Ultrastructural cytochemistry of atrial muscle cells. V. Characterization of specific granules in the human left atrium

JOURNAL OF ULTRASTRUCTURE RESEARCH 52,

179-192 (1975)

Ultrastructural Cytochemistry of Atrial Muscle Cells 1 V. Characterization of Specific Granules in the Human Left Atrium MARC CANTIN, SARITA BENCHIMOL, YVES CASTONGUAY, JEAN-CLAUDE BERLINGUET, AND MADELEINE HUET

D~partement de Pathologie, Universit~ de Montreal, and Service de Pathologie, Institut de Cardiologie de Montreal Received September !7, 1974 The carbohydrate content of specific granules in h u m a n left atrial appendages was assessed by cytochemical methods. The specific granules were found to be argentaphobic when ultrathin sections of Araldite-embedded atrial appendages were stained according to the periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery. The entire core of these granules was moderately positive after ultrathin sections of glutaraldehyde-fixed, glycol methacrylateembedded auricles were stained with phosphotungstic acid at a low pH. An analogous reaction w a s shown by the cell coat, residual bodies (C-granules), lysosomes, multivesicular bodies (mvb), and Z-disks as well as by a small portion of the Golgi complex. Cardiocytes of h u m a n left atrial appendages contain mvb with a dense core similar to those already noted in hypothalamic neurosecretory cells, mammotrophs, and adrenal medullary cells and are thought to be crinophagic. INTRODUCTION

In various animal species, specific granules, distinct from lipofuscin granules, are round and membrane-bound, having a homogeneous electron-dense core that makes them very similar to secretory granules in endocrine cells (4, 8, 25). Based on their morphology and localization, three types of specific granules have been recognized (8). A-granules possess a very electron-dense core that is sometimes slightly retracted from the inner aspect of the membrane, leaving a thin halo around the core. Most of these granules are found in the paranuclear region of cardiocytes. B-granules are characterized by a pale, fibrogranular core. Though present in the paranuclear region of the right atrial cardiocytes, they are more abundant in the left atrium. D-granules have a relatively small diameter and possess a dense core that is morphologically very similar to that of the norepi~Supported in part by the Medical Research Council of Canada (Grant MT-1973), The Quebec Heart Foundation, the Joseph C. Edwards Foundation, and the J. L. Levesque Foundation. 179 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

nephrine-containing granules in adrenergic nerve endings (6). D-granules, found mostly in the intermyofibrillar areas, are rare in the paranuclear regions. Investigations into the chemical nature of atrial specific granules in various animal species (9, 52) have focused mainly on their catecholamine content and finally have revealed that they do not contain any appreciable amounts of these substances (13). This finding is in agreement with certain autoradiographic studies (25, 57) where it was shown that atrial specific granules do not take up 3H-dopamine or bind ~H-norepinephrine in situ. In addition, fluorescence microscopy has indicated that these granules do not exhibit the characteristic fluorescence of catecholamines (1, 12, 36). In hamsters depleted of catecholamines by repeated injections of 6-hydroxydopamine, the catecholamine fluorescence of atrial tissue disappears, while, ultrastructurally, the specific granules remain intact (12). Cytochemistry has demonstrated that atrial specific granules are acid phosphatase-negative (25, 31), thus differing from

