Anatomical background of the sensory function in the urethra: involvement of endocrine paraneurons and afferent nerves in divergent urogenital functions. A review.

The urethra is ontogenetically derived from the cloaca together with distal parts of the large intestine, and serotonin cells are predominant among dispersed endocrine/paracrine cells in the epithelia of both tissues. Analysis of urethral endocrine cells thus helps us to understand the functions of gut endocrine cells and their communication with the nervous system, due to the fact that the urethra is a simple tubular organ, where only urine without microflora rapidly passes through. A certain number of urethral endocrine cells display unique, complicated shapes with dendritic processes, reminiscent of neurons. Characteristically, urethral endocrine cells-often called paraneurons-have direct contact with sensory nerves within the epithelium, unlike gut endocrine cells lacking in direct contact with nerves. These traits encourage us to focus on the urethral paraneurons as ideal endocrine/paracrine cells. A topographical complex of urethral paraneurons and afferent nerve fibers is sensitive to the passage of urine or the distention of the urethral lumen. The urethra-bladder excitatory reflex facilitates micturition via the release of serotonin from the paraneurons, ultimately ensuring complete voiding of the bladder. This reflex may also influence sexual behaviors such as ejaculation or the female orgasm. Urethral brush cells as well as paraneurons are responsible for continuous monitoring of the mucosal surface, especially for pathogens entering via the external urethral orifice.

Loss of CAPS2/Cadps2 leads to exocrine pancreatic cell injury and intracellular accumulation of secretory granules in mice.

The type 2 Ca2+-dependent activator protein for secretion (CAPS2/CADPS2) regulates dense-core vesicle trafficking and exocytosis and is involved in the regulated release of catecholamines, peptidergic hormones, and neuromodulators. CAPS2 is expressed in the pancreatic exocrine acinar cells that produce and secrete digestive enzymes. However, the functional role of CAPS2 in vesicular trafficking and/or exocytosis of non-regulatory proteins in the exocrine pancreas remains to be determined. Here, we analyzed the morpho-pathological indicators of the pancreatic exocrine pathway in Cadps2-deficient mouse models using histochemistry, biochemistry, and electron microscopy. We used whole exosome sequencing to identify CADPS2 variants in patients with chronic pancreatitis (CP). Caps2/Cadps2-knockout (KO) mice exhibited morphophysiological abnormalities in the exocrine pancreas, including excessive accumulation of secretory granules (zymogen granules) and their amylase content in the cytoplasm, deterioration of the fine intracellular membrane structures (disorganized rough endoplasmic reticulum, dilated Golgi cisternae, and the appearance of empty vesicles and autophagic-like vacuoles), as well as exocrine pancreatic cell injury, including acinar cell atrophy, increased fibrosis, and inflammatory cell infiltration. Pancreas-specific Cadps2 conditional KO mice exhibited pathological abnormalities in the exocrine pancreas similar to the global Cadps2 KO mice, indicating that these phenotypes were caused either directly or indirectly by CAPS2 deficiency in the pancreas. Furthermore, we identified a rare variant in the exon3 coding region of CADPS2 in a non-alcoholic patient with CP and showed that Cadps2-dex3 mice lacking CAPS2 exon3 exhibited symptoms similar to those exhibited by the Cadps2 KO and cKO mice. These results suggest that CAPS2 is critical for the proper functioning of the pancreatic exocrine pathway, and its deficiency is associated with a risk of pancreatic acinar cell pathology.

Structure and barrier functions of the perineurium and its relationship with associated sensory corpuscles: A review.

