Serial Section Array Scanning Electron Microscopy Analysis of Cells from Lung Autopsy Specimens following Fatal A/H1N1 2009 Pandemic Influenza Virus Infection.

A/H1N1 2009 pandemic influenza virus (A/H1N1/pdm09) was first identified as a novel pandemic influenza A virus (IAV) in 2009. Previously, we reported that many viral antigens were detected in type II alveolar epithelial cells (AEC-IIs) within autopsied lung tissue from a patient with A/H1N1/pdm09 pneumonia. It is important to identify the association between the virus and host cells to elucidate the pathogenesis of IAV pneumonia. To investigate the distribution of virus particles and morphological changes in host cells, the autopsied lung specimens from this patient were examined using transmission electron microscopy (TEM) and a novel scanning electron microscopy (SEM) method. We focused on AEC-IIs as viral antigen-positive cells and on monocytes/macrophages (Ms/Mϕs) and neutrophils (Neus) as innate immune cells. We identified virus particles and intranuclear dense tubules, which are associated with matrix 1 (M1) proteins from IAV. Large-scale two-dimensional observation was enabled by digitally "stitching" together contiguous SEM images. A single whole-cell analysis using a serial section array (SSA)-SEM identified virus particles in vesicles within the cytoplasm and/or around the surfaces of AEC-IIs, Ms/Mϕs, and Neus; however, intranuclear dense tubules were found only in AEC-IIs. Computer-assisted processing of SSA-SEM images from each cell type enabled three-dimensional (3D) modeling of the distribution of virus particles within an ACE-II, a M/Mϕ, and a Neu.IMPORTANCE Generally, it is difficult to observe IAV particles in postmortem samples from patients with seasonal influenza. In fact, only a few viral antigens are detected in bronchial epithelial cells from autopsied lung sections. Previously, we detected many viral antigens in AEC-IIs from the lung. This was because the majority of A/H1N1/pdm09 in the lung tissue harbored an aspartic acid-to-glycine substitution at position 222 (D222G) of the hemagglutinin protein. A/H1N1/pdm09 harboring the D222G substitution has a receptor-binding preference for α-2,3-linked sialic acids expressed on human AECs and infects them in the same way as H5N1 and H7N9 avian IAVs. Here, we report the first successful observation of virus particles, not only in AEC-IIs, but also in Ms/Mϕs and Neus, using electron microscopy. The finding of a M/Mϕ harboring numerous virus particles within vesicles and at the cell surface suggests that Ms/Mϕs are involved in the pathogenesis of IAV primary pneumonia.

Anomalous inferior mesenteric artery supplying the ascending, transverse, descending, and sigmoid colons.

We have encountered in our anatomical practice the first case and an extremely rare second case in which the ascending, transverse, descending, and sigmoid colons were supplied by the inferior mesenteric artery. The causes of colic artery anomalies are generally explained in conjunction with the development of the superior mesenteric artery, which is intimately related to embryonic elongation and midgut rotation. However, this embryological model was inapplicable to both cases. This difficulty motivated us to seek possible relationships with reported anomalous inferior mesenteric arteries in adults as well as their embryological causes. We consider that the aberrant right colic artery found in 2009 is an "intermesenteric artery" which anastomoses the superior (or its middle colic branch) and inferior mesenteric artery, but secondarily lost its origin from the superior mesenteric artery. The aberrant colic artery found in 2010 is a "middle-inferior mesenteric artery" in which the inferior mesenteric artery formed a common trunk with remnant middle mesenteric artery.

Inhibitory role of Munc13-1 in antigen-induced mast cell degranulation.

