Ca2+, calmodulin and phospholipids regulate nitricoxide synthase activity in the rabbit submandibular gland.

Nitric oxide (NO) plays an important role as an intra- and intercellular signaling molecule in mammalian tissues. In the submandibular gland, NO has been suggested to be involved in the regulation of secretion and in blood flow. NO is produced by activation of NO synthase (NOS). Here, we have investigated the regulation of NOS activity in the rabbit submandibular gland. NOS activity was detected in both the cytosolic and membrane fractions. Characteristics of NOS in the cytosolic and partially purified membrane fractions, such as Km values for l-arginine and EC(50) values for calmodulin and Ca(2+), were similar. A protein band that cross-reacted with anti-nNOS antibody was detected in both the cytosolic and membrane fractions. The membrane-fraction NOS activity increased 1.82-fold with treatment of Triton X-100, but the cytosolic-fraction NOS activity did not. The NOS activity was inhibited by phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate (PIP(2)). The inhibitory effects of phospholipids on the NOS activity were relieved by an increase in Ca(2+) concentrations. These results suggest that the Ca(2+)- and calmodulin-regulating enzyme nNOS occurs in cytosolic and membrane fractions, and PA and PIP(2) regulate the NOS activity in the membrane site by regulating the effect of Ca(2+) in the rabbit submandibular gland.

Association of FcgammaRII with low-density detergent-resistant membranes is important for cross-linking-dependent initiation of the tyrosine phosphorylation pathway and superoxide generation.

IgG immune complexes trigger humoral immune responses by cross-linking of FcRs for IgG (FcgammaRs). In the present study, we investigated role of lipid rafts, glycolipid- and cholesterol-rich membrane microdomains, in the FcgammaR-mediated responses. In retinoic acid-differentiated HL-60 cells, cross-linking of FcgammaRs resulted in a marked increase in the tyrosine phosphorylation of FcgammaRIIa, p58(lyn), and p120(c-cbl), which was inhibited by a specific inhibitor of Src family protein tyrosine kinases. After cross-linking, FcgammaRs and tyrosine-phosphorylated proteins including p120(c-cbl) were found in the low-density detergent-resistant membrane (DRM) fractions isolated by sucrose-density gradient ultracentrifugation. The association of FcgammaRs as well as p120(c-cbl) with DRMs did not depend on the tyrosine phosphorylation. When endogenous cholesterol was reduced with methyl-beta-cyclodextrin, the cross-linking did not induce the association of FcgammaRs as well as p120(c-cbl) with DRMs. In addition, although the physical association between FcgammaRIIa and p58(lyn) was not impaired, the cross-linking did not induce the tyrosine phosphorylation. In human neutrophils, superoxide generation induced by opsonized zymosan or chemoattractant fMLP was not affected or increased, respectively, after the methyl-beta-cyclodextrin treatment, but the superoxide generation induced by the insoluble immune complex via FcgammaRII was markedly reduced. Accordingly, we conclude that the cross-linking-dependent association of FcgammaRII to lipid rafts is important for the activation of FcgammaRII-associated Src family protein tyrosine kinases to initiate the tyrosine phosphorylation cascade leading to superoxide generation.

Complex gangliosides as cell surface inhibitors for the ecto-NAD+ glycohydrolase of CD38.

Leukocyte cell surface antigen CD38 is a single-transmembrane protein whose extracellular domain has catalytic activity for NAD(+) glycohydrolase (NADase). We previously reported that b-series gangliosides inhibit the NADase activity of the extracellular domain of CD38 expressed as a fusion protein [Hara-Yokoyama, M., Kukimoto, I., Nishina, H., Kontani, K., Hirabayashi, Y., Irie, F., Sugiya, H., Furuyama, S., and Katada, T. (1996) J. Biol. Chem. 271, 12951-12955]. In the present study, we examined the effect of exogenous gangliosides on the NADase activity of CD38 on the surface of retinoic acid-treated human leukemic HL60 cells and CD38-transfected THP-1 cells. After incubation of the cells with G(T1b), inhibition of NADase activity was observed. The time course of inhibition was slower than that of the incorporation of G(T1b) into the cells, suggesting that incorporation into the cell membranes is a prerequisite for inhibition. Inhibition occurred efficiently when G(T1b) and CD38 were present on the same cells (cis interaction) rather than on different cells (trans interaction). Although gangliosides may affect localization of cell surface proteins, indirect immunofluorescence intensity due to CD38 was not affected after G(T1b) treatment. Comparison of the effect of G(T1b) and G(D1a) indicates that the tandem sialic acid residues linked to the internal galactose residue of the gangliotetraose core are crucial to the inhibition. These results suggest a novel role of complex gangliosides for the first time as cell surface inhibitors of CD38 through specific and cis interaction between the oligosaccharide moiety and the extracellular domain.