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lysosomes and residual bodies (C-granu l e s ) . C y t o c h e m i c a l s t u d i e s h a v e also s h o w n t h a t s p e c i f i c g r a n u l e s are a r g e n t a phobic when stained by the periodic acids i l v e r m e t h e n a m i n e ( P A S M ) t e c h n i q u e (4, 6) o r t h e p e r i o d i c a c i d - t h i o c a r b o h y d r a z i d e s i l v e r p r o t e i n a t e m e t h o d of T h i e r y (10, 20, 22). H o w e v e r , in b o t h t h e r a t a n d m a n , they are weakly periodic acid-Schiff-posirive ( P A S ) in l i g h t m i c r o s c o p y a n d r e a c t to p h o s p h o t u n g s t i c a c i d ( P T A ) a t a low p H in e l e c t r o n m i c r o s c o p y . A c c o r d i n g to c u r r e n t c y t o c h e m i c a l k n o w l e d g e , t h e i r core m a y consequently contain complex carbohyd r a t e s (I0, 20, 22). I n t h e r a t a n d m a n (10, 19, 21, 23), a t r i a l s p e c i f i c g r a n u l e s also a p p e a r to be r i c h in p r o t e i n s since, in u l t r a t h i n s e c t i o n s of g l y c o l m e t h a c r y l a t e embedded (GMA) atria, they are readily and selectively digested by narrow and broad spectrum proteolytic enzymes. Furt h e r m o r e , in s e m i f i n e s e c t i o n s of A r a l d i t e embedded atria, these granules are stained b y v a r i o u s t e c h n i q u e s k n o w n to i d e n t i f y p r o t e i n s in s e c t i o n s (23). I n m a n , t h e e x i s t e n c e of s p e c i f i c g r a n u l e s in a u r i c u l a r c a r d i o c y t e s h a s a l r e a d y b e e n r e p o r t e d (3, 4, 17, 25, 26, 32, 40, 53, 56) a n d c y t o c h e m i c a l s t u d i e s (20, 21) h a v e b e e n p e r f o r m e d on c a r d i o c y t e s in h u m a n r i g h t auricular appendages. A systematic invest i g a t i o n i n t o h u m a n left a t r i a l a p p e n d a g e s was undertaken because the granule popul a t i o n in t h e left a t r i a of r a t s is d i f f e r e n t f r o m t h a t in t h e r i g h t (7, 8). T h e a i m of t h e p r e s e n t s t u d y w a s to e x a m i n e , b y v a r i o u s cytochemical methods, the characteristics a n d r e a c t i v i t y of t h e s p e c i f i c g r a n u l e s . T h e r e a c t i v i t y of o t h e r a u r i c u l a r cell o r g a n e l l e s w a s also s c r u t i n i z e d for c o n t r o l a n d c o m p a r a t i v e p u r p o s e s . In a d d i t i o n , t h e q u a s i constant occurrence of multivesicular b o d i e s ( m v b ) w i t h d e n s e cores is d e s c r i b e d and analyzed. MATERIALS AND METHODS Left atrial appendages from 11 patients (two men and nine women) were biopsied at the start of either aorta-to-coronary artery bypass grafting or mitral

valve surgery. The specimens were immediately minced in 2% glutaraldehyde (buffered with 0.1 M cacodylate-HC1 at pH 7.1) and left in the same fixative for 2 hr. They were then washed for three 15-min periods in cacodylate buffer to which 2% sucrose had been added. The specimens were left in the buffer (4°C) for periods varying from 12 hr to several days. Some fragments were postfixed for 1 hr in 2% osmium tetroxide (OsO4), buffered with veronal acetate. All the specimens (those fixed in glutaraldehyde alone as well as those postfixed in OsO~) were embedded in Araldite. A third set of fragments, fixed only in glutaraldehyde, was embedded in GMA. The standard technique (27), used previously for rat atria (10, 22, 23) is inadequate for human tissue because of poor penetration by the plastic. Therefore, the embedding procedure of Rosenberg et al, (44) was employed with the modifications introduced by Leduc and Bernhard (28). In addition, the tissue fragments were constantly agitated during the embedding sequence. Polymerization was carried out at 4°C between two ultraviolet lamps (P. W. Allen & Co., Liverpool, England) for 12-24 hr. Ultrathin sections, for routine electron microscopy, were cut from either Araldite or GMAembedded portions of auricular appendages in a LKB ultramicrotome with glass knives. These specimens were stained with uranyl acetate and lead citrate as described (10, 12, 2I, 22). The compounds employed in the following ultrastmctural-cytochemical procedures were all of reagent grade. The solutions were prepared with distilled water.

Thiery's Method The periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery (54, 55) was used, as previously described (10, 20, 22), for the demonstration of periodate-reactive vicinal glycols on tissues fixed in glutaraldehyde alone or in combination with Os04 and embedded in Araldite.