Secretory granules (SGs) of mast cells are lysosome-related organelles that contain various inflammatory molecules such as histamine, which are stored in the cytoplasm. Mast cell degranulation is the regulated exocytosis of SGs in response to external stimuli, such as the antigen-mediated cross-linking of the high-affinity IgE receptor, FcεRI. Upon stimulation, SGs undergo priming to become fusion-competent prior to fusing with the plasma membrane, which is mediated by Munc13-4, one of the five members of the vesicle-priming Munc13 protein family. Although Munc13-4 is shown to be crucial for mast cell degranulation, the functional involvement of other Munc13 isoform(s) remains unknown. Herein, this was investigated using the RBL-2H3 mast cell line. We found that Munc13-1 and Munc13-4 are the only Munc13 isoforms that are expressed in the RBL-2H3 cells, and Munc13-1 is distributed in the cytoplasm, but highly concentrated on the late endosome and/or lysosome. Unexpectedly, antigen-induced degranulation was considerably increased by Munc13-1 knockdown, but decreased by its overexpression. Further, we found that the hypersecretion phenotype of the Munc13-1-knockdown cells was attenuated by simultaneous Munc13-4 knockdown. These results suggested that Munc13-1 has an inhibitory role in antigen-induced mast cell degranulation, which is performed in a Munc13-4-dependent manner.

Role of pituitary adenylyl cyclase-activating polypeptide in intracellular calcium dynamics of neurons and satellite cells in rat superior cervical ganglia.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a bioactive peptide with diverse effects in the nervous system. The present study investigated whether stimulation of PACAP receptors (PACAPRs) induces responses in neurons and satellite cells of the superior cervical ganglia (SCG), with special reference to intracellular Ca2+ ([Ca2+]i) changes. The expression of PACAPRs in SCG was detected by reverse transcription-PCR. PACAP type 1 receptor (PAC1R), vasoactive intestinal peptide receptor type (VPAC)1R, and VPAC2R transcripts were expressed in SCG, with PAC1R showing the highest levels. Confocal microscopy analysis revealed that PACAP38 and PACAP27 induced an increase in [Ca2+]i in SCG, first in satellite cells and subsequently in neurons. Neither extracellular Ca2+ removal nor Ca2+ channel blockade affected the PACAP38-induced increase in [Ca2+]i in satellite cells; however, this was partly inhibited in neurons. U73122 or xestospongin C treatment completely and partly abrogated [Ca2+]i changes in satellite cells and in neurons, respectively, whereas VPAC1R and VPAC2R agonists increased [Ca2+]i in satellite cells only. This is the first report demonstrating the expression of PACAPRs specifically, VPAC1 and VPAC2 in SCG and providing evidence for PACAP38-induced [Ca2+]i changes in both satellite cells and neurons via Ca2+ mobilization.

Different effect of serotonin on intracellular calcium ion dynamics in the smooth muscle cells between rat posterior ciliary artery and vorticose vein.

5-hydroxytriptamine (5-HT: serotonin) is an important transmitter that causes vessel constriction, although few studies have examined the effect of 5-HT on venous smooth muscles. The intracellular Ca(2+) concentration ([Ca(2+)]i) plays an essential role in stimulus-response coupling in numerous tissue/cells including vascular smooth muscle cells. The present study was performed to examine whether differences between arteries and veins in the response to 5-HT can be detected under confocal microscope with respect to [Ca(2+)]i dynamics. In posterior ciliary arteries of rats, 5-HT induced a [Ca(2+)]i increase. The 5-HT-induced responses were caused by both Ca(2+) influx and mobilization. Agonist and antagonist experiments revealed that arterial smooth muscles possess 5-HT1a, 1b, 2 (Gprotein-coupled type) and 5-HT3 (ion channel type) receptors, and that 5-HT2 in particular plays a major role in these responses. For vorticose veins, the 5-HT-induced responses were also caused by both Ca(2+) influx and mobilization. However, the cAMP dependent pathway (5-HT4-7) was found to be significant in vasocontraction with respect to 5-HT in these vessels. Thus, Ca(2+) mobilization was induced by 5-HT2 and 5-HT4-7 in a vessel-dependent manner, whereas Ca(2+) influx universally was induced by 5-HT3. These results indicate that the posterior ciliary arteries and vorticose veins in the same tissue might differ greatly in their responses to stimulus.

