It is well known that the renin-angiotensin system (RAS) is involved in the control of blood pressure. Recently, all or part of the components concerning the RAS have been reported to be synthesized and secreted outside of classical organs or tissues, at sites including the brain, pituitary and pineal glands, eye, heart, adrenal gland, testes, ovary, placenta, and coagulating glands. The functions and roles of these local RAS are not well known. In the present review, the author explains the history of the RAS, the circulating RAS and the existence of local RAS in multiple organs and tissues, discussing especially the function of coagulating gland renin.
Renin protein, the triggering enzyme of the RAS, is distributed generally in certain fixed cells of several organs and tissues, exemplified by the gonadotrops in the pituitary glands and Leydig cells in the testis. Renin mRNA and its expressing cells can also be detected from the above cells as a whole. In some tissues, angiotensinogen-containing cells do not, however, correspond to its mRNA-expressing cells and potent activator angiotensin II-containing cells, as, for example, in the brain. These cases are explained by constitutive pathways of angiotensinogen processing. Coagulating gland renin, which the author is investigating vigorously, is the most recently discovered local renin, and represents significant subject for investigations. It is suggested that coagulating gland renin may play an unique function for sexual organs by exocrine mechanism.

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This review briefly introduces the principles of the atomic force microscope (AFM) and shows our own results of AFM application to biological samples. The AFM, invented in 1986, is an instrument that traces the surface topography of the sample with a sharp probe while monitoring the interaction forces working between the probe and sample surface. Thus, the AFM provides three-dimensional surface images of the sample with high resolution. The advantage of the AFM for biologists is that AFM can visualize non-conductive materials in a non-vacuous (i. e., air or liquid) environment.
AFM images of the plasmid DNA are comparable to those by transmission electron microscopy using a rotary shadowing technique, and have the advantage of examing directly the molecule without staining nor coating. The surface structure of human metaphase chromosomes and mouse collagen fibrils demonstrated in air by the non-contact mode AFM is comparable to that obtained by scanning electron microscopy. Quantitative information on the heights of structures is further obtainable from the AFM images. Embedmentfree thin tissue-sections are useful for observing intracellular structures by AFM.
The present review also shows AFM images of living cultured cells which have been collected in a contact mode in liquid. This technique afforded us three-dimensional observation of the cellular movement with high resolution. Although there are some innate limitations for AFM imaging, the AFM has great potential for providing valuable new information in histology and cytology.

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The occurrence, distribution and innervation of guinea pig vallate papillae were investigated by means of indirect immunofluorescence and immunoperoxidase methods using antibodies against: a neuronspecific protein, protein gene product 9.5 (PGP 9.5); various neuropeptides including calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal polypeptide (VIP) and galanin; a monoamine, serotonin (5-hydroxytryptamine; 5HT). Numerous PGP 9.5-immunoreactive nerve fibers were found to form plexuses in the lingual epithelium both intragemmally and extragemmally and to comprise dense bundles in the lamina propria just beneath the epithelium. Moderate numbers of PGP 9.5-immunoreactive cells were observed in the taste buds. These cells, typically spindle in shape, extended through the entire thickness of the taste bud. CGRP-immunoreactive nerve fibers were numerous in the subgemmal connective tissue and entered the epithelium to form intragemmal and extragemmal networks. A dense subgemmal SP-immunoreactive network in the vallate papilla can be linked to the presence of taste buds, even though SP-immunoreactive nerve fibers rarely occurred intragemmally. No taste cells immunoreactive for CGRP and for SP were observed. Immunoreactivity for VIP or galanin was not detected in nerve fibers and taste cells. In contrast, some taste cells and a few, fine networks of nerve fibers in the connective tissue were immunoreactive for 5HT; none of the intraepithelial fibers were 5HT-immunoreactive. We suggest that: 1) functionally, 5HT-containing cells and the CGRP-containing nerve fibers may be primarily involved in the neural transmission or its modulation of the taste sensation; and 2) VIP and galanin can be excluded from that group of substances which plays important roles in taste sensation.

