Yoshiaki Doi

Weibel‐Palade bodies as a storage site of calcitonin gene‐related peptide and endothelin‐1 in blood vessels of the rat carotid body

The vasculature of the carotid body has been considered to play a role in the regulation of blood flow into this organ. This light and electron microscope immunocytochemistry deals with endothelium‐dependent vasomotion by vasodilatory calcitonin gene‐related peptide (CGRP) and vasoconstrictive endothelin‐1 (ET‐1).

Immunohistochemical and ultrastructural observations of desmoplastic trichoepithelioma with a special reference to a morphological comparison with normal apocrine acrosyringeum.

Background: Desmoplastic trichoepithelioma is a benign neoplasm considered to have follicular differentiation. Its sweat gland‐ or sebaceous‐lines of differentiation have been also reported. There have been, however, only a few reports regarding extensive immunohistochemical and ultrastructural investigations of this neoplasm.

Synthesis and receptor sites of endothelin‐1 in the rat liver vasculature

Immunocytochemical localization of big endothelin‐1 (big ET‐1), ET‐1, and ET receptor A and B (ETA and ETB), and gene expression of prepro ET‐1 mRNA were examined on the rat liver vasculature. Immunoreactivities for big ET‐1 and ET‐1 were preferentially seen along the endothelium of interlobular veins (IV) and artery (IA), although the staining intensity was more pronounced in IV. Expression of preproET‐1 mRNA was detected in both vascular endothelia and the signal intensity was more prevalent in IV. Immunoelectron microscopy showed that rough endoplasmic cisterns were immunoreactive for big ET‐1, while Weibel‐Palade (WP) bodies, a storage site for ET‐1, were immunoreactive for ET‐1 in endothelial cells of IV. These results indicate that endothelial cells of IV are the major site of synthesis of ET‐1, which is extracellularly secreted by degranulation and/or exocytosis of WP bodies. Hepatic stellate cells (HSCs), especially of the plasma membrane of perisinusoidal and interhepatocellular processes, were immunoreactive for both ETA and ETB receptor antibodies. These findings suggest that ET‐1 receptor‐mediated HSC contraction is involved in the regulation of hepatic sinusoidal blood flow as previously cited in mammalian liver cirrhosis. We also showed that sarcolemma and caveoles in the smooth muscle cells of the media of IV, and its branches before reaching the hepatic sinusoids, were immunoreactive for ETA receptor antibody. The results suggest that such vessels, which contains a large amount of hepatic blood inflow, participate in pump mechanism toward hepatic sinusoidal circulation in a receptor‐mediated paracrine fashion. Anat Rec 259:437–445, 2000.

Expression of α-calcitonin gene-related peptide in the enteric nervous system of rat small intestine

Abstract We first detected α-calcitonin gene-related peptide (α-CGRP) precursor mRNA in the enteric nervous system (ENS) of rat small intestine by reverse transcriptase polymerase chain reaction (RT-PCR). The nucleotide sequence of the RT-PCR product was completely identical to that found in other organs. By in situ hybridization using digoxygenin-labeled α-CGRP precursor cRNA probe, we found that antisense probes detected a signal on nerve cell bodies of both submucosal and myenteric plexuses. Our findings indicate that the rat ENS participates in synthesis of α-CGRP precursor.

Effects of endothelin-1 on vasoactivity and its synthesis, storage, and acting sites in the rat superior mesenteric vasculature: an ultrastructural and immunocytochemical study.

Vasoactivity after treatment with endothelin (ET)-1 and immunoreactivity for ET-1 and its receptors were investigated in the rat superior mesenteric vasculature (SMV). By measurements of corrosion cast images of the SMVs, it was seen that ET-1 induces remarkable vasocontraction of the distal arterial branches, consisting of small arteries and arterioles, and localized vasoconstriction throughout the venous branches which possess localized medial thickenings. Immunoreactivity for ET-1 was preferentially seen along the endothelia of the proximal arterial branches. Cisterns of the rough endoplasmic reticulum and Weibel–Palade (WP) bodies of the endothelial cells were immunoreacted. Immunoreactivity for the ETa receptor was preferentially seen on the media of the distal arterial branches. These findings indicate that endothelial cells of the proximal arterial branches synthesize ET-1 and store it in the WP bodies. Because WP bodies are involved in the release of ET-1, this suggests that this endogenous ET-1, which is released from the proximal arterial branches, may be involved in the regulation of blood flow through the distal arterial branches by mediation of the ETa receptor. In addition, it seems likely that ET-1-induced vasoconstriction of the venous branches may act to impel the portal blood flow.

