Activation of cerebral sodium-glucose transporter type 1 function mediated by post-ischemic hyperglycemia exacerbates the development of cerebral ischemia.

The regulation of post-ischemic hyperglycemia plays an important role in suppressing neuronal damage in therapeutic strategies for cerebral ischemia. We previously reported that the cerebral sodium-glucose transporter (SGLT) was involved in the post-ischemic hyperglycemia-induced exacerbation of cerebral ischemic neuronal damage. Cortical SGLT-1, one of the cerebral SGLT isoforms, is dramatically increased by focal cerebral ischemia. In this study, we focused on the involvement of cerebral SGLT-1 in the development of cerebral ischemic neuronal damage. It was previously reported that activation of 5'-adenosine monophosphate-activated protein kinase (AMPK) increases SGLT-1 expression. Moreover, ischemic stress-induced activation of AMPK exacerbates cerebral ischemic neuronal damage. Therefore, we directly confirmed the relationship between cerebral SGLT-1 and cerebral AMPK activation using in vitro primary culture of mouse cortical neurons. An in vivo mouse model of focal cerebral ischemia was generated using a middle cerebral artery occlusion (MCAO). The development of infarct volume and behavioral abnormalities on day 3 after MCAO were ameliorated in cerebral SGLT-1 knock down mice. Cortical and striatal SGLT-1 expression levels were significantly increased at 12h after MCAO. Immunofluorescence revealed that SGLT-1 and the neuronal nuclear antigen (NeuN) were co-localized in the cortex and striatum of MCAO mice. In the in vitro study, primary cortical neurons were cultured for five days before each treatment with reagents. Concomitant treatment with hydrogen peroxide and glucose induced the elevation of SGLT-1 and phosphorylated AMPK/AMPK ratio, and this elevation was suppressed by compound C, an AMPK inhibitor in primary cortical neurons. Moreover, compound C suppressed neuronal cell death induced by concomitant hydrogen peroxide/glucose treatment in primary cortical neurons. Therefore, we concluded that enhanced cerebral SGLT-1 function mediated by post-ischemic hyperglycemia exacerbates the development of cerebral ischemic neuronal damage. One of the mechanisms of cerebral SGLT-1 up-regulation may be involved in the AMPK activation after cerebral ischemia.

Mechanism of atropine-resistant contraction induced by Dai-kenchu-to in guinea pig ileum.

To clarify the contractile mechanism of Dai-kenchu-to, the effects of hydroxy beta-sanshool (an ingredient of Zanthoxylum fruit), Zanthoxylum fruit (a constituent herb of Dai-kenchu-to) and Dai-kenchu-to were studied in mucosa-free longitudinal muscle of guinea pig ileum. Hydroxy beta-sanshool at 10(-7)-10(-5) g/ml induced dose-related contractions accompanied by autonomous contraction and produced an initial contraction at a concentration of 10(-4) g/ml or more. The contraction induced by hydroxy beta-sanshool (10(-5) g/ml) was significantly inhibited by tetrodotoxin or the capsaicin-receptor antagonist capsazepine. Although atropine or the substance P antagonist spantide tended to inhibit the contraction, a combination of atropine and spantide almost abolished the contraction by hydroxy beta-sanshool. The P2-purinoceptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid did not affect hydroxy beta-sanshool-induced contraction in the presence or absence of spantide. The tonic contractions by Zanthoxylum fruit (2 x 10(-4) g/ml) and Dai-kenchu-to (10(-3) g/ml) were significantly inhibited or tended to be inhibited by atropine, spantide, tetrodotoxin or capsazepine and were remarkably suppressed by the combination of atropine and spantide. These results suggested that acetylcholine release from intrinsic cholinergic nerves and tachykinins from sensory neurons are involved in the contractions induced by hydroxy beta-sanshool and that tachykinins may be involved in the atropine-resistant contraction by Dai-kenchu-to.

Effect of activin on cell growth in primary cultures of guinea pig gastric epithelial cells.