Mast cell degranulation is negatively regulated by the Munc13-4-binding small-guanosine triphosphatase Rab37.

Mast cell degranulation is regulated by the small guanosine triphosphatases (GTPases) Rab27a and Rab27b, which have distinct and opposing roles: Rab27b acts as a positive regulator through its effector protein Munc13-4, a non-neuronal isoform of the vesicle-priming Munc13 family of proteins, whereas Rab27a acts as a negative regulator through its effector protein melanophilin, by maintaining integrity of cortical filamentous actin (F-actin), a barrier to degranulation. Here we investigated the role of Rab37, one of the Rab GTPases assumed to be implicated in regulated secretion during mast cell degranulation. Using the RBL-2H3 mast cell line, we detected Rab37 on the secretory granules and found that antigen-induced degranulation was extensively increased by either knockdown of Rab37 or overexpression of a dominant-active Rab37 mutant. This hypersecretion phenotype in the Rab37-knockdown cells was suppressed by simultaneous knockdown of Rab27a and Rab27b or of Munc13-4, but not by disruption of cortical F-actin. We further found that Rab37 interacted with Munc13-4 in a GTP-independent manner and formed a Rab27-Munc13-4-Rab37 complex. These results suggest that Rab37 is a Munc13-4-binding protein that inhibits mast cell degranulation through its effector protein, by counteracting the vesicle-priming activity of the Rab27-Munc13-4 system.

P2Y purinoceptors mediate ATP-induced changes in intracellular calcium and amylase release in acinar cells of mouse parotid glands.

Adenosine 5'-triphosphate (ATP) can act as an extracellular signal that regulates various cellular functions. The present study aimed to determine which purinoceptors play a role in ATP-induced changes in intracellular Ca(2+) ([Ca(2+)]i) and amylase secretion in mouse parotid glands. ATP induced a steep increase in [Ca(2+)]i in acinar cells. The removal of extracellular Ca(2+) or the use of Ca(2+) channel blockers slightly inhibited this increase. Inhibition of PLCγ by U73122 and of IP3 by xestospongin C did not completely block this increase. The purinoceptor antagonists suramin and reactive blue-2 strongly inhibited the ATP-induced changes in [Ca(2+)]i. 2-MeSATP induced a strong increase in [Ca(2+)]i, while Bz-ATP induced a small [Ca(2+)]i increase, and UTP and α,ß-MeATP had no effect. The potency order of ATP analogs (2-MeSATP > ATP >> UTP) suggested that P2Y1 and P2Y12 play a significant role in the cellular response to ATP. RT-PCR revealed that P2X2,4,7 and P2Y1,2,10,12,14 were expressed in acinar cells. Ca(2+)-dependent exocytotic secretion of amylase was detected in parotid glands. These findings indicated that ATP activates P2Y receptors more than P2X receptors at low concentrations. Thus, P2Y receptors were found to be the main receptors involved in Ca(2+)-related cell homeostasis and amylase secretion in mouse parotid glands.

Results of Questionnaire Survey on Gross Anatomy Education (March 2014).

To understand the current situation of gross anatomy education anatomy classes. Regarding the influence of increased enrollment and to promote sharing of information on its improvement, we capacity in medical schools, many respondents were worried about conducted a questionnaire survey on gross anatomy education the impact on research activities due to the increase in teaching in September 2013. In most medical and dental schools, gross workload without expanding in teaching staff. In some schools, anatomy courses were offered to second-year students. The owing to the limitations of the facilities or the number of donated average numbers of gross anatomy practices were 34.6 in medical bodies, the number of students per cadaver had to be increased. schools and 27.4 in dental schools. The average total hours of We received various effective and practical measures for the practice in the curriculum was 125 in medical schools, and 97 improvement of gross anatomy education, such as improvement in dental schools. However, in about 80% of total schools, the of teaching materials and dissection methods, introduction of length of the actual gross anatomy practice was considerably lectures on clinical anatomy by clinicians, and implementation longer, because the students could not finish the work within of the second-round gross anatomy practice in the upper grades. the allotted class time. As to the effect of curriculum reform in Many respondents emphasized both the need for a training system respond to the introduction of the accreditation of medical and for young teaching staff, and the importance of opportunities for dental education programs, many respondents answered that sharing information on education. they had a minimal effect except earlier commencement of gross.

