Golgi Saccules Research Articles

Traffic through the golgi apparatus as studied by radioautography

The ability to radiolabel biological molecules, in conjunction with radioautographic or cell fractionation techniques, has brought about a revolution in our knowledge of dynamic cellular processes. This has been particularly true since the 1940's, when isotopes such as 35S and 14C became available, since these isotopes could be incorporated into a great variety of biologically important compounds. The first dynamic evidence for Golgi apparatus involvement in biosynthesis came from light microscope radioautographic studies by Jennings and Florey in the 1950's, in which label was localized to the supranuclear Golgi region of goblet cells soon after injection of 35S-sulfate. When the low energy isotope tritium became available, and when radioautography could be extended to the electron microscope level, a great improvement in spatial resolution was achieved. Studies using 3H-amino acids revealed that proteins were synthesized in the rough endoplasmic reticulum, migrated to the Golgi apparatus, and thence to secretion granules, lysosomes, or the plasma membrane. The work of Neutra and Leblond in the 1960's using 3H-glucose provided dramatic evidence that the Golgi apparatus was involved in glycosylation. Work with 3H-mannose (a core sugar in N-linked side chains), showed that this sugar was incorporated into glycoproteins in the rough endoplasmic reticulum, providing the first radioautographic evidence that glycosylation of proteins did not occur solely in the Golgi apparatus. Studies with the tritiated precursors of fucose, galactose, and sialic acid, on the other hand, showed that these terminal sugars are mainly added in the Golgi apparatus. With its limited spatial resolution, radioautography cannot discriminate between label in adjacent Golgi saccules. Nonetheless, in some cell types, radioautographic evidence (along with cytochemical and cell fractionation data) has indicated that the Golgi is subcompartmentalized in terms of glycosylation, with galactose and sialic acid being added to glycoproteins only within the trans-Golgi compartment. In the last ten years, radioautographic tracing of radioiodinated plasma membrane molecules has indicated a substantial recycling of such molecules to the Golgi apparatus.

Glucose-6-phosphatase activity of endoplasmic reticulum and Golgi apparatus in spermatocytes and spermatids of the rat: an electron microscopic cytochemical study.

The glucose-6-phosphatase (G6Pase) activity of cytoplasmic components of spermatocytes and spermatids of the rat was examined by electron microscope cytochemistry using cerium chloride as a capture agent. G6Pase activity, a recognized ER-resident enzyme, was present in all ER cisternae of spermatocytes. In spermatids, while some ER cisternae were G6Pase-reactive, others were negative or only slightly reactive, indicating an unequal distribution of the enzymatic activity throughout the network of ER cisternae in these cells. In spermatocytes, the cis- and trans-elements of the stacks of Golgi saccules were slightly but significantly reactive for G6Pase. In the Golgi apparatus of spermatids, the cis-element, 4 or 5 underlying saccules, as well as one or two thick trans Golgi elements were G6Pase reactive. The G6Pase activity of the various Golgi elements, like that of the ER cisternae was not affected by the pH of the medium and was completely inhibited by Na-vanadate, a known G6Pase inhibitor. Sertoli and Leydig cells, submitted to the same cytochemical conditions, showed complete G6Pase reactivity of their ER; however in Sertoli cells, all Golgi components were consistently negative while in Leydig cells the cis- and trans-elements of the Golgi stacks were slightly reactive, as in spermatocytes. Thus, the G6Pase reactivity of Golgi elements, appeared variable from one cell type to another. The compact juxtanuclear Golgi apparatuses of spermatocytes and spermatids were both associated with numerous G6Pase reactive ER cisternae; some were present at their surface, others crossed their cortices between Golgi stacks and formed elaborate networks in their cores.(ABSTRACT TRUNCATED AT 250 WORDS)

The Nature of Cytoplasmic Vacuoles in Chordoma Cells: A Correlative Enzyme and Electron Microscopic Histochemical Study