BACKGROUND: The effect of activin on differentiated cells is known to be different from that on undifferentiated cells. Cultured gastric epithelial cells in complete serum-free conditions grew into matured mucous cells after treatment with epidermal growth factor (EGF). AIM: To elucidate the effect of activin on the growth of differentiated and undifferentiated gastric mucosal cells. METHODS: Cultured guinea pig gastric epithelial cells were prepared using the method of Ogihara et al. Synthesis of activin was analysed by Western blot using monoclonal anti-activin A antibody. Cell proliferation was assessed by counting the number of cells. Mucin production was assessed by histochemical study using periodic acid-Schiff (PAS) reaction. RESULTS: Western blot analysis indicated that activin was synthesized in cultured guinea pig gastric mucosal cells. One hundred nanomolar EGF induced a 3-fold increase in cell count and the appearance of PAS-positive granules. Five nanograms activin per millilitre without EGF stimulated proliferation of the cells that showed almost negative PAS staining. When activin was added after treatment with 100 nM EGF for 24 h, cell proliferation induced by EGF was inhibited by activin at concentrations higher than 5 ng/mL. CONCLUSION: These results suggest that activin stimulates proliferation of undifferentiated cells and inhibits growth of differentiated cells.

Endocrine cell distribution and expression of tissue-associated antigens in human female paraurethral duct: possible clue to the origin of urethral diverticular cancer.

BACKGROUND: To investigate (i) what determines the histologic differences seen among female urethral diverticular cancers and (ii) the possible embryologic origin of the female paraurethral duct, we performed a distribution analysis of endocrine cells and a comparative study of tissue-associated antigens in the female paraurethral duct. METHODS: Six human female urethras were obtained from surgical and autopsy cases including two cases of urethral diverticular cancer (columnar/mucinous type adenocarcinoma). The urethral and paraurethral epithelia were examined histologically and immunohistochemically. RESULTS: Immunoreactive endocrine cells predominated and carcinoembryonic antigen (CEA) was strongly expressed in the larger portion of the paraurethral duct close to the urethral lumen. Conversely, prostate-specific antigen and prostatic acid phosphatase were positive only in the smaller distal duct. In two cases of adenocarcinoma including endocrine cells, cancer cells were strongly positive for CEA. CONCLUSIONS: This study suggests that the proximal and distal parts of the paraurethral duct have different histologic characteristics and that the pathologic differences seen among female diverticular cancers may result from their cancer-genesis from different parts of the paraurethral duct.

Actomyosin contraction of the posterior hemisphere is required for inversion of the Volvox embryo.

During inversion of a Volvox embryo, a series of cell shape changes causes the multicellular sheet to bend outward, and propagation of the bend from the anterior to the posterior pole eventually results in an inside-out spherical sheet of cells. We use fluorescent and electron microscopy to study the behavior of the cytoskeleton in cells undergoing shape changes. Microtubules are aligned parallel to the cell's long axis and become elongated in the bend. Myosin and actin filaments are arrayed perinuclearly before inversion. In inversion, actin and myosin are located in a subnuclear position throughout the uninverted region but this localization is gradually lost towards the bend. Actomyosin inhibitors cause enlargement of the embryo. The bend propagation is inhibited halfway and, as a consequence, the posterior hemisphere remains uninverted. The arrested posterior hemisphere will resume and complete inversion even in the presence of an actomyosin inhibitor if the anterior hemisphere is removed microsurgically. We conclude that the principal role of actomyosin in inversion is to cause a compaction of the posterior hemisphere; unless the equatorial diameter of the embryo is reduced in this manner, it is too large to pass through the opening defined by the already-inverted anterior hemisphere.

Carcinoembryonic antigen positive adenocarcinoma of a female urethral diverticulum: case report and review of the literature.

Paraurethral glands of the female urethra, which are assumed to be embryologically homologous to the male prostate gland, are possible origins for diverticular cancer of the urethra. A case of primary adenocarcinoma arising in a female urethral diverticulum is presented. Pathology revealed a columnar/mucinous type adenocarcinoma which stained positively for carcinoembryonic antigen (CEA) and negatively for PSA. Normal paraurethral ducts located near the urethra and normal urethral epithelium also stained positively for CEA. These findings suggest that the adenocarcinoma in our case originated from the paraurethral duct near the urethral lumen.