α1-Adrenoceptors relate Ca(2+) modulation and protein secretions in rat lacrimal gland.

Noradrenaline (NA) is a catecholamine with multiple roles including as a hormone and a neurotransmitter. Cellular secretory activities are enhanced by adrenergic stimuli as well as by cholinergic stimuli. The present study aimed to determine which adrenoceptors play a role in controlling intracellular calcium ion ([Ca(2+)]i) level in acinar cells of rat lacrimal glands. Expression of mRNA for adrenoceptor subtypes in the acinar cells was assessed using RT-PCR. All types except α2c, ß1, and ß3 were detected. NA induced a [Ca(2+)]i increase with a biphasic pattern in the acinar cells. Removal of extracellular Ca(2+) and use of Ca(2+)-channel blockers did not inhibit the NA-induced [Ca(2+)]i increases. In contrast, U73122 and suramin almost blocked these increases. The α1-adrenoceptor agonist phenylephrine induced a strong increase in [Ca(2+)]i. However, clonidine and isoproterenol failed to induce a [Ca(2+)]i increase. The peroxidase activity was quantified as a measure of mucin secretion. Ca(2+)-dependent exocytotic secretion of peroxidase was detected in rat lacrimal glands. The RT-PCR results showed that MUC1, MUC4, MUC5AC, MUC5B, and MUC16 were expressed in acinar cells. These findings indicated that NA activates α1-adrenoceptors, which were found to be the main receptors in Ca(2+)-related cell homeostasis and protein (including mucin) secretion in lacrimal glands.

World's first telepathology experiments employing WINDS ultra-high-speed internet satellite, nicknamed "KIZUNA".

BACKGROUND: Recent advances in information technology have allowed the development of a telepathology system involving high-speed transfer of high-volume histological figures via fiber optic landlines. However, at present there are geographical limits to landlines. The Japan Aerospace Exploration Agency (JAXA) has developed the "Kizuna" ultra-high speed internet satellite and has pursued its various applications. In this study we experimented with telepathology in collaboration with JAXA using Kizuna. To measure the functionality of the Wideband InterNet working engineering test and Demonstration Satellite (WINDS) ultra-high speed internet satellite in remote pathological diagnosis and consultation, we examined the adequate data transfer speed and stability to conduct telepathology (both diagnosis and conferencing) with functionality, and ease similar or equal to telepathology using fiber-optic landlines. MATERIALS AND METHODS: We performed experiments for 2 years. In year 1, we tested the usability of the WINDS for telepathology with real-time video and virtual slide systems. These are state-of-the-art technologies requiring massive volumes of data transfer. In year 2, we tested the usability of the WINDS for three-way teleconferencing with virtual slides. Facilities in Iwate (northern Japan), Tokyo, and Okinawa were connected via the WINDS and voice conferenced while remotely examining and manipulating virtual slides. RESULTS: Network function parameters measured using ping and Iperf were within acceptable limits. However; stage movement, zoom, and conversation suffered a lag of approximately 0.8 s when using real-time video, and a delay of 60-90 s was experienced when accessing the first virtual slide in a session. No significant lag or inconvenience was experienced during diagnosis and conferencing, and the results were satisfactory. Our hypothesis was confirmed for both remote diagnosis using real-time video and virtual slide systems, and also for teleconferencing using virtual slide systems with voice functionality. CONCLUSIONS: Our results demonstrate the feasibility of ultra-high-speed internet satellite networks for use in telepathology. Because communications satellites have less geographical and infrastructural requirements than landlines, ultra-high-speed internet satellite telepathology represents a major step toward alleviating regional disparity in the quality of medical care.