Enzyme histochemical study revealed that a sacrococcygeal chordoma not only was rich in oxidoreductive enzymes but also in the enzymes (phosphorylase, hexokinase, phosphoglucomutase, glucose phosphate isomerase and UDP-glucose dehydrogenase) leading to the synthesis of stromal glycosaminoglycans from glycogen. UDP-glucose dehydrogenase is particularly important in oxidizing UDP-glucose to UDP-glucuronic acid, the building block of hyaluronic acid and chondroitin sulfates. These enzymatic activities were consistent with the ultrastructural findings of abundant membrane-bound glycogen as well as large intracytoplasmic vacuoles with occasional residual glycogen particles. Furthermore, ultrastructural histochemical study using high iron diamine (HID) specifically localized the sulfated glycosaminoglycans (SG) extracellularly as well as intracellularly in distended Golgi saccules and 187-320 nm mature secretory vesicles. No HID staining was noted in the large intracytoplasmic vacuoles or rough endoplasmic reticulum. This study not only supports the hypothesis that the vacuoles of physaliphorous cells are the result of breakdown and utilization of membrane bound glycogen in the biosynthesis of SG, but also demonstrates that intracellular synthesis and storage of SG in chordoma are not in large vacuoles as previous investigators have believed.

Tetracycline administration normalizes the structure and acid phosphatase activity of osteoclasts in streptozotocin‐induced diabetic rats

Diabetes induces osteopenia, which is characterized by a deficiency of osteoid and decreased activity of osteoblasts. We recently found that tetracyclines prevent the loss of osteoid and bone matrix and the degeneration of osteoblasts in diabetic rats by a mechanism independent of their antimicrobial efficacy. However, bone remodeling requires the activity of osteoclasts as well as osteoblasts. To determine the in vivo effects of tetracycline on osteoclasts in long bones, either a tetracycline (minocycline, TC) or its chemically modified non-antibiotic analogue (CMT), 4-de-dimethylaminotetracycline, was administrated daily to streptozotocin-induced diabetic rats by oral intubation. After 21 days, the rats were perfusion-fixed with a mixture of formaldehyde and glutaraldehyde, and the humeri were dissected and processed for ultracytochemical demonstration of acid trimetaphosphatase (ACPase) activity. In untreated non-diabetic (control) rats, the osteoclasts at the zone of provisional ossification exhibited abundant mitochondria and cisterns of rough endoplasmic reticulum (RER) throughout the cytoplasm, prominent stacks of Golgi membranes, and lysosomes in the perinuclear cytoplasm, and numerous various pale vacuoles in the cytoplasmic area adjacent to well-developed ruffled border. Intense ACPase activity was observed in the Golgi saccules, lysosomes, pale vacuoles, and the extracellular canals of ruffled border. The reaction products were also noted along the resorbing bone surfaces associated with the osteoclast ruffled border. The osteoclasts in the untreated diabetic rats showed a cytoplasmic organization similar to that of the non-diabetic control rats, but showed little or no ruffled border which was replaced by a broad clear zone in some of these cells. However, most of the osteoclasts on bone matrix in the diabetics were devoid of both a ruffled border and a clear zone. ACPase activity was detected in the osteoclast cytoplasm of diabetic rat, as in the controls, but to a much lesser extent along the broad clear zone facing the resorbing bone surfaces. The osteoclasts in TC-treated diabetic rats possessed both a clear zone and a small ruffled border. However, in some cases, they lacked both structures reminiscent of the untreated diabetic cells. The osteoclasts of CMT-treated diabetic rats exhibited structural and enzymatic features essentially identical to those of the non-diabetic control rats. These results suggest that the diabetes-induced osteopenia results, at least in part, from degeneration of osteoclasts (as well as atrophic osteoblasts) and that tetracyclines may be effective in preventing these abnormalities by a mechanism not dependent on the drugs' antimicrobial properties.