State of actin in gastric parietal cells.

Remodeling of the apical membrane-cytoskeleton has been suggested to occur when gastric parietal cells are stimulated to secrete HCl. The present experiments assayed the relative amounts of F-actin and G-actin in gastric glands and parietal cells, as well as the changes in the state of actin on stimulation. Glands and cells were treated with a Nonidet P-40 extraction buffer for separation into detergent-soluble (supernatant) and detergent-insoluble (pellet) pools. Two actin assays were used to quantitate actin the deoxyribonuclease I binding assay to measure G-actin and F-actin content in the two pools and a simple Western blot assay to quantitate the relative amount of actin in the pools. Functional secretory responsiveness was assayed by aminopyrine accumulation. About 5% of the total parietal cell protein is actin, with about 90% of the actin present as F-actin. Stimulation of acid secretion resulted in no measurable change in the relative amounts of G-actin and cytoskeletal F-actin. Treatment of gastric glands with cytochalasin D inhibited acid secretion and resulted in a decrease in F-actin and an increase in G-actin. No inhibition of parietal cell secretion was observed when phalloidin was used to stabilize actin filaments. These data are consistent with the hypothesis that microfilamentous actin is essential for membrane recruitment underlying parietal cell secretion. Although the experiments do not eliminate the importance of rapid exchange between G- and F-actin for the secretory process, the parietal cell maintains actin in a highly polymerized state, and no measurable changes in the steady-state ratio of G-actin to F-actin are associated with stimulation to secrete acid.

Protrusion of cell surface coupled with single exocytotic events of secretion of the slime in Physarum plasmodia.

Exocytosis has been proposed to participate in the formation of pseudopods. Using video-enhanced microscopy, we directly visualized exocytosis of single vesicles in living Physarum plasmodia migrating on a substrate. Vesicles containing slime, the plasmodial extracellular matrix, of approximately 3.5 microm in diameter, shrank at the cell periphery at the average rate of approximately 1 microm/second, and became invisible. Immediately after exocytotic events, the neighboring cell surface extended to form a protrusion. The rate of extension was approximately 1 microm/second. The protrusion showed lamella-like morphology, and contained actin microfilaments. Electron microscopy suggested that the organization of microfilaments in such protrusions may be a random meshwork rather than straight bundles. These morphologies suggest that protruded regions are pseudopods. Importantly, only the slime-containing vesicle preferentially invaded the hyaline layer that consists of dense actin microfilaments while the other vesicular organelles remained in the granuloplasm. Quantitative analysis demonstrated a linear relationship in terms of their surface area, between individual protrusions and single slime-containing vesicles. It is, therefore, likely that most of the plasma membrane of the protrusion was supplied by fusion of the slime-containing vesicle during exocytosis.

Secretion of slime, the extracellular matrix of the plasmodium, as visualized with a fluorescent probe and its correlation with locomotion on the substratum.

Slime, the extracellular matrix of Physarum plasmodium, is secreted by the exocytosis of a vesicles that contain a slime precursor. Using an antibody raised against biochemically purified slime, we detected the intracellular localization of the slime vesicle. Slime vesicles are abundant in the advancing front of the plasmodium, as confirmed by electron microscopic observation in two different cross-sectional angles. Screening various reagents, we found that rhodamine-phosphatidylethanolamine (Rh-PE) binds specifically to slime in both its intravesicular and extracellular forms, as confirmed by immunoelectron microscopy using an antibody against fluorochrome rhodamine. The plasmodia vitally stained with Rh-PE exhibited dynamic fluorescent patterns during the course of locomotion. The fluorescence was conspicuous at the periphery of the leading pseudopods and oscillated according to the shuttle streaming that accompanied the relaxation and contraction of the periphery; it was intense in the relaxation phase when pseudopods extended, and became weak in the contraction phase when pseudopods contracted. The results collectively mean that the slime vesicles carried by the cytoplasmic streaming accumulated prior to secretion at the advancing margin of the plasmodium.

Insulin potentiates mitogenic effect of epidermal growth factor on cultured guinea pig gastric mucous cells.