Effects of protease-activated receptors (PARs) on intracellular calcium dynamics of acinar cells in rat lacrimal glands.

Protease-activated receptors (PARs) represent a novel class of seven transmembrane domain G-protein coupled receptors, which are activated by proteolytic cleavage. PARs are present in a variety of cells and have been prominently implicated in the regulation of a number of vital functions. Here, lacrimal gland acinar cell responses to PAR activation were examined, with special reference to intracellular Ca(2+) concentration ([Ca(2+)]i) dynamics. In the present study, detection of acinar cell mRNA specific to known PAR subtypes was determined by reverse transcriptase polymerase chain reaction. Only PAR2 mRNA was detected in acinar cells of lacrimal glands. Both trypsin and a PAR2-activating peptide (PAR2-AP), SLIGRL-NH2, induced an increase in [Ca(2+)]i in acinar cells. The removal of extracellular Ca(2+) and the use of Ca(2+) channel blockers did not inhibit PAR2-AP-induced [Ca(2+)]i increases. Furthermore, U73122 and xestospongin C failed to inhibit PAR2-induced increases in [Ca(2+)]i. The origin of the calcium influx observed after activated PAR2-induced Ca(2+) release from intracellular Ca(2+) stores was also evaluated. The NO donor, GEA 3162, mimicked the effects of PAR2 in activating non-capacitative calcium entry (NCCE). However, both calyculin A (100 nM) and a low concentration of Gd(3+) (5 µM) did not completely block the PAR2-AP-induced increase in [Ca(2+)]i. These findings indicated that PAR2 activation resulted primarily in Ca(2+) mobilization from intracellular Ca(2+) stores and that PAR2-mediated [Ca(2+)]i changes were mainly independent of IP3. RT-PCR indicated that TRPC 1, 3 and 6, which play a role in CCE and NCCE, are expressed in acinar cells. We suggest that PAR2-AP differentially regulates both NCCE and CCE, predominantly NCCE. Finally, our results suggested that PAR2 may function as a key receptor in calcium-related cell homeostasis under pathophysiological conditions such as tissue injury or inflammation.

P2Y purinoceptors induce changes in intracellular calcium in acinar cells of rat lacrimal glands.

Adenosine 5'-triphosphate (ATP) is an extracellular signal that regulates various cellular functions. Cellular secretory activities are enhanced by ATP as well as by cholinergic and adrenergic stimuli. The present study aimed to determine which purinoceptors play a role in ATP-induced changes in the intracellular concentration of calcium ions ([Ca²âº](i)) and in the fine structure of acinar cells of rat lacrimal glands. ATP induced exocytotic structures, vacuolation and an increase in [Ca²âº](i) in acinar cells. The removal of extracellular Ca²âº or the use of Ca²âº channel blockers partially inhibited the ATP-induced [Ca²âº](i) increase. U73122 (an antagonist of PLC) and heparin (an antagonist of IP3 receptors) did not completely inhibit the ATP-induced [Ca²âº](i) increase. P1 purinoceptor agonists did not induce any changes in [Ca²âº](i), whereas suramin (an antagonist of P2 receptors) completely inhibited ATP-induced changes in [Ca²âº](i). A P2Y receptor agonist, 2-MeSATP, induced a strong increase in [Ca²âº](i), although UTP (a P2Y2,4,6 receptor agonist) had no effect, and reactive blue 2 (a P2Y receptor antagonist) resulted in partial inhibition. The potency order of ATP analogs (2-MeSATP > ATP >>> UTP) suggested that P2Y1 played a significant role in the cellular response to ATP. BzATP (a P2X7 receptor agonist) induced a small increase in [Ca²âº](i), but α,ß-meATP (a P2X1,3 receptor agonist) had no effect. RT-PCR indicated that P2X2,3,4,5,6,7 and P2Y1,2,4,12,14 are expressed in acinar cells. In conclusion, the response of acinar cells to ATP is mediated by P2Y (especially P2Y1) as well as by P2X purinoceptors.