Immunoelectron microscopic observation of intrahepatic HBeAg in patients with chronic hepatitis B

Immune light and electron microscopic studies using monoclonal antibodies have been applied to localize HBeAg in liver biopsy specimens of 19 patients with chronic hepatitis B. Under the light microscope, HBeAg was demonstrated in nuclei, cytoplasm and on the cell surface of hepatocytes. The number of HBeAg-positive hepatocytes correlated well with the serum levels of HBeAg (enzyme immunoassay) and DNA-polymerase. Of 11 patients in whom high numbers of HBeAg-positive hepatocytes were found at the light microscopic level, HBeAg was also studied in hepatocytes at the electron microscopic level. The HBeAg in nuclei was either found as aggregates or dispersed diffusely. In the aggregates of HBeAg, the 27-nm core particles were frequently found. In addition, the antigen was found in the cytosol of some hepatocytes as amorphous mass and in some hepatocytes in the cisternae of perinuclear space, endoplasmic reticulum and Golgi saccules. Occasionally the antigen was found on the membranes of the cell organelles and on the plasma membranes that faced the intercellular and the Disse spaces. These findings suggest that cytoplasmic HBeAg in hepatocytes may be ultrastructurally classified into two different patterns by its distribution, mainly in endoplasmic reticulum or in cytosol. The ratio varied between hepatocytes with these two types of patterns. The titer of serum HBeAg tended to be higher when the corresponding liver biopsy specimens contained more hepatocytes with HBeAg in endoplasmic reticulum than those with HBeAg in the cytosol.

Multitubular bodies in intestinal cells of Amphiuma means/tridactylum (Urodela): ultrastructural characterization

The jejunal absorptive cells of the salamander Amphiuma, when examined using transmission electron microscopy, were found to possess a unique type of intracellular vacuole containing membranous tubules. These vacuoles, tentatively named multitubular bodies, were located in the cytoplasm between the nucleus and the brush-border membrane, and were seen with greatest frequency in the summer and fall. The vacuoles containing multitubular bodies had an average diameter of 0.6 microns, and the membranous tubules within had an average diameter of 30 nm. The tubules differed morphologically from the vesicles in the multivesicular bodies, and from the primary lysosomes in the polylysosomal vacuoles. The tubules did not exhibit acid phosphatase activity, and were of similar diameter and membrane thickness as the Golgi saccules. In contrast to the multivesicular bodies, the multitubular bodies did not take up exogenous horseradish peroxidase. Early forms of autophagosomes resembling these vacuoles were often seen in the para-Golgi region of the cell. The multitubular bodies may represent a distinct type of autophagosome. Although the exact origin of the tubules as well as their role in cellular activity is unclear, their seasonal appearance within the multitubular bodies of the absorptive cells suggests a unique means of selective down-regulation of Golgi-like organelles.

Endocytosis of wheat germ agglutinin binding sites from the cell surface into a tubular endosomal network

By using fluorescence and electron microscopy, the endocytic pathway encountered by cell surface components after they had bound wheat germ agglutinin (WGA) was visualized. The majority of these components are thought to consist of sialylated glycoproteins (HMWAG) that represent a subpopulation of the total cell surface proteins but most of the externally disposed plasma membrane proteins of the cell. Examination of semi-thin sections by medium- and high-voltage electron microscopy revealed the three-dimensional organization of vesicular and tubular endosomes. Binding of either fluorescein isothiocyanate-, horseradish peroxidase-, or ferritin-conjugated WGA to cells at 4 degrees C showed that the HMWAG were distributed uniformly over the cell surface. Warming of surface-labeled cells to 37 degrees C resulted in the endocytosis of WGA into peripheral endosomes via invagination of regions of both coated and uncoated membrane. The peripheral endosome appeared as isolated complexes comprising a vesicular element (300-400 nm diam.) surrounded by and continuous with tubular cisternae (45-60 nm diam.), which did not interconnect the endosomes. After 30 min or more label also became localized in a network of anastomosing tubules (45-60 nm diam.) that were located in the centrosomal region of the cell. Endocytosed WGA-HMWAG complexes did not become associated with cisternae of the Golgi apparatus, although tubular and vesicular endosomes were noted in the vicinity of the trans-Golgi region. The accumulation of WGA-HMWAG in the endosomes within the centrosomal region was inhibited when cells were incubated at 18 degrees C. None of these compartments contained acid phosphatase activity, a result that is consistent with other data that the HMWAG do not pass through lysosomes initially. The kinetics of labeling were consistent with the interpretation that recycling of most of the WGA binding surface glycoproteins occurred rapidly from early peripheral endosomes followed by the late trans-Golgi compartment. In conclusion, a portion of cell surface glycoproteins are routed to a complex arrangement of tubular and vesicular compartments following endocytosis that includes a putative post-endosomal, tubular reticulum that appears to be separate from the trans-most Golgi saccule.