Low pH Phosphotungstic Acid (PTA) Method This technique (29, 38, 42, 43) was applied for the visualization of hydroxyl groups in complex carbohydrates. Ultrathin (silver to gray) sections from glutaraldehyde-fixed, GMA-embedded tissues were stained (10, 20, 22) with a 1% solution of PTA in 1NHC1 (pH 0.3) for 8-10 min and then rapidly rinsed with distilled water. RESULTS

Ultrastructure of Auricular Specific Granules and mvb with a Dense Core In l o n g i t u d i n a l , Araldite-embedded

u l t r a t h i n s e c t i o n s of tissues stained with

SPECIFIC GRANULESOF HUMAN LEFT ATRIUM

181

uranyl and lead (Figs. 1-5), the cardiocytes to the dark type described by Nicander showed a central nucleus. The paranuclear (33). Tentatively labeled "intermediate" hyaloplasm, laterally bordered by myofila- types--between mvb with a dense core ments arranged in repeating sarcomeres and specific granules--were rare. They delimited by densely stained Z-disks, con- were much smaller than mvb and contained many mitochondria and Golgi com- tained a few vesicles between their limitplexes with ~acks of saccules and groups of ing membrane and dense core (Figs. 9 and vesicles and most of the specific granules 11). (Figs. 1 and 2). Dense A-granules were Ultrastructural Cytochemistry more numerous than fibrogranular B-granSpecific granules, lysosomes, residual ules. A and B-granules were also present bodies, and mvb with a dense core. The between sarcomeres throughout the cardiauricular specific granules, regardless of ocytes (Figs. 3 and 4). The small dense the method of fixation, were argentaphobic D-granules were mainly located between myofilaments and in the subsarcolemmal when stained according to Thiery's technique, even after 5 days of exposure to spaces. While regular mvb (i.e., membrane- thiocarbohydrazide (Figs. 12 and 13, Table bound groups of small vesicles) were rare in I). Marked variations in the number, size, the left auricle (Fig. 6), another type with and morphology of lysosomes and lipofusdense cores (Fig. 5) was encountered in cin granules (C-granules) were evident nine of the 11 patients examined. The from one patient to another and even in frequency of their occurrence varied from various sections of the same tissue. Typical section to section and from on patient to lipofuscin granules were especially numerthe next, but when present, they could ous in some cases (Fig. 2). These large easily be detected in several sections. granules consisted of more or less circular Some were located in the paranuclear electron-lucent areas surrounded by dense region next to the Golgi complexes (Fig. osmiophilic material. Some were delimited 5), while others were noted between by a single membrane while others were sarcomeres or near the sarcolemma but free in the hyaloplasm. These structures not in contact with the Golgi structures. contained silver deposits of variable denMost of the mvb had only one core (Figs. sity located in the above-described osmio6 and 8-11) in the center or periphery, philic areas (Fig. 12). They were more but a few showed two (Fig. 7). These cores intensely silver-positive after fixation in had the density, size, and contours of A both glutaraldehyde and OsO4 than after or B-granules but were devoid of a limiting fixation in glutaraldehyde alone. This reacmembrane. They were surrounded by a tion was evident even without previous variable number of small vesicles which, oxidation by periodic acid or exposure to in turn, were enclosed by a single mem- thiocarbohydrazide. Structures of various brane. sizes were defined as lysosomes because The matrix of the mvb with dense cores, they were silver-positive, even in specilike that of the classic type, was generally mens fixed in glutaraldehyde alone and electron-lucent, being practically without without previous periodic acid oxidation any clumps of finely particulate material (10, 20-22). The smaller lysosomes con(Fig. 6). Thus, in the myocardium, most of tained only a few electron-dense particles, the mvb with or without dense cores while the large ones were sometimes laden roughly correspond to the light kind ob- with irregular clumps of osmiophilic mateserved in the epididymis by Nicander (33). rial (Figs. 12 and 13). They appeared as Some had a dense, more or less uniform round structures of variable diameter covgranular matrix (Fig. 7) that was similar ered by fine silver grains.

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All the specific granules were positively stained by the P T A technique (Figs. 14-i6). T h e y were round, uniformly but lightly stained structures contrasting with the pale unstained background of myofilaments and mitochondria. The C-granules were large, pale, and irregularly stained. Other structures of variable d i a m e t e r - - c o r r e s p o n d i n g to lysosomes--were intensely stained (Figs. 14 and 15). Like the classic type of m v b (20), those with dense cores were silver-negative (Figs. 17 and 18). None of t h e m could be identified with certainty in G M A - e m b e d d e d specimens exposed to PTA, whereas regular mvb showed a stained matrix containing unstained vesicles (Fig. 19).