The fibril structure of type V collagen triple-helical domain.

Although the triple-helical structure of fibrillar collagen is regarded in general as being quite similar, each type of collagen molecule has inherent characteristics in the triple-helical domain. Few studies have ever been performed in terms of the aggregate structure of the triple-helical domain of fibrillar collagen. Reconstituted aggregates from the purified triple-helical domain of each type of fibrillar collagen might amplify the subtle differences in the structural characteristics of each type of collagen molecule. In this study, the reconstituted aggregate structure of pepsin-treated type V collagen (type Vp collagen), that is, virtually its triple-helical domain was characterized by transmission electron microscopy. Pepsin-treated type I (type Ip) and type II (type IIp) collagen were compared with type Vp collagen. Unique features of the aggregate structure of the triple-helical domain of the type V collagen can be summarized as follows:These results suggested that the lateral packing of the triple-helical domain of type V collagen is determined by its molecular structure. The characteristics of type Vp collagen fibrils might be explained by their characteristic amino acid composition. A significant feature of the triple-helical domain of type V collagen is the high content of glycosylated hydroxylysine residues. Molecular model building of the collagenous structure suggests that a change in surface roughness is conspicuous by incorporating the glycosylated hydroxylysine residues. More than a ten-fold content of bulky glycosylated hydroxylysine residues in type V collagen compared to that of type I might have a significant influence on both the intermolecular and interfibrillar interactions of the triple-helical domain of type V collagen molecule.

Structural analysis of red blood cell membrane with an atomic force microscope.

To evaluate the usefulness of the atomic force microscope (AFM) for structural analysis of biomedical samples and to determine suitable sample preparation methods for AFM observation, the membrane of human erythrocytes prepared by various methods for electron microscopy was examined by the AFM. Strand-like elevations with 20-50 nm in width, 30-80 nm in length and 3-5 nm in height were observed, which formed networks composed of squares, pentagons and hexagons on the cytoplasmic or back surface of the erythrocyte membrane. Using colloidal gold labelled antibody, this network was found to contain spectrin molecules. Therefore it was very likely that the undercoat molecules of the plasma membrane were imaged by AFM. A large number of gentle elevations 300-400 nm in diameter and 2 nm in height were found to be distributed uniformly on the extracellular or true surface of intact erythrocyte, presumably reflecting the presence of undercoat membrane skeleton on the cytoplasmic surface. However, no structure that seemed to be derived from glycocalyces was discernible on the true surface. Structure corresponding to the unit membrane or lipid bilayer structure observable by electron microscopy was not demonstrated in the cross-section of the membrane. In freeze-fractured samples, a large number of small particles that corresponded to the intramembranous particle were also demonstrated on the membrane halves. Since AFM allows depiction of the fine structures of biological samples with very simple sample processing at a resolution comparable to or exceeding that of SEM, imaging technology using AFM can be applied to obtain biomedical information. However, several problems have to be solved in future development of the equipment.

Review article fine structure of the exocrine cells of rat sublingual gland revealed by rapid freezing and freeze substitution method.

The ultrastructure of mucous cells of rat sublingual gland processed by rapid freezing, followed by freeze substitution, was compared with that obtained by the standard chemical fixation technique. The rapid freezing method gave a very good preservation of membrane structure with round and discrete mucous droplets (granules) not showing any sign of coalescence. The cisterns of the Golgi apparatus and the trans Golgi network also were well preserved. Upon secretory stimulation by pilocarpine, mucous droplets were discharged by the usual mechanism of exocytosis. From all these findings it emerged that mucous cells had the same structural characteristics as serous cells. In the endpieces of rat sublingual gland prepared by the rapid freezing method, serous cells aligned with mucous cells around the central lumen, and no cap-like arrangement of serous cells (demilunes) was observed. Furthermore, computer reconstruction of stereo images from serial section light micrographs prepared by the rapid freezing method showed that, within a given endpiece, all serous cells had direct access to the lumen and that they were disseminated throughout it and not only in its fundus. From our observations it seems very likely that, at least in rat sublingual gland, serous demilunes are an artificial product caused by the compression exerted on serous cells by the mucous cells distended during the conventional fixation procedure.