Alpha-granule pool of glycoprotein IIb-IIIa in normal and pathologic platelets and megakaryocytes

Using an immunogold staining technique and electron microscopy, we investigated the localization of the alpha-granule pool of glycoprotein (GP) IIb-IIIa in normal platelets and maturing megakaryocytes (MK), in pathologic platelets from a patient with type I Glanzmann's thrombasthenia (GT), and from three patients with the gray platelet syndrome (GPS). In normal resting platelets, GPIIb-IIIa was observed on the plasmatic side of the plasma membrane, the open canicular system (OCS) membranes, and along the internal face of the alpha-granule membrane. This location was found with three monospecific polyclonal antibodies: one anti-GPIIb-IIIa antibody, the second specific for GPIIb, and the third specific for GPIIIa. After thrombin stimulation, the alpha-granule labeling disappeared whereas membrane labeling increased. Platelets from GT did not display labeling on plasma membranes, OCS membranes, or alpha-granule membranes. Platelets from the three patients with GPS displayed intense labeling of the plasma membrane and the OCS membrane, as well as the abnormal small alpha- granules and along the inside of large vacuoles (which contain the granule membrane protein [GMP]-140). In cultured immature MK from normal progenitors, both peptide components of GPIIb-IIIa appeared in the Golgi saccules and vesicles, and in the small precursors of alpha- granules, labeling both their membranes and their matrix. It was then observed only on the membrane of the mature MK alpha-granules, although labeling was less consistent than on the platelet granules. The MK plasma membrane and demarcation membrane system also displayed GPIIb- IIIa labeling. In conclusion, this study demonstrates that GPIIb-IIIa is present on the internal face of the alpha-granule membranes of platelets (where it appears early during MK maturation) as well as in the abnormal alpha-granules of gray platelets; it is absent from GT type I platelets.

Alpha-Granule Pool of Glycoprotein Ilb-IIIa in Normal and Pathologic Platelets and Megakaryocytes

Using an immunogold staining technique and electron microscopy, we investigated the localization of the alpha-granule pool of glycoprotein (GP) IIb-IIIa in normal platelets and maturing megakaryocytes (MK), in pathologic platelets from a patient with type I Glanzmann's thrombasthenia (GT), and from three patients with the gray platelet syndrome (GPS). In normal resting platelets, GPIIb-IIIa was observed on the plasmatic side of the plasma membrane, the open canicular system (OCS) membranes, and along the internal face of the alpha-granule membrane. This location was found with three monospecific polyclonal antibodies: one anti-GPIIb-IIIa antibody, the second specific for GPIIb, and the third specific for GPIIIa. After thrombin stimulation, the alpha-granule labeling disappeared whereas membrane labeling increased. Platelets from GT did not display labeling on plasma membranes, OCS membranes, or alpha-granule membranes. Platelets from the three patients with GPS displayed intense labeling of the plasma membrane and the OCS membrane, as well as the abnormal small alpha-granules and along the inside of large vacuoles (which contain the granule membrane protein [GMP]-140). In cultured immature MK from normal progenitors, both peptide components of GPIIb-IIIa appeared in the Golgi saccules and vesicles, and in the small precursors of alpha-granules, labeling both their membranes and their matrix. It was then observed only on the membrane of the mature MK alpha-granules, although labeling was less consistent than on the platelet granules. The MK plasma membrane and demarcation membrane system also displayed GPIIb-IIIa labeling. In conclusion, this study demonstrates that GPIIb-IIIa is present on the internal face of the alpha-granule membranes of platelets (where it appears early during MK maturation) as well as in the abnormal alpha-granules of gray platelets; it is absent from GT type I platelets.