Surface coat, sarcolemma, and T-tubules. The surface or cell coat was barely visible after Araldite-embedded tissues were stained with uranyl and lead, nor could it be seen convincingly with the T h i e r y technique, even after 5 days of exposure to thiocarbohydrazide. It appeared as a discontinuous light gray line of variable width, separated from the sarcolemma by a clear space. The surface coat, however, was effectively shown by P T A as a dark line, apposed on the external surface of each cell (Figs. 14 and 15). In Araldite-embedded cardiocytes, stained either with uranyl and lead or according to Thiery's technique, the sarcolemma appeared as a dense, continuous, well-delimited line. T h e cell surface often exhibited regular identations at the level of the Z-disks. T - t u b u l e s were rare. T h e y appeared as vaguely triangular, blunt

invaginations of the cell surface with a continuous heavy line of PTA-positive material on their external aspect. In contrast, micropinocytosis was very active as judged by the extremely great n u m b e r of small vesicles in the subsarcolemmal spaces. Golgi complex. The Golgi complexes in the cardiocytes of the left atrial appendage were relatively well-developed (Figs. 1 and 5). T h e y were comprised of several stacks of saccules and n u m e r o u s vesicles often containing progranules. When stained by Thiery's technique, all the Golgi structures were silver-negative (Figs. 12 and 13), even in sections exposed to thiocarbohydrazide for 5 days. In contrast, some of the Golgi saccules and vesicles located near the nuclei were lightly stained by P T A (Fig. 16). Z-disks. T h e Z-disks were electron-dense in sections stained with uranyl and lead (Fig. 1). T h e y were silver-negative (Figs. 12 and 13). After s t a i n i n g wi~:h PTA, they appeared as thin, lightly positive, transversal lines (Figs. 14 and 15). In patients with mitral valve disease, the thickness of the Z-disks was slightly to markedly increased (Fig. 4). These thickened Z-disks were also stained by PTA. Glycogen. B-type glycogen was evidenced by only 30 rain of exposure to thiocarbohydrazide. The granules were spherical and distributed either singly or in groups between the organelles (Figs. 12 and 13). T h e y were not stained when the periodic acid or thiocarbohydrazide steps were omitted. Since glycogen was extracted during embedding in GMA (42), no reaction to P T A was seen.

FIGS. 1 11. represent sections stained with uranyl acetate and lead citrate. FIG. 1. Longitudinal section of cardiocyte with nucleus (N), myofilaments with Z-bands (Z), mitochondria (m), Golgi vesicles (G), dense A-granules (gi), pale B-granules (g2), small D-granules (g~) and lysosomes (L). × 36 000. FIG. 2. Portion of cardiocyte showing Golgi complex (G), a few specific granules (g), and numerous large residual bodies (C-granules) filled with osmiophilic, granular material and pale lipid droplets. × 29 000. FIG. 3. Dense A-granules (gl) and pale B-granules (g2) are present not only between sarcomeres but also ir the subsarcolemmal areas. A few small D-granules (ga) are also visible. × 15 000. FIG. 4. Cardiocyte from a patient with mitral stenosis showing thickened Z-disks and a few intermyofibrillaI specific granules. × 9 000.

G

!!~!!~i~¸~!~i~ ii!~ii~i iii

184

FIG. 5. Longitudinal section of cardiocyte. A multivesicular body with a dense core (arrow) can be seen near a large Golgi complex. A few A-granules (gl) are present, x 11 000. Fins. 6-11. Multivesicular bodies with dense cores from various patients either with coronary insufficiency or mitral valve disease. Fro. 6. Multivesicular body with excentrically-located dense core, n u m e r o u s small vesicles, light matrix, and regular contours. × 53 00O. Fro. 7. Multivesicular body with two dense cores, a few vesicles, and an electrondense matrix. Small vesicles (arrow) are evident near the body. × 55 000. FIG. 8. Multivesicular body with a dense core surrounded by n u m e r o u s small vesicles. × 54 000. Fla. 9. Small multivesicular body near a specific granule (arrow). The small vesicles form a row around the dense core. × 63 000. FIG. 10. Multivesicular body with a dense core. Some of the small vesicles have a dense content. × 120 000. Fla. 11. Possible " i n t e r m e d i a t e " form between multivesicular body with a dense core and specific granule. The limited space between the dense core and the outer m e m b r a n e contains only a few vesicles, x 27 000. 185

