Haruko Yanase

Salivary Secretion of Highly Concentrated Chromogranin a in Response to Noradrenaline and Acetylcholine in Isolated and Perfused Rat Submandibular Glands

Chromogranin A (CgA) is a member of a family of highly acidic proteins, chromogranins, which are co‐stored in the adrenergic neurons and paraneurons and co‐released with adrenaline and noradrenaline (NAd) in response to adequate stimulation. The present study provides novel evidence that CgA‐like immunoreactivity (IR) is stored in the exocrine cells in the granular convoluted tubule, and is secreted into saliva by stimulation with NAd and acetylcholine (ACh) in the isolated and perfused rat submandibular gland. NAd at 1 μM produced maximum secretion of CgA‐like IR (<< 0.9 mM) and a marked increase in salivary flow. Further increases in NAd concentration (10 or 100 μM) yielded concentration‐dependent decreases in both responses. ACh at 1 μM produced maximum salivary flow and a slight elevation of CgA‐like IR secretion (6 μM); 100 μM ACh decreased the salivary flow but increased the CgA‐like IR secretion (0.6 mM). Electron microscopic examination showed vigorous compound exocytosis of secretory granules in the cells of the granular convoluted tubule when the submandibular gland was stimulated with 1 μM NAd. These results provide an experimental basis for the view that the salivary CgA‐like IR secretion may be a sensitive and quantitative index of the activity of the sympathetic nervous system innervating the gland.

Epididymis is a novel site of erythropoietin production in mouse reproductive organs.

The epididymis consists of the interstitial tissue and the ductus epididymidis, an extremely tortuous duct, in which spermatozoa exported from the testis gain motility and fertilizing capacity. We found that the cultured mouse epididymis produces erythropoietin (Epo). The content of Epo mRNA in the epididymis from the adult mouse (8-week-old) amounts to 40% of that in the kidney. The epididymal Epo mRNA dramatically increased upon growth; its level increased 120-fold from the age of 3 weeks to 7 weeks when they complete sexual maturation, while the increase in the total RNA was 3-fold. Hypoxia induced a 5-fold increase in the epididymal Epo mRNA transiently, which is much lower than the induction in the kidney (28-fold). In situ hybridization technique elucidated that the site of Epo production was located in the interstitial space between ductus epididymidis. The epididymal Epo may have an unidentified function in the male reproductive organ.

Unique Localization of Protein Gene Product 9.5 in Type B Synoviocytes in the Joints of the Horse

Fibroblast-like (Type B) synoviocytes are cells in the synovial membrane that are responsible for production of both synovial fluid and the extracellular matrix in the synovial intima. Immunostaining of the horse synovial membrane for protein gene product (PGP) 9.5, which is a neuron-specific ubiquitin C-terminal hydrolase, demonstrated selective localization of the immunoreactivity in a synoviocyte population different from acid phosphatase-positive Type A synoviocytes. The immunoreactive cells were lined up in the synovial intima and extended dendritic processes towards the joint cavity to form a dense plexus on the surface. Electron microscopic examination clearly identified the PGP 9.5-immunoreactive cells as Type B synoviocytes characterized by developed rough endoplasmic reticulum and free ribosomes. Immunoreactivity for PGP 9.5 was diffusely distributed throughout the cytoplasm, including the tips of fine processes. Western and Northern blot analyses could not distinguish the corresponding protein and mRNA obtained from the brain and synovial membrane. The existence of the neuron-specific PGP 9.5 in Type B synoviocytes suggests a common mechanism regulating the protein metabolism between neurons and synoviocytes, and also provides a new cytochemical marker for identification of the cells.

Cellular expression of a sodium-dependent monocarboxylate transporter (Slc5a8) and the MCT family in the mouse kidney

Expression analysis of transporters selective for monocarboxylates such as lactate and ketone bodies in the kidney contributes to understanding the renal energy metabolism. Distribution and expression intensity of a sodium-dependent monocarboxylate transporter (SMCT) and proton-coupled monocarboxylate transporters (MCT) were examined in the mouse kidney. In situ hybridization survey detected significant mRNA expressions of SMCT and MCT-1, 2, 5, 8, 9, 10, and 12. Among these, signals for SMCT, MCT2 and MCT8 were predominant; transcripts of SMCT were restricted to the cortex and the outer stripe of outer medulla, while those of MCT2 and MCT8 gathered in the inner stripe of outer medulla and the cortex, respectively. Immunohistochemically, SMCT was present at the brush border in S2 and S3 of proximal tubules, suggesting the active uptake of luminal monocarboxylates here. MCT1 and MCT2 immunoreactivities were respectively found baso-laterally in S1 and thick ascending limbs of Henle’s loop. The cellular localization of transporters suggests the involvement of SMCT in the uptake of filtrated lactate and ketone bodies and that of MCTs in the transport of monocarboxylate metabolites between tubular cells and circulation, but the different distribution patterns do not support the notion of a functional linkage between SMCT and MCT1/MCT2.

Salivary secretion of chromogranin A. Control by autonomic nervous system.

We have provided novel evidence that CGA-LI is stored in the cells of granular convoluted tubule, and is secreted into saliva by stimulation with NAd in the isolated and perfused rat submandibular gland. The cells are also known to secrete various growth factors, which are released into blood stream. The cells may release CGA-LI into blood stream (endocrine secretion) in addition to salivary release (exocrine secretion). The endocrine- exocrine secretion of the cells resembles the secretion of renin in the cells of juxtaglomerular apparatus and coagulating gland.

Production and characterization of monoclonal antibodies against midgut of ixodid tick, Haemaphysalis longicornis

There are concerted efforts toward development of tick vaccines to replace current chemical control strategies that have serious limitations [Parasitologia 32 (1990) 145; Infectious Disease Clinics of North America (1999) 209-226]. In this study, monoclonal antibodies (mAbs) specific to Haemaphysalis longicornis midgut proteins were produced and characterized. Eight antibody-secreting hybridomas were cloned and the mAbs typed as IgG1, IgG2a and IgG2b. On immunoblots, all mAbs reacted with a midgut protein band of about 76 kDa. All mAbs uniformly immunogold-stained the surface or epithelial layers of H. longicornis midgut and endosomes. Adult ticks (50%) that fed on an ascitic mouse producing the IgGs developed a red coloration and did not oviposit. As such, the 76 kDa protein that reacted with the mAbs could, therefore, be a potential candidate for tick vaccine development.

Glucocorticoid inhibits expression of V-1, a catecholamine biosynthesis regulatory protein, in cultured adrenal medullary cells

V‐1 acts as a positive and coordinate regulator of gene expression of catecholamine biosynthetic enzymes in PC12D cells. The present study was conducted to investigate the mechanism controlling expression of V‐1 in the adrenal gland. Immunohistochemical analysis demonstrated that noradrenergic chromaffin cells more highly expressed V‐1 than adrenergic chromaffin cells preferentially expressing the glucocorticoid receptor in rat adrenal glands. Western blotting showed that in cultured bovine adrenal medullary cells, dexamethasone, a synthetic glucocorticoid, inhibited expression of V‐1, and that this inhibition was prevented by RU‐486, a glucocorticoid receptor antagonist. These results suggest that adrenal expression of V‐1 is differentially controlled by glucocorticoids through the specific receptor, and that thereby V‐1 regulates catecholamine biosynthesis in a catecholaminergic phenotype‐dependent manner.