Immunoelectron microscopic localization of thyrotropin-releasing hormone (TRH) precursor in the rat raphe nuclei

Using antisera to two pro-thyrotropin-releasing hormone (pro-TRH)-derived cryptic peptides, we have studies by immunocytochemistry the ultrastructural localization of pro-TRH in the rat raphe nuclei. The same results were obtained with both antisera. Immunostaining was found in cell bodies, dendrites and endings. In cell bodies, the reaction product was restricted to Golgi saccules and dense core vesicles which were very few in number. In dendrites, the staining was rather diffuse without any association with specific organelles. These results suggest that the Golgi apparatus might be involved in pro-TRH processing.

Ultrastructural Distribution of NADPase within the Golgi Apparatus and Lysosomes of Mammalian Cells

Cytochemical studies with over 40 different mammalian cell types have indicated that NADPase activity is associated with the Golgi apparatus and/or lysosomes of all cells. In the majority of cases, NADPase is restricted to saccular elements comprising the medial region of the Golgi stack and an occasional lysosome. There is often weak NADPase activity in other Golgi compartments such as the trans Golgi saccules and/or elements of the trans Golgi network. In some cells, however, strong NADPase activity is found within these latter compartments, either exclusively in trans Golgi saccules or elements of the trans Golgi network, or in combination with medial Golgi saccules and each other including (1) medial Golgi saccules + trans Golgi saccules, (2) medial Golgi saccules + trans Golgi saccules + trans Golgi network, or (3) trans Golgi saccules + trans Golgi network. In some rare cases, no NADPase activity is detectable in either Golgi saccules or elements of the trans Golgi network, but it is observed in an occasional lysosome or throughout the lysosomal system of these cells. It is unclear at present if these variations in the distribution of NADPase across the Golgi apparatus, and between the Golgi apparatus and lysosomal system, are due to differences in targeting mechanisms or to the existence of "bottlenecks" in the natural flow of NADPase along the biosynthetic pathway toward lysosomes. While no clear pattern in the association of strong NADPase activity with lysosomes was apparent relative to the ultrastructural distribution of NADPase activity in Golgi saccules or elements of the trans Golgi network, the results of this investigation suggested that cells having NADPase localized predominantly toward the trans aspect of the Golgi apparatus (in trans Golgi saccules or elements of the trans Golgi network or both) have few NADPase-positive lysosomes. The only exception is hepatocytes which were classified as predominantly trans but had noticeable NADPase activity within medial Golgi saccules and elements of the trans Golgi network as well, and highly reactive lysosomes. Other cells showing highly reactive lysosomes including (1) Kupffer cells of liver and those forming the proximal convoluted tubules of the kidney, both of which also had strong NADPase activity within medial and trans Golgi saccules and elements of the trans Golgi network, (2) Leydig cells of the testis and interstitial cells of the ovary, which also showed strong NADPase activity within medial Golgi saccules, and (3) macrophages from lung, spleen and testis, and Sertoli cells from the testis all of which showed no Golgi associated NADPase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Lectin bindings and diethylstilbestrol effects on the recognition of mullerian inhibiting substance (MIS) on chick mullerian ducts by MIS-antiserum