The effects of diuretics on intracellular Ca2+ dynamics of arteriole smooth muscles as revealed by laser confocal microscopy.

The regulation of cytosolic Ca(2+) homeostasis is essential for cells, including vascular smooth muscle cells. Arterial tone, which underlies the maintenance of peripheral resistance in the circulation, is a major contributor to the control of blood pressure. Diuretics may regulate intracellular Ca(2+) concentration ([Ca(2+)](i)) and have an effect on vascular tone. In order to investigate the influence of diuretics on peripheral resistance in circulation, we investigated the alteration of [Ca(2+)](i) in testicular arterioles with respect to several categories of diuretics using real-time confocal laser scanning microscopy. In this study, hydrochlorothiazide (100 microM) and furosemide (100 microM) had no effect on the [Ca(2+)](i) dynamics. However, when spironolactone (300 microM) was applied, the [Ca(2+)](i) of smooth muscles increased. The response was considerably inhibited under either extracellular Ca(2+)-free conditions, the presence of Gd(3+), or with a treatment of diltiazem. After the thapsigargin-induced depletion of internal Ca(2+) store, the spironolactone-induced [Ca(2+)](i) dynamics was slightly inhibited. Therefore, the spironolactone-induced dynamics of [Ca(2+)](i) can be caused by either a Ca(2+) influx from extracellular fluid or Ca(2+) mobilization from internal Ca(2+) store, with the former being dominant. As tetraethylammonium, an inhibitor of the K(+) channel, slightly inhibited the spironolactone-induced [Ca(2+)](i) dynamics, the K(+) channel might play a minor role in those dynamics. Tetrodotoxin, a neurotoxic Na(+) channel blocker, had no effect, therefore the spironolactone-induced dynamics is a direct effect to smooth muscles, rather than an indirect effect via vessel nerves.

Abeta42-to-Abeta40- and angiotensin-converting activities in different domains of angiotensin-converting enzyme.

Amyloid beta-protein 1-42 (Abeta42) is believed to play a causative role in the development of Alzheimer disease (AD), although it is a minor part of Abeta. In contrast, Abeta40 is the predominant secreted form of Abeta and recent studies have suggested that Abeta40 has neuroprotective effects and inhibits amyloid deposition. We have reported that angiotensin-converting enzyme (ACE) converts Abeta42 to Abeta40, and its inhibition enhances brain Abeta42 deposition (Zou, K., Yamaguchi, H., Akatsu, H., Sakamoto, T., Ko, M., Mizoguchi, K., Gong, J. S., Yu, W., Yamamoto, T., Kosaka, K., Yanagisawa, K., and Michikawa, M. (2007) J. Neurosci. 27, 8628-8635). ACE has two homologous domains, each having a functional active site. In the present study, we identified the domain of ACE, which is responsible for converting Abeta42 to Abeta40. Interestingly, Abeta42-to-Abeta40-converting activity is solely found in the N-domain of ACE and the angiotensin-converting activity is found predominantly in the C-domain of ACE. We also found that the N-linked glycosylation is essential for both Abeta42-to-Abeta40- and angiotensin-converting activities and that unglycosylated ACE rapidly degraded. The domain-specific converting activity of ACE suggests that ACE inhibitors could be designed to specifically target the angiotensin-converting C-domain, without inhibiting the Abeta42-to-Abeta40-converting activity of ACE or increasing neurotoxic Abeta42.

Spontaneous [Ca2+]i oscillations in G1/S phase-synchronized cells.