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In response to antigen inhalation, immune cells including alveolar macrophages expressing a VIP1 receptor subtype (VIP1R), lymphocytes and leukocytes participate in the inflammatory event, migrating into and from vascular regions in lung tissue of sensitized mice. To analyze these migratory mechanisms of immune cells, we immunohistochemically examined the expression of the following: cellular adhesion molecules, lymphocyte function-associated antigen-1 (LFA-1), very late activation antigen-4 (VLA-4), and the alpha V (∂v) subunit and their respective ligands, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and fibronectine; the examination was carried out in pulmonary tissue from days 0, 2, 6 and 12 following intratracheal administration of sheep red blood cells (SRBC) as an antigen to previously sensitized mice. Two days following the antigen challenge, VIP1R-positive macrophages strongly expressing the ∂v integrin subunit were found clustered on the endothelial surface and among the aggregates of perivascularly infiltrated leukocytes. On the endothelium of arteries, veins and capillaries, ∂v immunoreactivity was prominently reduced, whereas staining for fibronectin was enhanced more than the prechallenge control level. The blood vessel endothelium was also stained positive for VCAM-1 and ICAM-1, while many of the infiltrating lymphocytes were positive for VLA-4 and LFA-1 immunolabelings. By post-challenge, day 6, ∂v integrin subunit immunoreactivity was re-expressed on the blood vessel endothelium and only weakly expressed on VIP1R-positive macrophages, which were in retreat from the leukocyte-aggregating perivascular region and located in the alveoli. VLA-4 bearing lymphocytes conspicuously increased in number among the perivascular leukocytes, while immunoreactivity for LFA-1, VCAM-1, ICAM-1 and fibronectin was unchanged from that for post-challenge day 2. The results indicate that the expression of the ∂v-bearing integrin and its ligand fibronectin drastically changes as pulmonary inflammatory responses. These changes in expression of adhesion molecules during immune response may play an important role in the dynamic regulation of VIP1R-positive macrophage migration in the lung parenchymal compartment.

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The intercalated discs of working myocardium and Purkinje fibers of the monkey heart were examined by scanning and transmission electron microscopy. The NaOH/ultrasonication technique resulted in the digestion of connective tissue and a separation of the intercellular junctions of intercalated discs, such that these could be visualized three-dimensionally.
The intercalated discs of ventricular myocytes, atrial myocytes and Purkinje fibers vary considerably in number and configuration, as do the intercalated discs of the three different layers of the ventricular myocardium. Myocytes in the subepicardial, middle and subendocardial layers of the ventricle have 1-3, 4-5 and 5-6 intercalated discs at the end of these cells, respectively. Those in the endocardial layer are characterized by the presence of small laterally-placed intercalated discs. Atrial myocytes and Purkinje fibers usually only have 1-2 intercalated discs. Individual intercalated discs in ventricular myocytes have complicated stairs with 10-30 steps and corresponding risers, while those of atrial myocytes and Purkinje fibers have simple stairs with 1-3 steps and risers. Steps equivalent to the plicate segments are characterized by densely-packed microplicae and finger-like microprojections which greatly increase surface area in vertricular myocytes. Microprojections in atrial myocytes and Purkinje fibers are sparse by comparison. Risers equivalent to the interplicate segments containing large gap junctional areas are most numerous in left ventricular myocytes, followed by right ventricular myocytes, Purkinje fibers and atrial myocytes in decreasing order.
The geometric arrangement of the various types of myocytes may be related with impulse propagation. Large intercalated discs of cell trunks and series branches may participate in longitudinal propagation, while small laterally-placed ones may be the site of transverse propagation.

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Follicles displaying a positive periodic acid-Schiff reaction to a colloid substance in the anterior pituitary gland have been observed in many vertebrates, including humans. It is also known that these follicles greatly increase in number and size with age. We therefore performed histochemical analysis of these colloid follicles in the senescent porcine pituitary gland in order to clarify their nature and biological significance. Results from various histological stains suggest that the colloid contains some polysaccharides or glycoproteins. In addition to the histological stains, lectin histochemistry revealed that the colloid contains sialic acid, N-acetyl galactosamine and galactose. Also, lectin staining indicated that some glycoproteins, most likely Asn-linked sialoglycoproteins, are localized in the colloid. The cells (FS cells) surrounding the colloid were densely stained with an antibody to S-100 protein but were not stained with antibodies to any other anterior pituitary hormones. Frequently mammotrophs (PRL cells), and occasionally gonadotrophs (LH cells) were found closely associated with folliculo-stellate cells (FS cells) which lay next to the large colloid containing follicles. This suggests that not only are the FS cells important in the production of the colloids, but the adjacent LH and PRL cells in some way also contribute to their formation.

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A transmission-electron microscope study of the rat circumvallate taste bud revealed that occasionally one and the same gustatory or Type III cell received innervation associated with subsurface cisterns as the Type II cell did, in addition to the ordinary afferent synapse.
The subsurface cistern, a flattened, smooth-surfaced saccular membrane system, hugged the plasma membrane of the gustatory cell along the boundary against the nerve terminal. The nerve terminal attaching to the cell was occupied with mitochondria and synaptic type vesicles.
As these structural features resemble those of efferent synapses in certain other sensory cells including the inner ear hair cells, the gustatory cells dually innervated as demonstrated in the present study are presumably involved not only in the afferent but also in the efferent projection of nerves.