The serous demilune of rat sublingual gland is an artificial structure produced by conventional fixation.

The ultrastructure of the secretory end-piece of the rat sublingual gland was examined in samples prepared by rapid freezing and freeze-substitution method, and results were analyzed in combination with 3-D images reconstructed by computer graphics from light micrographs of serial sections. Fixation by rapid freezing followed by freeze-substitution preserved cellular ultrastructures, especially the membrane structure, in perfect condition, and demonstrated the terminal portion of the sublingual gland to be a compound branched tubulo-alveolar gland with serous cells distributed throughout the end-pieces. All the serous cells aligned with mucous cells to surround a common lumen, leaving no demilune structure. In contrast, samples fixed by the conventional immersion method showed distended mucous cells displacing the serous cells toward the basal portion of the acinus to form the demilune structure. The luminal space was also compressed and appeared disconnected from the serous cells. From these observations, the serous demilune that for more than 130 years has been believed to be an actual histological entity was proved to be an artificial structure produced through compression by the hydrated and expanded mucous cells during immersion fixation.

Collagen II containing a Cys substitution for Arg-alpha1-519. Analysis by atomic force microscopy demonstrates that mutated monomers alter the topography of the surface of collagen II fibrils.

A recombinant human procollagen II was prepared that contained a substitution of Cys for Arg at alpha1-519 and that was found in five families with early onset generalized osteoarthritis with or without features of a mild chondrodysplasia. Previously, the presence of mutated monomers in mixtures with wildtype collagen II was shown to increase the lag period for fibril assembly. Also, the fibrils were more loosely packed and some thick fibrils lacked a D-periodic banding pattern. Here we re-examined the fibrils using a combination of transmission electron microscopy and atomic force microscopy. The presence of the mutated monomers increased the diameter of the thin filaments that were consistently formed in association with the thick fibrils of collagen II. In addition, the presence of the mutated monomers increased the depth of the gap regions in all fibrils with a distinct D-periodic banding pattern. The results, therefore, may indicate that the mutated monomers formed two or three additional outer layers of monomers in 0D-period staggers on the surface of the fibrils. Apparently, the mutated monomers were bound on the surface through intermolecular disulfide bonds.

Significant roles of inducible cyclooxygenase (COX)-2 in angiogenesis in rat sponge implants.

Angiogenesis in rat sponge implants, as determined from the concentration of hemoglobin in the sponge granuloma tissues, was gradually increased over a 14-day experimental period. The inducible cyclooxygenase COX-2 was detected in the sponge granuloma tissues at day 4 by Western blot analysis using specific mouse COX-2 antibody. Angiogenesis in the sponge implants was enhanced by daily topical injections of human recombinant basic fibroblast growth factor (bFGF) or human recombinant epidermal growth factor (EGF) (100 or 1000 ng/sponge/day) for 4 days. These treatments clearly enhanced the expression of COX-2 in the sponge granuloma tissues. In immunohistochemical studies, COX-2-positive staining was mainly observed in the endothelial cells of the neovasculature and in the fibroblasts of the granuloma capsule. Administration of the selective COX-2 inhibitor NS-398 (p.o., 3 mg/kg, 3 times a day) for 14 days significantly inhibited the angiogenesis. The angiogenesis enhanced with bFGF or EGF (day 4) was inhibited by administration of indomethacin or NS-398, both in the above regimen, and fell to the level obtained without growth factor treatment. These results suggest that COX-2 induced in the sponge granuloma tissues may participate in neovascularization through prostaglandin formation.

Substructures of the acinar basement membrane of rat submandibular gland as shown by alcian blue staining and cryo-fixation followed by freeze-substitution.

The ultrastructure of the epithelial basement membrane was studied in the acinar cells of adult rat submandibular glands after: (i) immersion fixation in 0.1 M sodium cacodylate buffer (CB, pH 7.2) containing 2.5% glutaraldehyde (GA) and alcian blue (0.5%) in conjunction with microwave irradiation, (ii) perfusion fixation with 2.5% GA in CB, (iii) rapid freezing followed by freeze-substitution (RF-FS) with 1% GA in acetone, and (iv) RF-FS with 2% OsO4 in acetone. The specimens were post-fixed with 1% OsO4 in CB after methods (i) and (ii) but not (iii) and (iv). Fixed specimens were embedded in epoxy resin and the ultrathin sections were cut, stained with both lead and uranium, and observed under a transmission electron microscope. Various substructural components could be seen in the acinar basement membrane. In the specimens processed by method (iv), a clear meshwork structure could be found just beneath the basal plasma membrane. This meshwork could not be seen in the specimens processed by method (iii) but thin filaments of approximately 100 nm in length extending from the epithelial base toward the connective tissue space were evident. By methods (i) and (ii), an electron dense 30-75 nm layer could be seen subjacent to basal cell membranes. By method (ii), particularly, thick threads connecting this layer to collagen fibres in the connective tissue were stained with alcian blue. Lamina lucida was not identified.