Epidermal growth factor (EGF) stimulated proliferation of gastric mucous epithelial cells from guinea pigs in serum-free culture conditions. Western blot analysis with antiphosphotyrosine antibody showed that EGF initiated tyrosine phosphorylation of a 170-kDa protein, and this protein was identical to the EGF receptor. Insulin was not mitogenic, but it potentiated the mitogenic effect of EGF. Tyrosine phosphorylation of additional proteins was not induced by the combined actions of insulin and EGF. Stimulation by EGF and/or insulin did not cause a calcium response. However, when insulin was added to cells pretreated with EGF for > 6 h, it elicited a rapid intracellular calcium concentration rise that was reproducible in both cell suspension and single cell analyses. This calcium response coincided with the translocation of protein kinase C (PKC) from the cytosolic to the particulate fraction. Phorbol 12-myristate 13-acetate also caused the translocation and stimulated proliferation of the cells. These results suggest that the calcium-dependent activation of PKC may participate in the potentiation of the mitogenic effect of EGF by insulin.

Salivacin 140, a novel bacteriocin from Lactobacillus salivarius subsp. salicinius T140 active against pathogenic bacteria.

Fifteen of 353 environmental isolates of lactic acid bacteria consistently showed activity against Listeria monocytogenes, Streptococcus mutans, Actinomyces viscosus, and/or Propionibacterium acnes. Strain T140, isolated from the surface of Japanese pampas grass leaves and identified as Lactobacillus salivarius subsp. salicinius, also had activity against several Lactobacillus species, Staphylococcus aureus and Yersinia enterocolitica. Since the antagonistic factor(s) produced by T140 was sensitive to a proteolytic enzyme, it was concluded that a bacteriocin (named salivacin 140) was involved in the inhibition activity. Strain T140 required a high initial pH (7.5-8.5) in agar plates for bacteriocin production.

ATP is required in platelet serotonin exocytosis for protein phosphorylation and priming of secretory vesicles docked on the plasma membrane.

Calcium-evoked secretion generally requires the presence of millimolar concentrations of Mg-ATP. We investigated the role of Mg-ATP in the secretion of serotonin from electropermeabilized bovine platelets. The secretion of serotonin was lost within 5 minutes when the Mg-ATP concentration was diluted to less than 0.1 mM, but was maintained when ATP-gamma S (adenosine 5'-O-3-thiotriphosphate) was used instead of ATP. Okadaic acid, a potent inhibitor of protein phosphatase, could also maintain the exocytotic activity even when ATP was diluted. Decrease in the secretory activity was paralleled by a decrease in phosphorylation level of four proteins after dilution of ATP, but the activity was maintained when the thiophosphorylation level of these proteins was maintained. Two of these proteins were digested by a protease, calpain, which has been shown to lead to a loss in the exocytotic activity. Electron microscopic studies showed that calcium did not induce the formation of distinct bridge-like structures between the granule membrane and the plasma membrane in Mg-ATP-diluted cells, previously shown as the structure transiently formed prior to fusion of the two membranes. Anchorage of the secretory dense granules to the plasma membrane and the presence of the amorphous structures between the granules and the plasma membrane were unchanged by dilution of ATP. These results indicate that ATP is not required for the anchorage itself, but is required to prime anchored granules for calcium-triggered secretion. Maintenance of the phosphorylated state of proteins by ATP enables the calcium trigger to form the bridge-like structures preceding membrane fusion events.

Microtubules are required in amoeba chemotaxis for preferential stabilization of appropriate pseudopods.