Ca(2+) signaling controls a wide range of cellular functions such as division, fertilization, apoptosis and necrosis. Specifically, calcium signaling is thought to play a crucial role in driving cells through the different stages of the cell-division cycle. In most cells, however, this fact is far from being established. Few studies have examined this question from a different perspective: whether cells exhibit some characteristic cell cycle-dependent intracellular calcium-signaling patterns. This approach is effective in discerning the causal relationship between Ca(2+) signaling and the cell cycle. Through synchronization of the cell cycle, flow cytometry and confocal scanning microscopic intracellular calcium ion concentration ([Ca(2+)](i)) imaging, the present study shows that the G1/S phase transition is uniquely characterized by spontaneous [Ca(2+)](i) oscillations that last for up to 40 min. Most likely, these oscillations emanate from the [Ca(2+)](i) signaling that accompanies DNA replication as the cell prepares for the next division cycle. These temporal signals further affirm the significance of Ca(2+) in the cell cycle.

In vivo calcium imaging of OFF-responding ASK chemosensory neurons in C. elegans.

BACKGROUND: How neurons and neuronal circuits transform sensory input into behavior is not well understood. Because of its well-described, simple nervous system, Caenorhabditis elegans is an ideal model organism to study this issue. Transformation of sensory signals into neural activity is a crucial first step in the sensory-motor transformation pathway in an animal's nervous system. We examined the properties of chemosensory ASK neurons of C. elegans during sensory stimulation. METHOD: A genetically encoded calcium sensor protein, G-CaMP, was expressed in ASK neurons of C. elegans, and the intracellular calcium dynamics of the neurons were observed. RESULTS: After application of the attractants l-lysine or food-related stimuli, the level of calcium in ASK neurons decreased. In contrast, responses increased upon stimulus removal. Opposite responses were observed after application and removal of a repellent. CONCLUSION: The observed changes in response to external stimuli suggest that the activity of ASK neurons may impact stimulus-evoked worm behavior. The stimulus-ON/activity-OFF properties of ASK neurons are similar to those of vertebrate retinal photoreceptors. GENERAL SIGNIFICANCE: Analysis of sensory-motor transformation pathways based on the activity and structure of neuronal circuits is an important goal in neurobiology and is practical in C. elegans. Our study provides insights into the mechanism of such transformation in the animal.

Isolated rat alveolar type II cells protrude intracellular lamellar bodies by forming bubble-like structures during surfactant secretion.

Pulmonary surfactant is synthesized and secreted by pulmonary alveolar type II epithelial cells (type II cells). It passes through the alveolar lining fluid and adsorbs to the air-liquid interface. The process from secretion to adsorption is not yet entirely understood. To acquire a detailed understanding of this process, we used multiple observations of type II cells isolated from rat lungs under electron microscopy (EM) and confocal laser scanning microscopy (CLSM). Transmission EM observation demonstrated a loosening process of the intracellular lamellar bodies from the inside to the outside of the cell. Scanning EM observation revealed bubble-like protrusions from the cell surface, and differential interference contrast microscopy illustrated the protrusions expanding with time. CLSM observation with FM 1-43, a fluorescent membrane probe, revealed that the bubble-like protrusions were composed of phospholipids. Thus, we have demonstrated that isolated rat type II cells protrude intracellular lamellar bodies by forming bubble-like structures, possibly enabling them to adsorb to the air-liquid interface directly. These observations suggest a new mechanism for surfactant secretion from type II cells.

Dipyridamole inhibits intracellular calcium transients in isolated rat arteriole smooth muscle cells.