Peripheral nerves are provided with a blood-nerve barrier which prevents the invasion of harmful substances and pathogens, and also regulates metabolic and ionic homeostasis within nerve fascicles. The barrier functions are attributed to both the concentric layer of flattened cells in the perineurium and blood vessels running in the endoneurium. The perineurial cells develop continuous tight junctions as a diffusion barrier. In order to take up a predominant nutrient, glucose, the perineurium as well as endoneurial capillaries expresses GLUT1, a glucose transporter. An axon-Schwann cell complex within peripheral nerves utilizes glucose as a major energy source via the GLUT1, as does the brain. Under conditions of a reduced utilization of glucose, only the perineurial cells can transfer other nutrients, namely monocarboxylates such as ketone bodies and lactate via MCT1. Thus, MCT1 colocalizes with GLUT1 in the perineurium but not in endoneurial capillaries. To identify the cellular origins of the nerve sheath, marker proteins such as glial specific S100 protein, GLUT1, endoneurial CD34, and EMA (epithelial membrane antigen) are useful. Immunohistochemical findings for these markers are reviewed in this paper, focusing on the perineurium and endoneurium and their derivatives, Pacinian and Meissner corpuscles. Growing evidence throws light on the critical involvement of the nerve sheaths in the development, maintenance, and diseases of peripheral nerves.

Disposal of intestinal apoptotic epithelial cells and their fate via divergent routes.

Gut epithelial cells are characterized by rapid, constant cell renewal. The disposal of aging epithelial cells around the villus tips of the small intestine occurs so regularly that it has been regarded as a consequence of well-controlled cell death, designated as apoptosis. However, the notion of live cell extrusion in the intestine has been intensively built among researchers, and the disposal processes of effete epithelial cells display species and regional differences. Chemical mediators and mechanical forces rising from surrounding cells contribute to the regulated cell replacement. Cytotoxic intraepithelial lymphocytes and lamina propria macrophages play a leading role in the selection of disposal cells and their extrusion to maintain fully the epithelial homeostasis in tandem with the dynamic reconstruction of junctional devices. Lymphocyte-mediated cell killing is predominant in the mouse and rat, while the disposal of epithelial cells in the guinea pig, monkey, and human is characterized by active phagocytosis by subepithelially gathering macrophages. The fenestrated basement membrane formed by immune cells supports their involvement and explains species differences in the disposal of epithelial cells. Via these fenestrations, macrophages and dendritic cells can engulf apoptotic epithelial cells and debris and convey substantial information to regional lymph nodes. In this review, we attempt to focus on morphological aspects concerning the apoptosis and disposal process of effete epithelial cells; in vitro or ex vivo analyses using cultured monolayer has become predominant in recent studies concerning the exfoliation of apoptotic enterocytes. Furthermore, we give attention to their species differences, which is controversial but crucial to our understanding.

Osteoprotegerin-dependent M cell self-regulation balances gut infection and immunity.

Microfold cells (M cells) are responsible for antigen uptake to initiate immune responses in the gut-associated lymphoid tissue (GALT). Receptor activator of nuclear factor-κB ligand (RANKL) is essential for M cell differentiation. Follicle-associated epithelium (FAE) covers the GALT and is continuously exposed to RANKL from stromal cells underneath the FAE, yet only a subset of FAE cells undergoes differentiation into M cells. Here, we show that M cells express osteoprotegerin (OPG), a soluble inhibitor of RANKL, which suppresses the differentiation of adjacent FAE cells into M cells. Notably, OPG deficiency increases M cell number in the GALT and enhances commensal bacterium-specific immunoglobulin production, resulting in the amelioration of disease symptoms in mice with experimental colitis. By contrast, OPG-deficient mice are highly susceptible to Salmonella infection. Thus, OPG-dependent self-regulation of M cell differentiation is essential for the balance between the infectious risk and the ability to perform immunosurveillance at the mucosal surface.

Bush-like integrin filament networks associated with hyaloid vasculature in murine neonate eyes.

The vitreous of perinatal mice temporarily develops a unique vascular system, called the vasa hyaloidea propria (VHP). Observations showed the vessels possessed an extracellular matrix including the basement membrane in their entire length. Immunostaining of whole mount preparations of VHP with integrin ß1 antibody displayed a bush-like network consisting of long and straight fibers which were associated with the VHP but extended apart from the blood vessels. Electron microscopically, each fiber was composed of a bundle of thin filaments different from collagen fibrils. Macrophages associated with the VHP appeared to be arrested by the integrin bushes. The integrin bushes fragmented and disappeared by postnatal day 10, just before the regression of the VHP. Macrophages were involved in the digestion and clearance of integrin bushes. The vitreous integrin bushes appear to provide a scaffold for architectural maintenance of the hyaloid vessels and macrophages.