186

C A N T I N E T AL. TABLE I CYTOCHEMISTRY OF HUMAN LEFT ATRIAL CARDIOCYTES

Embedding

Fixative

Araldite

Glutaraldehyde, osmium tetroxide

Araldite

Glutaraldehyde, osmium tetroxide Glutaraldehyde, osmium tetroxide Glutaraldehyde

Araldite Araldite Glycol methacrylate

Glutaraldehyde

Staining

Specific granules

Periodic acid, thiocarbohydrazide (TCH), silver proteinate TCH, silver proteinate Periodic acid, silver proteinate Periodic acid, TCH, silver proteinate Phosphotungstic acid-hydrochloric acid (pH:0.3)

+ = weak; + + = moderate; + + + = strong reaction;

Collagen. Collagen was not stained by Thiery's technique, but reacted easily to PTA (Fig. 14). DISCUSSION

The material in the present study was taken from nine women and two men. There was no sex difference in the number, type, or localization of either specific granules or mvb with a dense core. In nine cases, surgery was done to correct or replace the mitral valve. One male and one female patient suffered from coronary insufficiency and were biopsied at the start of aorta-to-coronary artery bypass grafting. Here again, the pattern of distribution and the characteristics of the specific granules and mvb with a dense core were similar to that in the other cases. Mvb with a dense core were found in eight patients with

Residual Lysosomes MultiMultibodies vesicular vesicular (C-granules) bodies bodies with dense cores

0

+++

++

0

0

0

+++

++

0

0

0

Trace

Trace

0

0

0

++

++

0

0

+

+ to + +

+++

-b++

= not seen.

mitral valve disease and in one subject with coronary insufficiency. In all cases of mitral valve disease, electron microscopy revealed completely normal areas generally resembling corresponding regions in the right atrial appendages (20, 21). Other fields, however, showed alterations that are characteristic of mitral valve disease (45), i.e., two types of Z-band changes. The first took the form of generalized or local broadening of the Z-bands; the second consisted of Z-band material located along the sarcolemma and in close contact with it. The numerous residual bodies (C-granules) in the paranuclear areas of several cells in all biopsies were also indicative of previous cellular alterations (17, 20, 21). The general architecture of the cardiocytes in the present investigation was similar to that described by others (3, 4, 17,

FIG. 12. Longitudinal section of cardiocyte fixed in glutaraldehyde and OsO4, and stained according to Thiery's technique (floated on thiocarbohydrazide for 24 hr). An intense reaction is shown by B-glycogen, which is free in the cytoplasm and appears as single particles or clusters of electron-dense silver grains. While the specific granules (g) are silver-negative, lysosomes (L) of various sizes are covered by fine silver grains. The lipid content of the lipofuscin granules (LI) is electron-lucent, whereas their periphery is markedly electrondense. All the other structures, including the mitochondria (m), myofilaments with Z-bands (Z), and the nucleus (N), are silver-negative. × 24 000. Fro. 13. Portion of cardiocyte processed as in Fig. 12 b u t floated on thiocarbohydrazide for 5 days. The glycogen particles are intensely positive. The lysosomes (L) are covered by silver grains. The specific granules (arrow) and Golgi structures (G) are silver-negative. × 24 000.

187

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25, 26, 32, 40, 53, 56). T h e T - t u b u l e s in

