, is then taken up into the absorptive cell by endocytosis (Fig. 8). The endocytotic uptake of macromolecules (proteins) and ions (iron), and their subsequent confinement in membrane bound vesicles, is known to occur during their intestinal absorption (Cornell et al., 1971; Lev and Orlic, 1972: Rodewald, 1971; Deutschlander et al., 1975). Next, the vesicular structures, delimited by a unit membrane and containing CaBP and its bound calcium load, fuse with supranuclear primary lysosomes to form secondary lysosomes. Many vesicles may fuse with each other; similarly, several CaBP-Ca vesicles may fuse with a single primary lysosome resulting in the larger compound secondary vacuolar lysosomes we have described (Fig. 7). The process of endocytosis-lysosome fusion is not unique. It has been clearly demonstrated in many systems (De Duve and Wattiaux, 1966), including intestinal epithelia (Cornell et al., 1971). Because of their cytoplasmic location, these calcium containing secondary lysosomes may have been responsible for the discrete supranuclear calcium foci detected by microprobe analysis (Warner and Coleman, 1975). After fusion of the CaBP-Ca vesicles with lysosomes, the acid hydrolases (proteases) in the secondary lysosome come in contact with, and eventually denature CaBP. As a result of the denaturation, calcium is released within the membrane constraints of the secondary lysosome. While this is occurring, the secondary lysosome is migrating laterally toward the lateral plasma membrane; its supranuclear location being maintained at all times. Here, the lysosome membrane coalesces with the lateral plasma membrane. The contents of the vacuole, i.e. calcium, denatured protein (amino acids) lipid, are later liberated into the lateral intercellular space from which they gain easy access to the blood. Such an event would explain the numerous calcium foci identified at the lateral supranuclear cell borders of intestinal absorptive cells observed by microprobe analysis (Warner and Coleman, 1975), as well as the frequent association (fusation) of calcium containing secondary lysosomes with the lateral plasma membrane reported here (Fig. 3). The extracellular unloading (defecation) of lysosomes is known to occur by exocytosis (De Duve and Wattiaux, 1966).
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Fig. 13. Schematic depicting the possible pathways involved in the transcellular flux of calcium across the intestinal absorptive cell. (1) The mitochondrial pathway whereby calcium ions (Ca) enter the cell, gain access to the mitochondrial (Mi) matrix space, and are subsequently passed back into the cytosol from which they are pumped across the lateral plasma membrane into the extracellular fluid compartment (Ecf). With such a mechanism, several points are difficult to explain: First, the precise intracellular role for the glycocalyx-associated intestinal calcium binding protein (CaBP); secondly, how does calcium gain access to the mitochondrial matrix from either the intestinal lumen or from the absorptive cell cytoplasm; thirdly, how does calcium leave the mitochondrial matrix and eventually enter the extracellular fluid? We are postulating an alternate route (2) involving pinocytotic vesicles and lysosomes, the latter shown here to demonstrate a marked ability to accumulate calcium. In this proposed pathway, calcium (Ca) bound to the calcium binding protein component of the glycocalyx (CaBP-Ca), is taken up into the cell as a CaBP-Ca complex in pinocytotic (endocytotic) vesicles (PV). Similarly formed vesicles (V) may fuse with each other; all such vesicles (V) eventually fuse with primary lysosomes (LY-I) to form secondary lysosomes (LY-2), the so-called granular vesicles described in this report. In the secondary lysosomes (LY-2) specific acid hydrolytic enzymes (E) denature CaBP to cleave calcium from its binding agent. During the process of vesicle fusion or exocytosis (EX), the free calcium (Ca) is liberated into the extracellular space (Ecf). Some calcium may also become associated with lipids (L) within the secondary lysosomes (LY-a). A certain amount of interplay may exist between the vesicles (V) and/or the lysosomes (LY-2) and the mitochondrial calcium stores and vice versa. Thus, any vesicular calcium ‘spillover’ may be taken up by the mitochondrial calcium buffer system (Mi); alternatively, any mitochondrial calcium ‘spillover’ may be sequestered by either the vesicles (V) or lysosomes (LY-2), or both. N, nucleus; M, microvilli.
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While this proposed intestinal calcium transport pathway is intriguing and explains some heretofore isolated pieces of information such as the intracellular role of CaBP and the known inhibitory effects of steroids such as glucocorticoids (lysosome stabilizing agents) on calcium transport (Kimberg, 1969), such a mechanism must await further scrutiny. What is the effect of rachitogenic diets of intestinal lysosomes? What effect does vitamin D and its metabolites have on intestinal lysosomes? These and other experiments are currently in progress. At this time, however, it is tempting to speculate that intestinal lysosomes might function in calcium homeostatic mechanisms like their counterparts in bone resorbing osteoclasts.
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However, less provocative functions for these calcium containing lysosomes cannot at present be ruled out. These include: intracellular calcium storage: calcium accumulation via the intracellular autophagy of calcium containing organelles (mitochondria); and calcium secretion (lumenal) via the desquamation of absorptive cells rich in calcium containing lysosomes.
Acknowledgements The authors gratefully acknowledge the continued support of the Research Committee, Baylor College of Dentistry. Excellent manuscript preparation and secretarial assistance provided by Jane Coleman.
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