Cell- and stage-specific localization of galectin-3, a ß-galactoside-binding lectin, in a mouse model of experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune disease in which pathogenic T cells play an important role, and an experimental autoimmune encephalomyelitis (EAE) is used as an animal model of MS. Galectins are ß-galactoside-binding lectins and involved in various physiological and pathological events. Among fifteen members of galectins, galectin-1, -8, and -9 play immunosuppressive roles in MS and EAE; however, the role of galectin-3 (gal-3) is complex and controversial. We examined expression of gal-3 in the spinal cord and nerve roots of EAE mice. No immunohistochemical signals were detected in naïve mice, whereas gal-3 appeared at lower lumbar levels of the spinal cord and nerve roots in EAE mice. In the spinal cord, gal-3-positive cells were activated microglia and/or infiltrating macrophages, which were round in shape and intensified for the lysosomal enzyme, cathepsin D, indicating elevated phagocytic activity. Gal-3-positive cells in the spinal cord were most abundant during the peak symptomatic period. In the recovery period, they disappeared from the spinal parenchyma but remained at moderate levels in the pia mater. Interestingly, gal-3-positive cells selectively appeared in ventral, but not dorsal, nerve roots running through the spinal canal, with expression peaking during the recovery period. In ventral nerve roots, the major cell type expressing gal-3 was a specific population of Schwann cells that surround unmyelinated axons and express the biosynthetic enzyme for l-serine, a potent neurotrophic amino acid. Gal-3 was also induced in Iba1/F4/80-positive macrophages, which engulf damaged myelin and axon debris. Thus, gal-3 is induced in distinct cell types that are engaged in removal of damaged axons and cell debris and axon regeneration and remyelination, suggesting a potential neuroprotective role of gal-3 in EAE mice.

Histochemical characteristics of regressing vessels in the hyaloid vascular system of neonatal mice: Novel implication for vascular atrophy.

The hyaloid vasculature constitutes a transitory system nourishing the internal structures of the developing eye, but the mechanism of vascular regression and its cell biological characteristics are not fully understood. The present study aimed to reveal the specificity of the hyaloid vessels by a systematic immunohistochemical approach for marker substances of myeloid cells and the extracellular matrix (ECM) in neonatal mice. Macrophages immunoreactive for F4/80, cathepsin D, and LYVE-1 gathered around the vasa hyaloidea propria (VHP), while small round cells in vascular lumen of VHP were selectively immunoreactive for galectin-3; their segmented nuclei and immunoreactivities for Ly-6G, CD11b, and myeloperoxidase indicated their neutrophilic origin. VHP possessed thick ECM and a dense pericyte envelope as demonstrated by immunostaining for laminin, type IV collagen, integrin ß1, and NG2. The galectin-3+ cells loosely aggregated with numerous erythrocytes in the lumen of hyaloid vessels in a manner reminiscent of vascular congestion. Galectin-3 is known to polymerize and form a complex with ECM and NG2 as well as recruit leukocytes on the endothelium. Observation of galectin-3 KO mice implicated the involvement of galectin-3 in the regression of hyaloid vasculature. Since macrophages may play central roles including blocking of the blood flow and the induction of apoptosis in the regression, galectin-3+ neutrophils may play a supportive role in the macrophage-mediated involution of the hyaloid vascular system.

Intrarenal signaling mediated by CCK plays a role in salt intake-induced natriuresis.