Immunoreactivity of endothelins and endothelin receptor in the stria vascularis of the mouse cochlea

Immunoreactivities of endothelin-1, endothelin-3, endothelin receptor type A, and Na,K-ATPase were investigated in the stria vascularis of adult male WBB6F1 +/+ mice and in that of W/Wv mutants lacking strial intermediate cells. In the +/+ mice, electron microscopic immunoreactivity for the endothelins was seen on the rough endoplasmic reticulum, Golgi apparatus, cytoplasmic vesicles and lysosomes exclusively in the strial intermediate cells by the postembedment method. Immunoreactive endothelin receptor A was localized along the plasma membrane of strial marginal cells of both wild and mutant types although the immunoreactivity of the latter was much less than that of the former by the preembedment method. These findings suggest that the endothelins, which are produced in the strial intermediate cells, may play a role in the maintenance of the stria vascularis function in the +/+ mice. Since the plasma membrane of the marginal cells of the W/Wv mice, which do not generate a high positive endocochlear potential, also showed immunoreactivity for Na,K-ATPase, it seems likely that the endothelins are involved in the activation of sodium pump of the strial marginal cells by mediation of endothelin receptor A. In addition, the role of lysosomes in the crinophagy of the endothelins in the strial intermediate cells is proposed in the +/+ mice.

Expressions of endothelin-1, fibronectin, and interleukin-1α of human umbilical vein endothelial cells under prolonged culture

We examined human umbilical vein endothelial cells (HUVECs) under prolonged culture by electron microscopy and by light and electron immunocytochemistry including double immunolabeling. Based on the cell area of HUVECs through multiple passages, we divided the cells into first, second, and third stages, which exhibited distinct morphological and immunocytochemical characteristics. During the first stage, HUVECs were polygonal in shape and had already formed the monolayer confluence. During the second stage, they were characterized by an increased number of Weibel–Palade (WP) bodies, which were actively segregated from Golgi cisterns. Endothelin (ET)-1 and von Willebrand factor, an endothelial cell marker, were occasionally colocalized in WP bodies. The increase in WP bodies correlated with high ET-1 concentration in the cultured medium, suggesting that these inclusions are involved in storage and release of ET-1 in a manner indicating a regulatory pathway. During the third stage, fibronectin and interleukin (IL)-1α were expressed in HUVECs as well as in multinucleated giant cells, which originated from HUVECs, but WP bodies decreased in number in association with a decrease in ET-1 immunoreactivity and concentration. The foregoing changes in immunoreactivities for ET-1, fibronectin, and IL-1α affecting HUVECs under prolonged culture may reflect a senescent process of these cells.

The role of endothelin-1 in regulation of rat mesenteric microcirculation.

Immunocytochemistry of endothelin-1 (ET-1) and the ETA receptor, and scanning electron microscopy using cast resin after treatment with ET-1, were carried out in the rat mesenteric microvasculature. Immunoreaction of ET-1 was preferentially seen along the endothelium of the proximal portion of the anterior mesenteric artery, the endothelial cells of which contain abundant Weibel-Palade (WP) bodies. However, the arterioles and small veins distal to the artery showed little immunoreactivity but showed heavy immunoreaction of ETA receptors in the media. By scanning electron microscopy after treatment with ET-1, the mesenteric microvasculature became slightly narrower compared to the control exhibited of localized constricted areas, especially in the region of the small veins. Light microscopy of such areas revealed localized thickening of the medial muscle cells. Because the release of ET-1 from endothelial cells depends in part on extracellular discharge of the WP bodies, the results indicate that ET-1, discharged from the proximal portion of the anterior mesenteric artery, induces vasoconstriction of the arterioles and small veins, mediated by ETA receptors. The localized thickening in areas in the media of the small veins may participate in the maintenance of blood flow through the portal circulation.

Immunocytochemistry of Fibronectin and Endothelin-1 in the Cavernous Body of Postnatal Rabbit Penises

The differentiating cavernous body (CB) of postnatal rabbit penises was examined with a special reference to immunolocalizations for fibronectin (FN) and endothelin-1 (ET-1). At postnatal day 1, the CBs were embedded by an abundance of mesenchymal cells (MCs), and some of them were closely associated with endothelial cells of preexisting capillaries. Our electron micrographs indicated that such MCs are successively incorporated into the capillary endothelium as vasoformative cells. At this period, vascular sprouts of the helicine artery (HA), which were associated with the MCs, arose from the deep penile artery, and the transformation of such cells to endothelial and medial muscle ones was also indicated, and some MCs appeared to differentiate to epithelioid cells in the media. Immunoreactions for FN were preferentially localized in the rough endoplasmic reticulum (rER) and along the plasma membrane of such vasoformative MCs, and on the extracellular matrix components which connect these MCs with sprouts of both growing capillaries and HA. These findings suggest that FN, which is produced in the rER of the MCs, plays a crucial role in the mechanical linkage during the incorporation of vasoformative MCs into these penile vessels. Immunoreactions for ET-1 were preferentially localized on Weibel-Palade bodies in endothelial cells of the HA, implying the involvement of this peptide in the regulation of the local blood flow in this vessel.