Sarita Benchimol

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

The carbohydrate content of specific granules in human 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 was 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 human 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.

Ultrastructural cytochemistry of atrial muscle cells

SummarySections of atrial cardiocytes from young rats were subjected to radioautography after a single intravenous injection of L-leucine-4,5 3H to identify the sites of synthesis and to follow the migration of newly-formed proteins. As early as 2 min after injection of L-leucine 3H, the label was highest in the rough endoplasmic reticulum (RER), suggesting that cisternal ribosomes are sites of protein synthesis. By 5 min, most of the label had migrated from the RER to the Golgi complex. Some label was already present over specific granules by 2 min but the peak was reached at 1 h. By 4 h, the label over the specific granules had diminished, possibly indicating a release of newly-synthetized secretory material outside the cell. The label over myofilaments and Z-bands was relatively high at most time intervals, suggesting an early and important incorporation of leucine into the contractile and structural proteins of these organelles. The label over the cytosol was initially high and increased even further at 5 and 20 min but decreased to a very low level at 4 h. In contrast, the label over the cell surface rose continuously and peaked at 4 h. The pattern of increment of the label over the cell surface suggests that the newly-formed proteins of these sites are also synthetized in the RER, pass through the Golgi complex and are transported in the cytosol before reaching their destination.

Ultrastructural cytochemistry of atrial muscle cells: IX. Reactivity of specific granules in cultured cardiocytes

Abstract A technique for the culture of neonatal (2- to 3-day-old) rat cardiocytes is described. With this technique, ventricular cardiocytes started beating earlier and lived longer, atrial cardiocytes degenerating after 10 days of culture. Specific granules, mostly of the A type, were present in atrial but not ventricular cardiocytes at all time intervals. These specific granules were argentaphobic when stained according to the periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery. The core of these granules was moderately positive after staining fine sections with phosphotungstic acid at low pH. A similar reaction was shown in both atria and ventricle by the cell coat, residual bodies (C-granules) and Z-discs. The Golgi complex was more extensively stained by phosphotungstic acid in atrial than in ventricular cultured cardiocytes or in atrial and ventricular cardiocytes of young (3- and 13-day-old) rats in situ. Multivesicular bodies with a dense core, identical to those already noted in the cells of various endocrine glands, and thought to be crinophagic, were present, as in atrial cardiocytes in situ, in cultured atrial but not ventricular cardiocytes. They were silver negative. Their dense core, as in situ, reacted to phosphotungstic acid but their matrix, contrary to classical multivesicular bodies without a dense core, did not. Numerous, small, phosphotungstic acid-positive vesicles were present in cultured atrial and, to a lesser extent, in cultured ventricular cardiocytes. Vesicles of the same type were rare in either atrial of ventricular cardiocytes of young rats in situ. Glycogen, as revealed by the periodic acid Schiff and Thiery techniques, was equally abundant in cardiocytes in situ and in 1-day-old cultures of cultured cardiocytes from atria and ventricle. These results indicate that specific granules of cultured atrial cardiocytes are probably made up, as in situ, of glycoproteins.

Ultrastructural cytochemistry of atrial muscle cells. VII. Radioautographic study of synthesis and migration of glycoproteins

Rats were injected intravenously with l-fucose 3H to study the synthesis and migration of glycoproteins in atrial cardiocytes and to evaluate the fate of this radioactive sugar in both serum and right atrium at various time intervals. Radioactivity was decreased in serum by 50% within 20 min of the injection and by a 100-fold within 1 h. There was a low uptake of radioactivity by the right atrium and it showed a 5-fold reduction between 20 min and 1 h post-injection. Ultrastructural radioautography revealed a high relative specific radioactivity in the Golgi complex at 5 min and a rapid decrease thereafter. The radioactivity increased steadily in specific granules and cell surface to reach maxima at 4 h. Low levels of radioactivity were present at all time intervals in mitochondria, cytosol, myofilaments and Z-bands. These results indicate that atrial specific granules contain glycoproteins synthetized in the Golgi complex of atrial cardiocytes.

Ultrastructural radioautography of synthesis and migration of proteins and catecholamines in the rat adrenal medulla.

SummaryThe synthetic pathways of proteins and catecholamines in the rat adrenal medullary cells were compared systematically at the ultrastructural level, within a 24 h period, with 2 tracers, L-tyrosine 3,5-3H and L-3,4-dihydroxy [ring 2,5,6-3H] phenylalanine (L-dopa3H). Young rats were injected with either of these tracers and sacrificed in pairs at close time intervals. With L-tyrosine 3H, the label was about equal over rough endoplasmic reticulum (RER) and secretory granules at 2 min after injection and remained almost constant in intensity over the secretory granules throughout the period of observation. A peak of radioactivity was also observed in the Golgi complex between 5 and 20 min after injection. This indicates that L-tyrosine 3H participates in the synthesis of both granule proteins and catecholamines as confirmed by the results obtained after injection of L-dopa 3H. With this tracer, radioactivity over RER, Golgi complex, cytosol and cell surface remained very low at all times and was undetectable at several time intervals. In contrast, radioactivity over secretory granules was very high at all time intervals. The present results thus confirm that in both adrenaline- and noradrenaline-storing cells, the protein moiety of chromaffin granules is synthetized in the RER, packaged in the Golgi complex and rapidly found in newly formed secretory granules. Following either L-tyrosine 3H or L-dopa 3H injection, catecholamine synthesis occurs only in or in close vicinity to chromaffin granules in both cell types at all time intervals.