The natriuretic hormone CCK exhibits its gene transcripts in total kidney extracts. To test the possibility of CCK acting as an intrarenal mediator of sodium excretion, we examined mouse kidneys by 1) an in situ hybridization technique for CCK mRNA in animals fed a normal- or a high-sodium diet; 2) immuno-electron microscopy for the CCK peptide, 3) an in situ hybridization method and immunohistochemistry for the CCK-specific receptor CCKAR; 4) confocal image analysis of receptor-mediated Ca2+ responses in isolated renal tubules; and 5) metabolic cage experiments for the measurement of urinary sodium excretion in high-salt-fed mice either treated or untreated with the CCKAR antagonist lorglumide. Results showed the CCK gene to be expressed intensely in the inner medulla and moderately in the inner stripe of the outer medulla, with the expression in the latter being enhanced by high sodium intake. Immunoreactivity for the CCK peptide was localized to the rough endoplasmic reticulum of the medullary interstitial cells in corresponding renal regions, confirming it to be a secretory protein. Gene transcripts, protein products, and the functional activity for CCKAR were consistently localized to the late proximal tubule segments (S2 and S3) in the medullary rays, and the outer stripe of the outer medulla. Lorglumide significantly diminished natriuretic responses of mice to a dietary sodium load without altering the glomerular filtration rate. These findings suggest that the medullary interstitial cells respond to body fluid expansion by CCK release for feedback regulation of the late proximal tubular reabsorption.

IRBIT controls apoptosis by interacting with the Bcl-2 homolog, Bcl2l10, and by promoting ER-mitochondria contact.

IRBIT is a molecule that interacts with the inositol 1,4,5-trisphosphate (IP3)-binding pocket of the IP3 receptor (IP3R), whereas the antiapoptotic protein, Bcl2l10, binds to another part of the IP3-binding domain. Here we show that Bcl2l10 and IRBIT interact and exert an additive inhibition of IP3R in the physiological state. Moreover, we found that these proteins associate in a complex in mitochondria-associated membranes (MAMs) and that their interplay is involved in apoptosis regulation. MAMs are a hotspot for Ca2+ transfer between endoplasmic reticulum (ER) and mitochondria, and massive Ca2+ release through IP3R in mitochondria induces cell death. We found that upon apoptotic stress, IRBIT is dephosphorylated, becoming an inhibitor of Bcl2l10. Moreover, IRBIT promotes ER mitochondria contact. Our results suggest that by inhibiting Bcl2l10 activity and promoting contact between ER and mitochondria, IRBIT facilitates massive Ca2+ transfer to mitochondria and promotes apoptosis. This work then describes IRBIT as a new regulator of cell death.

Differential expression of endothelial nutrient transporters (MCT1 and GLUT1) in the developing eyes of mice.

The blood-brain barrier in the neonatal brain expresses the monocarboxylate transporter (MCT)-1 rather than the glucose transporter (GLUT)-1, due to the special energy supply during the suckling period. The hyaloid vascular system, consisting of the vasa hyaloidea propria and tunica vasculosa lentis, is a temporary vasculature present only during the early development of mammalian eyes and later regresses. Although the ocular vasculature manifests such a unique developmental process, no information is available concerning the expression of endothelial nutrient transporters in the developing eye. The present immunohistochemical study using whole mount preparations of murine eyes found that the hyaloid vascular system predominantly expressed GLUT1 in the endothelium, in contrast to the brain endothelium. Characteristically, the endothelium in peripheral regions of the neonatal hyaloid vessels displayed a mosaic pattern of MCT1-immunoreactive cells scattered within the GLUT1-expressing endothelium. The proper retinal vessels first developed by sprouting angiogenesis endowed with filopodia, which were absolutely free from the immunoreactivities of GLUT1 and MCT1. The remodeling retinal capillary networks and veins in the surface layer of the retina mainly expressed MCT1 until the weaning period. Immunostaining of MCT1 in the retina revealed fine radicular processes projecting from the endothelium, differing from the MCT1-immunonegative filopodia. These findings suggest that the expression of nutrient transporters in the ocular blood vessels is differentially regulated at a cellular level and that the neonatal eyes provide an interesting model for research on nutrient transporters in the endothelium.

RANKL regulates differentiation of microfold cells in mouse nasopharynx-associated lymphoid tissue (NALT).