Failure of the oxytocin-induced increase in secretion of urinary kallikrein in young spontaneously hypertensive rats.

Urinary kallikrein excretion during oxytocin (OT) infusion were studied in anesthetized (sodium pentobarbital, 50 mg/kg, i.p.) young (4-weeks-old) spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). OT-infusion (30 nmol/kg/30 min) to WKY significantly increased urinary excretion of the active kallikrein from the basal levels (25.4 +/- 5.6 10(-2) x AU/15 min, n = 5) to 37.3 +/- 5.0 10(-2) x AU/15 min (P < 0.05, n = 5) and 50.7 +/- 17.1 10(-2) x AU/15 min (P < 0.05, n = 5) 15 and 30 min after the start of OT-infusion, respectively. In SHR, OT-infusion did not increase the urinary excretion of active kallikrein, but did decrease the urine volume and sodium excretion. The concentration of the active kallikrein in the kidney of WKY was not changed by OT-infusion, but that of SHR was slightly increased. The OT-infusion resulted in significantly higher concentrations of the active kallikrein in SHR kidney than in WKY kidney. These results suggest that less excretion of urinary kallikrein in SHR during OT-infusion may be attributable to a lower response in the secretion of kallikrein from the kidney.

Morphological effects of brefeldin A on the intracellular transport of secretory materials in parotid acinar cells.

The morphological effects of Brefeldin A (BFA) on the parotid acinar cells of a rat were investigated at the stage of active resynthesis of secretory materials following administration of the secretogogue, isoproterenol. Incubation with BFA resulted in: a) marked dilation of the rough endoplasmic reticulum (RER), b) involution of the Golgi complex to rudimentary forms which disseminated throughout the cytoplasm, and c) agenesis of secretion granules. It appears that the primary action of BFA is inhibition of the export of secretory materials from the RER toward the Golgi complexes. Histochemical staining indicated the thiamine pyrophosphatase (TPPase) positive saccules of the Golgi stack to undergo degradation in autophagic vacuoles. In contrast, small vesicles showing the osmium reducing activity characteristic of cis elements, including osmium negative vesicles, continued to be present throughout a 4-h period of investigation, indicating the cis and, most likely, medial elements to be the components of the rudimentary Golgi complexes. On removal of the drug, a large number of transport vesicles appeared immediately from the RER and carried secretory materials to the rudimentary Golgi complex, so that the organelles were rapidly reconstructed within 30-60 min, followed by the reaccumulation of secretory granules by 90 min. It is thus indicated that the size and configuration of the Golgi complex is regulated by a dynamic equilibrium of the transport of secretory materials, and that the rudimentary Golgi complex containing cis and probably medial elements may function as the smallest units of the Golgi complex for full development as seen under normal conditions.

Electron microscopic localization of 5'-nucleotidase in rat salivary glands. Comparative enzyme- and immunohistochemical studies.

The localization of 5'-nucleotidase in rat parotid and submandibular glands was investigated at the electron microscope level by an immunohistochemical technique using a highly specific antibody, and the results were compared with those obtained using the newley developed cerium method for enzyme histochemistry. Both methods demonstrated that 5'-nucleotidase is located on the external surface of the luminal plasma membranes of acinar cells as well as on intercalated and striated ductal cells. In the basolateral membranes of these cells, the portions adjacent to myoepithelial cells exhibited intense reaction products, but the other areas of plasma membranes contained only trace amounts of the reaction products. Both cerium-based enzyme histochemistry and immunohistochemistry showed that myoepithelial cells retain the enzyme on their plasma membranes. Neither method produced reaction products in the intracytoplasmic structure of constitutive cells of the salivary glands. We discuss the usefulness of the cerium-ion method for the demonstration of 5'-nucleotidase activity and compare it with the traditional lead-ion method.