A high intensity of lectin bindings was demonstrated on the epithelial cells and serosa cells of the regressing right Mullerian ducts (Mds) in the female chick embryos. The strong lectin bindings occurs on, or in the regressing Md cells along with marked surface MIS bindings at the age of day 13. However, at the age of days 5-7 1/2, bindings of lectins were weak. Neither Wheat-germ agglutinin (WGA) or Concanavalin A (Con-A) labelings before MIS-antiserum (MIS-Ab) incubation can block antibody recognitions to the antigens, including MIS and growth hormone at the age of day 13. Our previous studies indicated that after WGA labeling on the surfaces of Md epithelial cells prior to the incubation of MIS-Ab at day 10 did not prevent the recognition of MIS-Ab (Wang 1989). On the contrary, at day 7 1/2, the specific binding of MIS was eliminated after preincubations with lectins and prenatal diethylstilbestrol (DES) treatment at the age of day 5. It is suggested that DES provides a protection to the Mds from MIS-induced regression by preventing the MIS binding to its specific membrane receptors. An increase of extra- and intracellular glycoproteins or carbohydrates of regressing Md epithelial cells were suggested. Internalization of WGA but not MIS molecules was found in Md epithelial cells. The Golgi saccules were negative of lectin bindings.

Ultrastructural and Cytochemical Studies of the Subcommissural Organ in the Domestic Chicken with Special Reference to the Apical Secretion

Ultrastructural and cytochemical studies were undertaken with special reference to the apical secretion in the ependymal cells of the subcommissural organ in the domestic chicken. Glucose-6-phosphatase (G6Pase) and acid phosphatase (AcPae) were used as cytochemical markers for the endoplasmic reticulum (ER) and lysosomes, respectively. Periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) cytochemistry was employed to detect glycoconjugates. Most dense bodies were negative with AcPase staining. Dilated cisternal of ER were reacted with G6Pase cytochemistry. Golgi saccules and several kinds of spherical bodies were stained with PA-TCH-SP cytochemistry. Our results suggest that: 1) most dense bodies in the apical cytoplasm belong to the secretory granules discharged into the ventricular cavity, 2) dilated cisternae of the ER serve as the storage site of the substance which eventually becomes the secretory product, however, contents of the ER are not directly released into the ventricular cavity. Findings of this studies lead us to speculate that there are two intracellular transportation via different compartments: one is a transport via small vesicles from the perinuclear ER to the Golgi apparatus and the other is a transport bypassing the Golgi apparatus via cisternae of ER from the perinuclear ER to the supra-Golgi region. The substances in these two structures seem to be intermingled with each other and matured into secretory granules. This granules may be discharged into the ventricular cavity by exocytosis.

High-voltage electron microscopic observation of golgi complex in the rat small ganglion cell. Thiamine metabolism.

Thiamine mono- and pyrophosphatase (TMPase, TPPase) activities were demonstrated in the rat small ganglion cell under high-voltage electron microscopy. From a series of micrographs of the same field tilted in increments, a trans tubular network (TTN) consisting of looping and anastomosing tubules was found; this network showed relatively higher intense TMPase activity, and exhibited connections with TMPase-positive vesicle-like bodies. TPPase activity was localized in the trans Golgi saccules, and the TPPase-positive transmost saccule had a structural resemblance to the TTN. These results indicate that the TTN plays an important role in thiamine metabolism.

Cosmos 1887: morphology, histochemistry, and vasculature of the growing rat tibia

Light microscopy, electron microscopy, and enzyme histochemistry were used to study the effects of spaceflight on metaphyseal and cortical bone of the rat tibia. Cortical cross-sectional area and perimeter were not altered by a 12.5-day spaceflight in 3-month-old male rats. The endosteal osteoblast population and the vasculature near the periosteal surface in flight rats compared with ground controls showed more pronounced changes in cortical bone than in metaphyseal bone. The osteoblasts demonstrated greater numbers of transitional Golgi vesicles, possibly caused by a decreased cellular metabolic energy source, but no difference in the large Golgi saccules or the cell membrane-associated alkaline phosphatase activity. The periosteal vasculature in the diaphysis of flight rats often showed lipid accumulations within the lumen of the vessels, occasional degeneration of the vascular wall, and degeneration of osteocytes adjacent to vessels containing intraluminal deposits. These changes were not found in the metaphyseal region of flight animals. The focal vascular changes may be due to ischemia of bone or a developing fragility of the vessel walls as a result of spaceflight.