Ultrastructural cytochemistry of the human adrenal medulla.

SummaryA cytochemical study of the human adrenal medulla showed that it is made up of two cell types, the adrenaline (A-) and noradrenaline (N-) storing cells. A- and N-storing granules were argentaphobic when ultrathin sections of Araldite-embedded medullae were stained according to the periodic acid-thiocarbohydrazide silver proteinate technique of Thiery. A small amount of glycogen (which disappeared after digestion with alpha amylase) in the form of B-particles, as well as lysosomes were, however, visualized by this technique. The entire core of A granules was markedly positive after ultrathin sections of glutaraldehyde-fixed, glycol methacrylate-(GMA-) embedded medullae were stained with phosphotungstic acid (PTA) at a low pH (0.3). The N granules, in contrast, were mostly unreactive. PTA stained a large part of the Golgi complex of A cells, whereas it generally had no such effect on that of the N cells. In both cell types, the cell coat, lysosomes and multivesicular bodies reacted to PTA. The periodic acid —schiff (PAS) technique showed A but not N granules in semithin sections of GMA- or Araldite-embedded medullae. The PTA and PAS stains were abolished by acetylation, restored by saponification, unchanged by methylation and greatly diminished by sulfation or by digestion with beta glucuronidase after oxidation by perchloric acid. These results indicate that in man the A granules and the Golgi complex of A cells, unlike the same structures in N cells, are rich in glycoproteins.

Ultrastructural radioautography of the incorporation of tritiated leucine by the rat adrenal medulla in vivo

SummarySections of tissues from the adrenal medullae of young rats were subjected to radioautography after a single intravenous injection of L-leucine 4,5 3H to identify the sites of synthesis and follow the migration of newly-formed proteins in both adrenaline-storing (A) and noradrenaline-storing (N) cells. As early as 2 min after injection of leucine 3H, the label was highest in the rough endoplasmic reticulum (RER) of A and N cells, suggesting that cisternal ribosomes are sites of protein synthesis. By 5 and 10 min, much of the label had migrated from the RER into the Golgi complex of both cell types. Some label was already present over the secretory granule matrix (chromogranins) by 2 min but the peak was reached at 1 h in both A and N cells. By 4 h, the label over the secretory granules had diminished, indicating a release of newly-synthetized chromogranins outside the cells. The label over the hyaloplasm was relatively high at 2 min but it decreased rapidly to low levels. In contrast, the label over the cell surface continually increased to reach the highest levels among all organelles at 4 h in both cell types. The pattern of increment of the label over the cell surface suggests that the newly-formed proteins of these sites are also synthetized in the RER, pass through the Golgi complex and are transported in the hyaloplasm, before reaching the surface of A and N cells.

On the lysosomal function of juxtaglomerular granules

Summary The presence of acid phosphatase, β-glucuronidase and aryl sulfatase in juxtaglomerular cell granules (JGG) as well as the uptake and concentration of certain low molecular weight dyes by these granules have repeatedly suggested that they are akin to lysosomes. In the present experiments, rats were injected with three substances of widely different molecular weight and physicochemical properties - sucrose, iron sorbitol-citric acid complex (Jectofer) and horseradish peroxidase - that are well known to selectively concentrate in renal tubular cell lysosomes. None of these substances was found to enter the JGG to any significant degree, although both sucrose and Jectofer were evident in juxtaglomerular cells. Contrary to previous reports, thorium dioxide (Thorotrast) particles were not detected in the JGG after parenteral injection. These results indicate that JGG do not possess any significant lysosomal function and raise the question of the role of hydrolytic enzymes in the physiology of these granules.

Synthesis and migration of proteins and glycoproteins in juxtaglomerular cells of sodium-deficient rats

SummarySections of juxtaglomerular cells from sodium-deficient rats were subjected to radioautography after a single intravenous injection of L-tyrosine3,5 3H or of L-fucose 3H to identify the sites of synthesis and to follow the migration of newly-formed proteins and glycoproteins. As early as 2 min after injection of L-tyrosine 3H, the label was highest in the rough endoplasmic reticulum (RER), suggesting that cisternal ribosomes are sites of protein synthesis. By 60 min, much of the label had migrated from the RER to the Golgi complex. Some radioactivity was already present over specific granules by 2 min but a peak was reached at 4h. The label over myofilaments was evident at all time intervals, indicating a certain incorporation of tyrosine into their contractile and/or structural proteins. The label over the cell surface peaked at 4h. After injection of L-fucose 3H, there was an early and important relative specific radioactivity in the Golgi complex at 5 min with a peak at 20 min and a decrease thereafter. The label increased slightly but steadily in secretory granules and cell surface to reach maxima at 4 h. A low level of radioactivity was recorded in mitochondria at all time intervals. After injection of both fucose 3H and tyrosine 3H, the label was detected at relatively low levels in the cytosol. These results suggest that renin, as the major secretory glycoprotein of juxtaglomerular cells, is synthetized in the RER, packaged in the Golgi complex and found relatively rapidly in newly-formed secretory granules. Part of the fucose and tyrosine labels is also associated with the thick cell coat of these cells.