Dipyridamole, an inhibitor of adenosine uptake as well as a cGMP phosphodiesterase inhibitor, is commonly used in prophylactic therapy for patients with angina pectoris. However, the effects of dipyridamole on systemic blood vessels, especially on the peripheral vascular system, are not well understood. Therefore, the effect of dipyridamole on ATP-induced arteriole contraction was examined with special reference to intracellular Ca(2+) concentration ([Ca(2+)](i)) using real-time confocal microscopy. In cases of 0.1-10microM range, dipyridamole induced only slight [Ca(2+)](i) decreases in smooth muscle cells of both testicular and cerebral arterioles. However, 100microM dipyridamole induced substantial [Ca(2+)](i) decreases in the cells. In the presence of 10microM dipyridamole, changes in ATP-induced [Ca(2+)](i) were found to be inhibited in smooth muscle cells of testicular arterioles but not in those of cerebral arterioles. In addition, alpha, beta-methylene ATP-induced [Ca(2+)](i) increases in testicular arteriole smooth muscle cells were also partially inhibited in the presence of dipyridamole. When testicular arterioles were perfused with dipyridamole, no increases in nitric oxide levels were detected. High levels of K(+) induced a [Ca(2+)](i) increase in testicular arterioles that was also partially inhibited by dipyridamole. In the presence of substances that affect protein kinase A or G, ATP-induced [Ca(2+)](i) was not completely inhibited. These findings suggest that dipyridamole may act not only as an inhibitor of adenosine uptake and as a cGMP phosphodiesterase inhibitor, but also as a calcium channel blocker in arteriole smooth muscle cells.

Microtubule remodeling mediates the inhibition of store-operated calcium entry (SOCE) during mitosis in COS-7 cells.

Regulation of the intracellular calcium ion concentration ([Ca(2+)](i)) is critical, because calcium signaling controls diverse and vital cellular processes such as secretion, proliferation, division, gene transcription, and apoptosis. Store-operated calcium entry (SOCE) is the main mechanism through which non-excitable cells replenish and thus maintain this delicate balance. There is limited evidence which indicates that SOCE may be inhibited during mitosis, and the mechanisms leading to the presumed inhibition has not been elucidated. In the present study, we examined and compared the [Ca(2+)](i) dynamics of COS-7 cells in mitotic and non-mitotic phases with special reference paid to SOCE. Laser scanning confocal microscopy to monitor [Ca(2+)](i) dynamics revealed that SOCE was progressively inhibited in mitosis and became virtually absent during the metaphase. We used various cytoskeletal modifying drugs and immunofluorescence to assess the contribution of microtubule and actin filaments in SOCE signaling. Nocodazole treatment caused microtubule reorganization and retraction from the cell periphery that mimicked the natural mitotic microtubule remodeling that was also accompanied by SOCE inhibition. Short exposure to paclitaxel, a microtubule-stabilizing drug, bolstered SOCE, whereas long exposure resulted in microtubule disruption and SOCE inhibition. Actin-modifying drugs did not affect SOCE. These findings indicate that mitotic microtubule remodeling plays a significant role in the inhibition of SOCE during mitosis.

Regional differences in 5-HT receptors in cerebral and testicular arterioles of the rat as revealed by Ca2+ imaging of real-time confocal microscopy: variances by artery size and organ specificity.

5-hydroxytriptamine (5-HT) is an important transmitter for vessel constriction. The present study was performed to clarify the effect of 5-HT on smooth muscles in large- and small-sized cerebral and testicular arterioles by confocal microscopy, with special reference to intracellular Ca2+ concentration ([Ca2+]i) dynamics. In cerebral vessels, 5-HT induced a [Ca2+]i increase and the contraction of smooth muscle cells in large- and midsized arterioles (external diameters>50 microm) but not in small-sized arterioles. Conspicuous [Ca2+]i changes by 5-HT were especially observed in the portions close to the cerebral arterial circle, and the 5-HT-induced responses were caused by both Ca2+ influx and mobilization. Experiments using agonists and antagonists also revealed that cerebral arteriole smooth muscles possess 5-HT1a, 1b, 2 (G-protein-coupled type), and 3 (ion channel type) receptors; specifically, 5-HT2 plays a major role in these responses. On the other hand, in testicular vessels, there were few regional differences among responses to 5-HT, and both large- and small-sized arterioles responded to 5-HT. The responses were caused by only Ca2+ mobilization mediated 5-HT1a and 2. These results indicate that arterioles in different tissues may respond to 5-HT in different manners. Regional differences and the size-dependent manner of responses to 5-HT in cerebral blood vessels also indicate that the regulatory mechanism of blood circulation is highly differentiated in each region of the central nervous system.