Effects of Secretory Stimulation on the Golgi Apparatus and Gerl of Rat Tracheal Gland Cells

The morphological and cytochemical changes of the Golgi apparatus and GERL of rat tracheal serous cells were studied after in vivo secretory stimulation with pilocarpine. Discharge of PAS-positive secretory granules had started within 30 min and complete discharge was finished within 2 h after stimulation. Reaccumulation of secretory granules became evident at 6 h and the volume of granules was similar to that of unstimulated control cells at 8 h. At 6 h after pilocarpine administration, acid phosphatase (AcPase) activity was markedly increased in GERL and immature secretory granules as compared to uninjected controls and this phenomenon continued 12 h after stimulation. GERL appeared to increase in extent and numerous continuities with immature granules were observed. Thiamine pyrophosphatase (TPPase) activity, normally present in trans Golgi saccules was not altered during the secretory cycle. At 2 h after stimulation, tubular structures of GERL, which also react to AcPase, were seldom observed. At 6 h, these structures were more numerous than those of control cells. These results demonstrate that, in rat tracheal serous cells, pilocarpine induces alteration of only GERL, and reconstitution followed by resynthesis of secretory granules is performed without participation of the Golgi stack.

Density gradient separation of two populations of lysosomes from rat parotid acinar cells.

Exocrine acinar cells possess two cytochemically distinct populations of secondary lysosomes. One population is Golgi associated and has demonstrable acid phosphatase (AcPase) activity, whereas the second is basally located and lacks AcPase activity but has trimetaphosphatase (TMPase) activity. The basal lysosomes are tubular in shape and rapidly label with horseradish peroxidase (HRP) after intravenous injection. In the present study using isolated rat parotid acinar cells, the two lysosomal populations were separated by cell fractionation on Percoll density gradients and were analyzed biochemically and by EM cytochemistry. On 35% Percoll gradients, two peaks of AcPase and beta-hexosaminidase, both lysosomal marker enzymes, and succinic dehydrogenase, an enzyme marker for mitochondria, could be resolved. The major peaks of beta-hexosaminidase and succinic dehydrogenase and the minor peak of AcPase corresponded with the dense lysosome fraction. The major peak of AcPase and the minor peaks for beta-hexosaminidase and succinic dehydrogenase coincided with the light membrane fraction. Galactosyl transferase (a marker enzyme for Golgi saccules) and 5'-nucleotidase (a plasma membrane marker) were also associated with this fraction. By electron microscopy, the light membrane fraction was seen to contain tubular elements, multivesicular bodies (MVB), Golgi saccules, GERL, immature secretory granules, and some mitochondria. Electron microscopic cytochemical examination showed that these tubular structures were lysosomes. The dense lysosome fraction contained lysosomes positive for both AcPase and TMPase. After continuous incubation of isolated acinar cells with HRP, reaction product was rapidly localized to the light membrane fraction (greater than 2 min), where it was found in vesicles and tubular lysosomes. By 10 min it was present in MVB and tubular lysosomes, but by 60 min no HRP reaction product had appeared in the dense lysosomes. These results demonstrate that the tubular lysosomes are separable from dense lysosomes, typical secondary lysosomes, and are involved in the initial stages of endocytosis.

Cytochemical demonstration of TPPase in myelinated fibers in the central and peripheral nervous system of the rat

Thiamine pyrophosphatase (TPPase) activity was demonstrated by means of cytochemistry and electron microscopy in association with myelinated fibers in the central and peripheral nervous system of the rat. The areas studied included corpus callosum, hippocampus, cerebral cortex, cervical spinal cord and sciatic nerves. In the myelin sheaths, the enzymatic activity was found in 3 locations: (1) within oligodendroglial and Schwann cytoplasmic clefts between myelin lamellae; (2) in the major dense line of myelin; and (3) within the periaxonal space. In addition to this myelin-associated TPPase, enzymatic activity was also observed in specific cytoplasmic localizations in myelinogenic cells. Oligodendrocytes displayed TPPase activity within the nuclear envelope and the endoplasmic reticulum cisternae, whereas Schwann cells displayed TPPase activity within the endoplasmic reticulum and Golgi saccules. The results are discussed in relation to the role that TPPase might play in myelinated fibers, including roles in the conduction of nerve impulses or roles in the maintenance of structural configuration of myelin sheaths.