Murine nasopharynx-associated lymphoid tissue (NALT), located at the base of the nasal cavity, serves as a major site for the induction of mucosal immune responses against airway antigens. The follicle-associated epithelium (FAE) covering the luminal surface of NALT is characterized by the presence of microfold cells (M cells), which take up and transport luminal antigens to lymphocytes. Glycoprotein 2 (GP2) has recently been identified as a reliable marker for M cells in Peyer's patches of the intestine. However, the expression of GP2 and other functional molecules in the M cells of NALT has not yet been examined. We have immunohistochemically detected GP2-expressing cells in the FAE of NALT and the simultaneous expression of other intestinal M-cell markers, namely Tnfaip2, CCL9, and Spi-B. These cells have been further identified as M cells because of their higher uptake capacity of luminal microbeads. Electron microscopic observations have shown that GP2-expressing cells on the FAE display morphological features typical of M cells: they possess short microvilli and microfolds on the luminal surface and are closely associated with intraepithelial lymphocytes. We have also found that the receptor activator of nuclear factor kappa-B ligand (RANKL) is expressed by stromal cells underneath the FAE, which provides its receptor RANK. The administration of RANKL markedly increases the number of GP2(+)Tnfaip2(+) cells on the NALT FAE and that of intestinal M cells. These results suggest that GP2(+)Tnfaip2(+) cells in NALT are equivalent to intestinal M cells, and that RANKL-RANK signaling induces their differentiation.

Three-dimensional microanatomy of the pericapillary mesangial tissues in the renal glomerulus: Comparative observations in four vertebrate classes.

The renal glomeruli in lower vertebrates display mesangium-like cells and matrices interposed between the capillary endothelium and the basement membrane, while those in mammals reportedly lack such interpositions except in pathological conditions. By combined scanning and transmission electron microscopic observations, the pericapillary mesangial tissues were comparatively analyzed in four vertebrate classes: mammals (rats and rabbits), reptiles (green iguanas), amphibians (bullfrogs), and teleosts (carps). The observations discriminated three types of pericapillary interposition. The first, acellular interpositions, occurred universally, with mammalians displaying rudimental ones. This tissue type corresponded with extracellular matrices held in subendothelial grooves which were supported by fine endothelial projections anchored to the basement membrane. In lower vertebrates these grooves constituted an anastomosed system of subendothelial channels that communicated with the mesangial region, to favor cleaning of the glomerular filter. The second, compound type was specific to reptiles and amphibians, affecting the entire capillary circumference in the latter. In this tissue type, fine mesangial processes--which accompanied considerable amounts of fibrillar matrices--were loosely associated with the endothelial bases, indicating their possible nature as a kind of myofibroblast. Occurrence of the third, cellular interpositions was confined to small incidental loci in mammalian and teleost glomeruli. This tissue type was mostly occupied by thick processes or main bodies of the mesangial cells that tightly interlocked their short marginal microvilli with corresponding indentations on the endothelial bases.

GP2-expressing cells in the conjunctiva and tear ducts of mice: identification of a novel type of cells in the squamous stratified epithelium.

GP2 is a membrane-associated secretory protein originally identified in zymogen granules of pancreatic acinar cells. Recently, this glycoprotein has attracted attention as a marker substance of M cells of Peyer's patches and for its involvement in the selective uptake of pathological bacteria via M cells. When we stained the conjunctiva and tear ducts of mice using a GP2 antibody, all goblet cells in the squamous stratified epithelium of the conjunctiva were intensely immunolabeled, while goblet cells in the intestine and airway were devoid of the immunoreactivity, indicating that the conjunctiva contains a special type of goblet cell. Further immunostaining for GP-2 labeled dispersed cells of peculiar shapes within the stratified squamous epithelium in the lacrimal canaliculi, lacrimal sac, and nasolacrimal duct. The GP2-immunoreactive cells in the tear duct projected arched or branched processes toward the basement membrane. Electron-microscopically, immunogold particles for GP2 outlined the basolateral plasma membrane of both the conjuntival goblet cells and the peculiarly shaped cells in the tear duct. Intracellularly, GP2 products of the goblet cells were localized around secretory granules in the apical cytoplasm and those of the tear duct cells inside the vesicles. The luminal contents close to apical plasma membrane were heavily labeled with immunogold particles, suggesting an exocytosis-based targeting of GP2 to the plasma membrane and its release into the lumen. The possible function of GP2 in tear ducts is discussed in relation to a defense system against invasive microoranisms and antigens.