Amoebae of Physarum polycephalum exhibit chemotactic responses to glucose and to cAMP. The chemotaxing amoebae exhibit alternating locomotive movements: relatively linear locomotion and movements that change the direction of the locomotion. Such locomotive activity is tightly coupled with the changes in the number and the positions of the pseudopods; cells have one pseudopod at the leading edge during their linear locomotion, while they have multiple pseudopods when they are changing the direction of locomotion. Treatment of cells with microtubule-disrupting reagents inhibited the chemotaxis of the cells. To characterize the role of the microtubule system in chemotaxis, we quantitatively analyzed the relationship between the positions of multiple pseudopods of the amoebae and the relative stability of the pseudopods during reorientation. No significant differences were observed in the pseudopod dynamics between the untreated and the treated amoebae. In both cases, one pseudopod at the leading edge continued to expand during linear locomotion. It then split into two to three pseudopods in the reorientation phase, and the positions of the multiple pseudopods were random. Among multiple pseudopods, however, the pseudopods closer to the microneedle tip were selectively stabilized more often than those distant from the tip in the presence of the microtubule system. By contrast, such preferential stabilization of the appropriate pseudopods was completely abolished by microtubule inhibitors. The microtubule-dependent selection of appropriately located pseudopods enables amoebae to turn correctly at the reorientation step.

[Calcium-dependent signaling of acid secretion in isolated parietal cells from guinea pigs and its modification by ethanol].

Treatment of isolated parietal cells from guinea pig gastric mucosa with ethanol caused a rapid increase in [Ca2+]i and concomitant decrease in the capacity for carbachol-stimulated acid secretion in a dose dependent manner. Carbachol rapidly increased the [Ca2+]i from trimethoxybenzoic acid 8-(diethylamino)-octyl ester sensitive intracellular pool. In contrast, the increase with ethanol was through La3+ sensitive Ca2+ channel from external source, which suppressed the Ca2+ response subsequently stimulated with carbachol. Pretreatment of the cells with EGTA or La3+ completely prevented the elevation of [Ca2+]i with ethanol and preserved the Ca2+ response to carbachol. These findings indicate that ethanol-induced elevation of [Ca2+]i may desensitize the stimulation of carbachol. Furthermore, treatment of the parietal cells with ethanol increased the activity of protein kinase C in both cytosolic and membrane fractions of the cells. Activation of protein kinase C with phorbol diester suppressed the capacity for acid secretion. These results suggest that ethanol may inhibit the carbachol-stimulated acid secretion through the desensitization of Ca2+ response and the activation of protein kinase C.

Twenty-eight-kilodalton phosphorylatable calcium- and lipid-binding proteins purified from Physarum plasmodium.

Two 28-kDa calcium- and lipid-binding proteins were isolated from a detergent-insoluble fraction of the Physarum plasmodium. Both proteins have molecular masses of approximately 28 kDa by SDS-PAGE. The protein designated 28K-I has a slightly lower mobility than that designated 28K-II. The purified 28K-I has a dissociation constant of 1.0 microM for Ca2+ ions, while the 28K-II has two different dissociation constants: one of 0.32 microM and the other of 3.2 mM. The 28K-I binds to liposomes at Ca2+ concentrations higher than 1.0 microM and has a dissociation constant for lipid of 34 micrograms/ml at 10 microM Ca2+. The 28K-II binds to liposomes at concentrations of Ca2+ above the mM range and has a dissociation constant of 36 micrograms/ml for lipid at 2 mM Ca2+. There is no evidence of actin-binding activity by either of the 28-kDa (28K) proteins. The 28K proteins crossreacted with an antiserum against chicken brush border calpactin I. The two proteins have quite different phosphorylation levels between a fraction prepared from the cytosolic endoplasm and a fraction prepared from the whole cell. The 28K proteins may play some role in the membrane structure dynamics of the cortical gel layer.

Interaction of the low-molecular-mass, guanine-nucleotide-binding protein with the actin-binding protein and its modulation by the cAMP-dependent protein kinase in bovine platelets.

Platelets have been shown to possess several, different, low-molecular-mass, guanine-nucleotide-binding proteins (G-proteins) with molecular masses about 20-30 kDa. We report here that a 25-kDa G-protein copurified with the bovine platelet actin-binding protein (ABP), a cross-linker of actin filaments which is known to generate the three-dimensional network of actin. Both the G-protein and ABP were recovered in a fraction that was insoluble in Triton X-100 and were extracted in 0.6 M NaCl. Gel-filtration chromatography of the high-salt extract and rechromatography in a low-salt solution indicated that the two proteins may be associated with each other. The association of the two proteins was suggested by cosedimentation of the G-protein with the actin gel formed by actin and ABP. The amounts of the cosedimented G-protein and ABP was unaffected by guanosine-5'-O-[beta-thio]diphosphate and guanosine-5'-O-[gamma-thio]triphosphate, but the G-protein, not ABP, was partially released from the actin gel by phosphorylating ABP with cAMP-dependent protein kinase. Thus, the association of the two proteins was affected by modification of ABP, but not by modification of G-proteins. The physiological significance of the possible association of the two proteins might be that the membrane skeleton functions as a modulator of the G-protein, rather than that the G-protein modulates the function of the membrane skeleton which comprises ABP.