Subcellular localization of benzodiazepine/GABAA receptors in the cerebellum of rat, cat, and monkey using monoclonal antibodies

Two monoclonal antibodies, bd-17 and bd-24, specific for the beta- and alpha-subunit of the GABAA/benzodiazepine receptor/chloride channel complex, respectively, were used to determine the subcellular distribution of immunoreactivity in the cerebellum by electron microscopy. The 2 antibodies showed similar antigen distribution on the plasma membrane (except in the rat; bd-24 does not recognize the rat antigen), but intracellular immunoreactivity was more prevalent for the alpha-subunit. The plasma membrane of all neuronal types was immunopositive. The degree of immunoreactivity varied greatly between different types of cell, but it was stereotyped among individual cells of the same type. Granule cells showed the strongest immunoreactivity, not only on their dendrites which receive synapses from GABA-containing Golgi cell terminals, but also on their somata which do not receive synapses. Stellate and basket cells were somewhat weaker in immunoreactivity. Purkinje cells were only weakly positive on their somatic membrane but stronger on their dendritic shafts and spines. Golgi cells showed negligible if any immunoreactivity. Neurons of the deep cerebellar nuclei were strongly immunopositive along their plasma membrane. Immunoreactivity was strong in cisternae of the endoplasmic reticulum and in the Golgi saccules of stellate and basket cells, variable in Purkinje cells, while granule cells were rarely immunoreactive intracellularly. It is suggested that these differences reflect differences in the turnover of the receptor complex in the different cell types. The synaptic clefts established by boutons of the GABAergic stellate, basket, and Golgi cells were immunopositive, as were many synapses in the deep cerebellar nuclei. However, immunoreactivity was also present along the nonjunctional plasma membrane, and it was concluded that this reflected the distribution of the antigen. The synaptic clefts at the presumed glutamate-releasing parallel and mossy fiber terminals were almost always immunonegative. No immunoreactivity was detected on axons, nerve terminals, or glial cells. The results demonstrate that different neuronal types express the GABAA/benzodiazepine receptor/chloride channel complex to different degrees. The distribution of the receptor complex suggests that the cellular topography of GABAergic influence is not governed by the precise spatial arrangement of the receptors but by the precise placement of the GABA-releasing terminals, a characteristic of the cerebellar circuit.

Polyol and vacuole formation in cultured canine lens epithelial cells

Polyol accumulation and myo-inositol depletion were accompanied by extensive vacuole formation in cultured canine lens epithelial cells that were incubated for up to 96 hr in growth medium supplemented with 30 m m d-galactose or 30 m m d-glucose. These changes did not occur in cells incubated in a hypergalactosemic or hyperglycemic medium which also contained an aldose reductase inhibitor (20 μ m sorbinil). In addition, these changes were not observed in lens cells incubated in growth medium supplemented with either 30 m m mannitol, which is known to enter cells only slowly, or in 30 m m l-galactose, which is not a substrate for aldose reductase. The vacuoles were visible at the ultrastructural level after 6 hr of incubation in 30 m m >d-galactose and increased in both number and size with time. These vacuoles had a unique fine structure. They did not result from swelling of mitochondria or other cell organelles. As demonstrated cytochemically, they did not represent either lysosomes or Golgi saccules. The proliferation pattern of cells incubated with 30 m m d-galactose was clearly different from that of control cells, but approached normal when an aldose reductase inhibitor was added to the incubation medium. Together these findings suggest that vacuole formation and altered cell proliferation were caused by polyol accumulation and/or myo-inositol loss, both of which result from aldose reductase activity.