rarely be identified in the present material. p a r t i c u l a r were not very n u m e r o u s . In all Nevertheless, it is possible t h a t m v b with cases, their visible portions were covered dense cores represent an initial stage of on their external surface by a cell coat and, specific granule formation. T h e close cytoas already noted (20, 21, 26), the first chemical kinship m a n i f e s t e d by Golgi vesiregion (i.e., the area closest to the periph- cles a n d m v b (5, i5) also favors this asery of the cell) s o m e t i m e s c o n t a i n e d fine s u m p t i o n . If so, they ought to be present collagenous fibrils. Micropinocytosis was not only in the left b u t also in the right intense in virtually all the s u b s a r c o l e m m a l a t r i u m and in other species as well. Howzones. ever, in the right a t r i u m of rats, as in T h e general distribution of specific gran- h u m a n right atrial a p p e n d a g e s , m v b are ules in the left a t r i u m was different from exceedingly rare (I1). t h a t prevailing in the right atrial a p p e n d B a s e d on presently available evidence, it ages of rats and m a n (20, 21) and resem- is m u c h more likely t h a t m v b with dense bled the p a t t e r n discerned in the left cores represent an autolytic, crinophagic a t r i u m of the rat (8). F i b r o g r a n u l a r B- process analogous to t h a t occurring in varigranules were more a b u n d a n t and both A ous endocrine glands (I4, 18, 34, 35, 37). In and B-granules were present not only in the the pituitary, for instance, m v b with dense p a r a n u c l e a r r e g i o n - - a s in the right a t r i u m cores are frequently found in m a m m o - - b u t also between sarcomeres t h r o u g h o u t trophs upon a b r u p t cessation of suckling the h y a l o p l a s m . T h e D-granules in the left (I4, 5I). T h e y are also visible in h y p o t h a a t r i u m were similar to those in the right lamic neurosecretory cells (37) and adrenal with regard to n u m b e r and location. T h e m e d u l l a r y cells (I8) where a crinophagic presence of m v b with dense cores, a fre- function has been t e n t a t i v e l y a t t r i b u t e d to q u e n t feature of the left h u m a n atrium, has t h e m (14, 18). Since this process occurs, not been noted in other m a m m a l s . How- albeit at a m u c h reduced rate, in otherwise ever, these structures h a v e been seen in the n o r m a l endocrine glands (14, 37, 5I), the cardiac ventricle of the frog (47), where s a m e possibility m a y exist in atrial cardit h e y were t h o u g h t to be p a r t of the mor- ocytes. In our material, the absence of a n y phogenetic process of specific granules. In f o r m where partial digestion of granules the frog, m u c h more t h a n in m a m m a l s , the has occurred precludes a definitive conclum a t u r e A-granule possesses a clear halo sion. It r e m a i n s to be d e t e r m i n e d whether, between the s u r r o u n d i n g m e m b r a n e and in the left auricular cardiocytes, the presthe dense core. " I n t e r m e d i a t e " forms of ence of' relatively n u m e r o u s m v b with a m v b , i.e., small residual vesicles between dense core is a physiologic process or the the dense core and the m e m b r a n e , lend pathologic consequence of mitral valve diss u p p o r t to this hypothesis. T h e y could ease or coronary insufficiency. In a n y

FIG. 14. Cardiocyte embedded in glycol methacry!ate and exposed to phosphotungstic-hydrochloric acid showing cell coat (vertical arrow), Z-bands (Z), small densely stained lysosomes (L), and a few lightly stained specific granules (g). The mitochondria (m) and myofilaments are unstained. Collagen is intensely reactive. × 10 000. Fro. 15. Portion of auricle embedded in glycol methacrylate. Exposure to phosphotungstic-hydrochloric acid reveals a cardiocyte in close contact with a capillary containing a red blood cell (R); the cardiocyte shows a cell coat, densely stained small lysosomes (L), moderately-stained specific granules (g), and lightly positive Z-bands (Z). The mitochondria (m) and myofilaments are unstained. A large lysosome exhibits a rim of positivity around an unstained core (horizontal arrow). Nucleus (N). × 21 000. Fro. 16. Portion of cardiocyte embedded in glycol methacrylate and stained with phosphotungstic-hydrochloric acid. The Golgi saccules and vesicles (arrow) located near the nucleus (N) are lightly positive as are the specific granules (g). Mitochondria (m). × 39 000.

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Fics. 17-19. Cytochemistry or multivesicular bodies with or without dense cores. FIG. 17. Part of cardiocyte stained according to Thiery's technique (floated on thiocarbohydrazide for 24 hr) showing an intensely reactive fraction of a residual body (top of picture), a lysosome covered by fine silver grains (bottom left), glycogen particles, and two multivesicular bodies, one with (arrow) and the other without a dense core. Both are unstained. × 19 000. Fro. 18. Unstained multivesicular body with dense core, treated as in Fig. 17. The lysomomes (arrow) are intensely reactive. Glycogen particles are scattered between the organelles. × 120 000. Fro. 19. Multivesieular body from eardiocyte embedded in glycol methaerylate and exposed to phosphotungstic-hydrochloric acid. The matrix is markedly stained, whereas the small vesicles are not. × 59 000. event, m v b with a dense core were often seen in otherwise entirely n o r m a l - a p p e a r ing cardiocytes. Previous studies on the right atrial cardiocytes of rats (10, 22) and h u m a n s (20, 21) have p r o d u c e d m u t u a l l y c o m p l e m e n t a r y results with the P A S - t e c h n i q u e (light microscopy) and with P T A (electron microscopy): P A S a n d P T A - p o s i t i v e reactions were exhibited b y collagen, the surface coat, Z-disks, lysosomes, and specific granules. As regards staining with PTA, identical observations were m a d e in the present work. T h e reactions of lysosomes and m v b to P T A also agree with previous findings in other cell types (16, 43). T h e direct staining of hydroxyl groups in complex c a r b o h y d r a t e s has been achieved with a high degree of specificity with P T A (at a very low pH) in G M A - e m b e d d e d sections of g l u t a r a l d e h y d e - f i x e d tissues (29, 30, 37, 42). However, other studies (41, 48, 49) cast doubts on the validity of this m e t h o d for the detection of complex carb o h y d r a t e s . T h e results of these experim e n t s indicate t h a t P T A is an anionic