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RNA blot analysis has shown that an ionotropic ATP receptor P2X₄ is widely distributed, as compared with other P2X subtypes. Cellular distribution of the P2X₄ ATP receptor mRNA was investigated by in situ hybridization technique in the brain and non-neuronal organs. In the brain, transcripts of the P2X₄ were widely distributed in various regions, with the highest signals in the cerebellar Purkinje cells and granule cells. In other organs, intense and selective expression was detected in seminiferous tubules of the testis and intestinal crypts of the colon, suggesting an involvement of ATP in spermatogenesis and intestinal secretion. A low but significant level of the P2X₄ mRNA was detected in the adenohypophysis and the cortex of the adrenal gland. The tissue- or cell-specific expression of the P2X₄ mRNA is of importance to understand the functional roles of the purinergic transmission system.

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The distribution and origins of nitric oxide (NO)-producing nerves in the dog tongue with reference to calcitonin gene-related peptide (CGRP)-containing sensory fibers were investigated using NADPH-diaphorase (NADPH-d) histochemistry and immunohistochemistry for CGRP and NO synthase combined with retrograde axonal tracing and denervation experiments. The ultrastructural relationships between NADPH-d-positive and CGRP-immunoreactive neuronal elements were also examined electron microscopically. NADPH-d-positive and CGRP-immunoreactive varicose fibers were found within the taste buds and surrounding the epithelia of the fungiform papillae, and they disappeared completely after severance of the lingual nerve. Following injection of fast blue into the subepithelial layer of the anterior two thirds of the tongue, retrogradely labeled neurons possessing NO synthase and/or CGRP immunoreactivities were mainly detected in the trigeminal ganglion. Some of the retrogradely labeled trigeminal cells showed the coexistence of NADPH-d reactivity and CGRP immunoreactivity, but in the geniculate ganglion neither NADPH-d reactivity nor NO synthase immunoreactivity was found instead of retrogradely labeled CGRP-immunoreactive neurons.
The lingual artery and its branches, including the arteriovenous anastomoses, showed dense distributions of NADPH-d-positive fibers, most of which were unaffected by the denervation experiments. There were many small ganglia in the tongue, and virtually all ganglionic neurons were NADPH-d reactive. CGRP-immunoreactive varicose fibers were also found around the vascular walls and within the intralingual ganglia. Ultrastructural analysis revealed a close distribution of NADPH-d-positive and CGRP-immunoreactive varicose fibers within the arterial walls, and synaptic contacts between CGRP-immunoreactive terminals and NADPH-d-positive intralingual ganglionic neurons. These results indicated that the taste buds and epithelia of fungiform papillae in the anterior two thirds of the dog tongue receive NADPH-d-positive and CGRP-immunoreactive sensory fibers from the trigeminal ganglion, and that perivascular NADPH-d-positive fibers mainly originate from intrinsic ganglia in the tongue. The ultrastructural findings suggest an intrinsic peripheral nerve-reflex mechanism in the regulation of the lingual vascular function by NO-producing postganglionic parasympathetic neurons and CGRP-containing sensory fibers.

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Recent analyses of tears indicate the presence of glycosaminoglycans as their components, but their origin remains unknown. To further understand the origin of these tear components, we investigated by immunohistochemical techniques the localization of glycosaminoglycans and CD44 human lacrimal glands obtained from 20 cadavers at autopsy. Monoclonal antibodies to CD44, a receptor for hyaluronic acid, dermatan sulfate, chondroitin sulfate, and keratan sulfate were applied to the tissue. Hyaluronic acid binding region was also used for the staining of hyaluronic acid. By light microscopy, immunoreactivity for CD44 was mostly detected on the baso-lateral membrane of acinar and ductal cells, and the vascular endothelium in the interstitium. Positive staining of hyaluronic acid was associated intensely with the basal membrane of acinar and ductal cells and weakly, faintly or not at all with their lateral membrane. Positive staining of hyaluronic acid and immunoreactivity for dermatan sulfate were detected in interstitial fibrous structures; particularly, the former was intense in the perivascular fibrous structures, and the latter along the periparenchimal fibrous structures. Immunoreactivity for chondroitin sulfate and keratan sulfate was seen in some acinar cells and the acinar and ductal lumen. By electron microscopy, immunogold particles indicating chondroitin sulfate or keratan sulfate labeled sercretory granules of the acinar cells.
Considering the fact that CD44 is a receptor molecule for hyaluronic acid, the association of hyaluronic acid with the basal membrane and weakly or faintly with the lateral membrane of acinar and ductal cells may be attributed to the expression of CD44 on the baso-lateral membrane of the cells. Moreover, the presence of immunoreactivity for chondroitin sulfate and keratan sulfate in secretory granules of acinar cells and their lumens suggests that tears from the lacrimal gland contain these glycosaminoglycans.

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