ERp44 Exerts Redox-Dependent Control of Blood Pressure at the ER.

Blood pressure maintenance is vital for systemic homeostasis, and angiotensin II is a critical regulator. The upstream mechanisms that regulate angiotensin II are not completely understood. Here, we show that angiotensin II is regulated by ERp44, a factor involved in disulfide bond formation in the ER. In mice, genetic loss of ERp44 destabilizes angiotensin II and causes hypotension. We show that ERp44 forms a mixed disulfide bond with ERAP1, an aminopeptidase that cleaves angiotensin II. ERp44 controls the release of ERAP1 in a redox-dependent manner to control blood pressure. Additionally, we found that systemic inflammation triggers ERAP1 retention in the ER to inhibit hypotension. These findings suggest that the ER redox state calibrates serum angiotensin II levels via regulation of the ERp44-ERAP1 complex. Our results reveal a link between ER function and normotension and implicate the ER redox state as a potential risk factor in the development of cardiovascular disease.

Cellular and subcellular localization of cholecystokinin (CCK)-1 receptors in the pancreas, gallbladder, and stomach of mice.

Information concerning the cellular localization of cholecystokinin (CCK)-1 receptors has been discrepant and remained scanty at ultrastructural levels. The present immunohistochemical study at light and electron microscopic levels revealed the distinct localization of CCK1 receptors in visceral organs. Immunohistochemistry by use of a purified antibody against mouse CCK1 receptor was applied to fixed tissue sections of the pancreas, gallbladder, stomach, and intestine of mice. A silver-intensified immunogold method revealed the subcellular localization under electron microscope. The immunoreactivity for CCK1 receptors was selectively found in the basolateral membrane of pancreatic acinar cells and gastric chief cells but was absent in pancreatic islets and gastric D cells. Another intense expression in the gut was seen in the myenteric nerve plexus of the antro-duodenal region and some populations of c-Kit-expressing pacemaker cells in the duodenal musculature. The gallbladder contained smooth muscle fibers with an intense immunoreactivity of CCK1 receptors on cell surfaces. The restricted localization of CCK1 receptors on the basolateral membrane of pancreatic acinar cells and gastric chief cells, along with their absence in the islets of Langerhans and gastric D cells, provides definitive information concerning the regulatory mechanism by circulating CCK. Especially, the subcellular localization in the acinar cells completes the investigation for the detection of circulating CCK by the basolateral membrane.

Three types of macrophagic cells in the mesentery of mice with special reference to LYVE-1-immunoreactive cells.

Immunohistochemistry using whole mount preparations of the murine mesentery revealed two types of LYVE-1-immunoreactive cells with dendritic morphology other than F4/80(+) typical macrophages.The two types of LYVE-1(+) cells were regularly distributed with constant intervals throughout the mesentery and appeared to possess their own territory. Both types of LYVE-1(+) cells were weakly or moderately immunopositive for F4/80 antibody, a marker of macrophages,while F4/80(+) round macrophages were absolutely free from the LYVE-1 immunoreactivity. Only macrophages could ingest latex particles of 20 nm in diameter 3 h after a peritoneal injection.Peritoneal administration of lipopolysaccharide (LPS) induced a rapid reduction of LYVE-1 immunoreactivity in the cells with dendritic morphology followed by an increased immunoreactivity to F4/80 antibody, and simultaneously by dynamic changes in their shape. Under normal conditions,F4/80(+) macrophages in various connective tissues expressed LYVE-1, in contrast to lack of LYVE-1 in F4/80(+) macrophages within the parenchyma of visceral organs and macrophages residing in hepatic sinusoids and pulmonary alveoli. LYVE-1 may play a role in cell adhesion and migration of macrophagic cells within connective tissues rich in hyaluronan, and loss of LYVE-1 becomes a reliable sign of activated conditions in inflammation.