stain for certain positive groups of proteins. T h e fact t h a t P T A does not react with s o m e p o l y s a c c h a r i d e s (viz., glycogen) would be an a r g u m e n t against the specificity of this stain (50). W h a t e v e r the m e c h a n i s m of the staining reaction, there is a general consensus t h a t - - i n fine sections of glutaraldehyde-fixed, G M A - e m b e d d e d tissues, or fine sections o b t a i n e d by ultrac r y o t o m y - - P T A (at a low pH) reveals complex c a r b o h y d r a t e s that, light microscopically, a r e PAS-positive. Glycogen, which is not stained in G M A - e m b e d d e d tissues because it is dissolved during e m bedding, is intensely reactive in fine sections p r e p a r e d by u l t r a c r y o t o m y (2, 24). Although the left auricular specific granules were PTA-positive, t h e y were also silver-negative when stained according to T h i e r y ' s technique. This confirms previous findings, which showed t h a t specific granules of the rat atrium, after P A S M staining, were a r g e n t a p h o b i c , even when practically all the other cellular s t r u c t u r e s were covered b y silver p r e c i p i t a t e s (4, 6). Analogous results, with T h i e r y ' s technique, h a v e

SPECIFIC GRANULES OF HUMAN LEFT ATRIUM

been obtained in rat (10, 22) and human (20, 21) right atrial cardiocytes. In the same manner, special tubuloreticular structures, observed in certain lymphocytes, are silver-negative when stained according to Thiery's method, but they also react to PTA (46). Such discrepancies between the results obtained with PTA and those following staining with silver grains may be due to the greater sensitivity of the former technique. In fact, autoradiographic studies have shown that 3H-fucose is incorporated not only into gonadotrophic cell granules that are PAS-positive but also into somatotrophic cell granules that are PAS-negative (39). Thus, granules predominantly containing proteins, like those of somatotrophs and--as judged from digestion experiments (10, 19, 21, 23)--the protein-containing granules of atrial cardiocytes, could hold sufficient amounts of glyeoproteins in their matrices for a weak PTA-positive reaction but not enough or in such available forms as to be silver-positive. In the right and left atrial cardiocytes of rats (10, 22) and man (20, 2I), the presence of cytochemically-demonstrable complex carbohydrates in the Golgi apparatus (where progranules are often found) suggests that glycoproteins are indeed incorporated into the matrices of specific granules. REFERENCES 1. ANGELAKOS,E. T., FUXE, K., AND TORCHIANA, M. L., Acta Physiol. Scand. 59, 184 (1963). 2. BABAI, F., AND BERNHARD, W., J. Ultrastruct. Res. 37, 601 (1971). 3. BATTIG,C. C., ANDLOW, F. N., Amer. J. Anat. 108, 199 (1961). 4. BENCOSME, S. A., AND BERGER, J. M., in BAJuSZ, E., AND JASMIN, G. (Eds.), Meth. Achiev. Exp. Pathol., Vol. V., p. 173. Karger, Basel, 1971. 5. BENNETT, C., AND LEBLOND, C. P., J. Ceil Biol. 51, 875 (1971). 6. BERGER, J. M., AND BENCOSMn, S. A., J. Mol. Cell. Cardiol. 3, 111 (1971). 7. BERGnR, J. M., AND RONA, G., Anat. Rec. 166, 278 (1970). 8. BERGER, J. M., AND RONA, G., in BAJUSZ, E., AND JASMIN, G. (Eds.), Meth. Aehiev. Exp. Path., Vol. V, p. 540. Karger, Basel, 1971.

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