Repression of serotonin secretion by an endogenous Ca2(+)-activated protease in electropermeabilized bovine platelets.

Micromolar levels of free calcium ions added to the extracellular medium elicit secretion of serotonin from electropermeabilized bovine platelets in the presence of millimolar levels of Mg-ATP. Such Ca2(+)-dependent secretion of serotonin was almost completely impaired when the permeabilized platelets were preincubated for 1 min at 35 degrees C in 100 microM Ca2+ without Mg-ATP. The half-maximal effect was observed with about 45 microM Ca2+ in the preincubation medium. Inhibitors of serine-thiol protease, such as leupeptin and antipain, suppressed the impairment of the secretion of serotonin by the preincubation with Ca2+. Electron microscopic observation revealed that disorganization of the cytoskeletal structures, in particular of the membrane undercoat and the network of microfilaments, accompanied the impairment of secretion of serotonin. Microfilaments were also found to be dissociated from dense granules that contained serotonin. These morphological changes were also suppressed when antipain was included in the Ca2(+)-preincubation medium. Coincident with these morphological changes, the following biochemical changes were observed in 100 microM Ca2+ but not in the presence of Ca2+ and antipain. The amount of Triton-insoluble cytoskeleton and the acto-myosin content of the dense-granule fraction were markedly decreased. The decrease in Triton-insoluble cytoskeletons was quantitatively correlated with the degree of impairment of secretion of serotonin. Immunoblot analysis of EGTA extracts of the cells showed that the 240-kDa spectrin in platelets was degraded to a 235-kDa fragment, and a 260-kDa actin-binding protein (ABP) in platelets was partially degraded to 190- and 110-kDa components.(ABSTRACT TRUNCATED AT 250 WORDS)

Anchorage of secretion-competent dense granules on the plasma membrane of bovine platelets in the absence of secretory stimulation.

The ultrastructural changes in electropermeabilized bovine platelets that accompany the Ca2(+)-induced secretion of serotonin were investigated in ultra-thin sections of chemically fixed cells. Such preparations permitted us to study both the localization of and the structures associated with serotonin-containing dense granules. Localization of dense granules within cells was examined by measuring the shortest distances between the granular membranes and the plasma membrane. About 40% of total granules were located close to the plasma membrane at an average distance of 10.8 +/- 1.6 nm. 71% of the total number of granules were localized at a similar average distance of 12.5 +/- 2.7 nm in intact platelets. The percentage of granules apposed to the plasma membrane corresponded closely to the percentage of total serotonin that was maximally secreted after stimulation of the permeabilized (38 +/- 4.9%) and the intact platelets (72 +/- 3.6%). Furthermore, the percentage of granules anchored to the membrane, but not of those in other regions of permeabilized cells, decreased markedly when cells were stimulated for 30 s by extracellularly added Ca2+. The decrease in the numbers of granules in the vicinity of the plasma membrane corresponded to approximately 22% of the total number of dense granules that were used for measurements of the distances between the two membranes and corresponded roughly to the overall decrease (15%) in the average number of the granules per cell. Most dense granules were found to be associated with meshwork structures of microfilaments. Upon secretory stimulation, nonfilamentous, amorphous structures found between the plasma membrane and the apposed granules formed a bridge-like structure that connected both membranes without any obvious accompanying changes in the microfilament structures. These results suggest that the dense granules that are susceptible to secretory stimulation are anchored to the plasma membrane before stimulation, and that the formation of the bridge-like structure may participate in the Ca2(+)-regulated exocytosis.