The three-dimensional microanatomy of the pancreatic duct system in the Japanese monkey, Macaca fuscata, with special reference to fine proximal passages of a high species-specificity.

We have previously shown the duct system in the rat pancreas to consist of two parts: a fine proximal (intercalated) duct and thicker distal (intralobular and interlobular) duct, with the latter part displaying morphological signs indicative of a bicarbonate-rich fluid secretion. In this study the pancreatic duct system in the Japanese monkey Macaca fuscata was observed by scanning electron microscopy after the hydrolytic exposure of cell surfaces as well as by transmission electron microscopy of ultrathin sections. Cellular expression of the water channel aquaporin 1 (AQP1) was also examined immunohistochemically. In contrast to the segmented duct system in the rat, all the duct cells in the monkey pancreas consistently displayed rich mitochondria in the cytoplasm, elaborate interdigitations of cell processes, and an intense immunoreactivity for AQP1 on the apical and basolateral cell membrane to favor active ion transport and osmotic water movement across the epithelium. Both the existence of secretory canaliculi and basal trabeculae in the duct epithelium and randomized localization of primary cilia on the luminal cell surfaces were demonstrated for the first time in monkeys, and the physiological implications of these phenomena are discussed.

Regulation of hair shedding by the type 3 IP3 receptor.

Here we showed that the type 3 IP(3) receptor (IP(3)R3) is specifically expressed in hair follicles of the skin and plays an important role in the regulation of the hair cycle. We found that IP(3)R3-deficient (Itpr3(-/-)) mice had prominent alopecia, which was characterized by repeated hair loss and regrowth. The alopecic stripe runs along the body axis like a wave, suggesting disturbed hair-cycle regulation. Indeed, the hair follicles of the alopecic region were in the early anagen stage. Although the hair growth and proliferation activity of the hair matrix cells in the anagen phase were normal in Itpr3(-/-) mice, telogen club hairs in the telogen-anagen transition phase were loosely attached to the hair follicles and were easily removed in contrast to the more tightly attached club hairs of Itpr3(+/+) mice. Itpr3(-/-) keratinocytes surrounding the telogen club hairs have sparse cytokeratin filaments extending in random directions, as well as less developed desmosomes. Furthermore, nuclear factor of activated T cells c1 (NFATc1) failed to translocate into the nucleus of keratin 6-positive bulge cells in Itpr3(-/-) telogen follicles. We propose that hair shedding is actively controlled by the IP(3)R3/NFAT-dependent signaling pathway, possibly through the regulation of cytokeratin filaments in keratinocytes.

Accumulated caveolae constitute subcellular compartments for glial calcium signaling in lanceolate sensory endings innervating rat vibrissae.

The terminal Schwann cells that accompany lanceolate sensory endings in the rat vibrissal follicle are known to display the small plasma membrane invaginations termed caveolae, which concentrate Ca(2+) signaling molecules. We have previously shown that these cells generate Ca(2+) signals at the lamellar processes covering the receptor axons through activation of the metabotropic purinoceptor P2Y(2). To investigate the roles of caveolae in the spatiotemporal organization of Ca(2+) signals, terminal Schwann cells were observed by immunohistochemistry for the caveola protein caveolin-1, and by transmission and scanning electron microscopy. In addition, immunohistochemical detection of P2Y(2) and its coupling partner G(q/11) along with confocal image analysis of the purinergically induced glial Ca(2+) responses was performed in isolated tissue preparations either treated or untreated with the caveolae eliminator methyl-ß-cyclodextrin. Results showed the Schwann lamellae to be characterized by the presence of dense caveolae accompanying a fine tubular network of the endoplasmic reticulum Ca(2+) store and by intense expression of the signaling molecules P2Y(2) and G(q/11). Loss of caveolae diffusely redistributed these molecules throughout the entire cell and impaired the lamellar Ca(2+) signals, both in chronological priority (preceding the global cell response) and in spatial integrity (involving the entire length of the processes). To our knowledge, this is the first report of a subcellular accumulation of caveolae underlying compartmentalized glial Ca(2+) signals that can couple with local effects on the accompanying axon terminals.