Expanded quantum vortex liquid regimes in the electron nematic superconductors FeSe1-xSx and FeSe1-xTex.

The quantum vortex liquid (QVL) is an intriguing state of type-II superconductors in which intense quantum fluctuations of the superconducting (SC) order parameter destroy the Abrikosov lattice even at very low temperatures. Such a state has only rarely been observed, however, and remains poorly understood. One of the key questions is the precise origin of such intense quantum fluctuations and the role of nearby non-SC phases or quantum critical points in amplifying these effects. Here we report a high-field magnetotransport study of FeSe1-xSx and FeSe1-xTex which show a broad QVL regime both within and beyond their respective electron nematic phases. A clear correlation is found between the extent of the QVL and the strength of the superconductivity. This comparative study enables us to identify the essential elements that promote the QVL regime in unconventional superconductors and to demonstrate that the QVL regime itself is most extended wherever superconductivity is weakest.

Association between blood omega-3 polyunsaturated fatty acids and the gut microbiota among breast cancer survivors.

Omega-3 polyunsaturated fatty acids (PUFAs) are essential nutrients demonstrated to have health benefits, such as decreasing the risk of coronary heart disease, improving parameters associated with metabolic syndrome, and decreasing anxiety symptoms and depression risk. Previous intervention studies indicated the association between blood or tissue PUFA levels and the gut microbiota; however, the details remain incompletely elucidated. We conducted a cross-sectional study to examine the association between PUFAs and the gut microbiota among breast cancer survivors. Adults who had been diagnosed with invasive breast cancer more than one year ago and were not currently undergoing chemotherapy were enrolled. Capillary blood and faecal samples were obtained to assess the blood PUFA levels and gut microbiota compositions. The mean age (n=124) was 58.7 years, and 46% of the participants had a history of chemotherapy. Multiple regression analysis controlling for possible confounders indicated that an increased relative abundance of Actinobacteria was significantly associated with increased levels of docosahexaenoic acid (DHA, beta=0.304, q<0.01). At the genus level, the abundance of Bifidobacterium was positively associated with the level of DHA (beta=0.307, q<0.01). No significant association between omega-6 PUFAs and the relative abundances of gut microbiota members was observed. In addition, analyses stratified by the history of chemotherapy indicated significant associations of PUFA levels with the abundance of some bacterial taxa, including the phylum Actinobacteria (DHA, beta=0.365, q<0.01) and Bacteroidetes (EPA, beta=-0.339, q<0.01) and the genus Bifidobacterium (DHA, beta=0.368, q<0.01) only among participants without a history of chemotherapy. These findings provide the first evidence of positive associations between the abundances of Bifidobacterium among the gut microbiota and the levels of omega-3 PUFAs in the blood. Further studies are required to gain additional insight into these associations in healthy subjects as well as into the causality of the relationship.

Transient receptor potential ankyrin 1 agonists improve intestinal transit in a murine model of postoperative ileus.

BACKGROUND: Stimulation of transient receptor potential ankyrin 1 (TRPA1), which abundantly expressed in enterochromaffin cells (ECC), has been reported to exert apparently contradictory results in in vitro contractility and in vivo gastrointestinal (GI) transit evaluations. The pharmaceutical-grade Japanese traditional medicine daikenchuto (TU-100) has been reported to be beneficial for postoperative ileus (POI) and accelerate GI transit in animals and humans. TU-100 was recently shown to increase intestinal blood flow via stimulation of TRPA1 in the epithelial cells of the small intestine (SI). METHODS: The effects of various TRPA1 agonists on motility were examined in a manipulation-induced murine POI model, in vitro culture of SI segments and an ECC model cell line, RIN-14B. KEY RESULTS: Orally administered TRPA1 agonists, aryl isothiocyanate (AITC) and cinnamaldehyde (CA), TU-100 ingredients, [6]-shogaol (6S) and γ-sanshool (GS), improved SI transit in a POI model. The effects of AITC, 6S and GS but not CA were abrogated in TRPA1-deficient mice. SI segments show periodic peristaltic motor activity whose periodicity disappeared in TRPA1-deficient mice. TU-100 augmented the motility. AITC, CA and 6S increased 5-HT release from isolated SI segments and the effects of all these compounds except for CA were lost in TRPA1-deficient mice. 6S and GS induced a release of 5-HT from RIN-14B cells in a dose- and TRPA1-dependent manner. CONCLUSIONS & INFERENCES: Intraluminal TRPA1 stimulation is a potential therapeutic strategy for GI motility disorders. Further investigation is required to determine whether 5-HT and/or ECC are involved in the effect of TRPA1 on motility.

Increased ghrelin signaling prolongs survival in mouse models of human aging through activation of sirtuin1.

Caloric restriction (CR) is known to retard aging and delay functional decline as well as the onset of diseases in most organisms. Ghrelin is secreted from the stomach in response to CR and regulates energy metabolism. We hypothesized that in CR ghrelin has a role in protecting aging-related diseases. We examined the physiological mechanisms underlying the ghrelin system during the aging process in three mouse strains with different genetic and biochemical backgrounds as animal models of accelerated or normal human aging. The elevated plasma ghrelin concentration was observed in both klotho-deficient and senescence-accelerated mouse prone/8 (SAMP8) mice. Ghrelin treatment failed to stimulate appetite and prolong survival in klotho-deficient mice, suggesting the existence of ghrelin resistance in the process of aging. However, ghrelin antagonist hastened death and ghrelin signaling potentiators rikkunshito and atractylodin ameliorated several age-related diseases with decreased microglial activation in the brain and prolonged survival in klotho-deficient, SAMP8 and aged ICR mice. In vitro experiments, the elevated sirtuin1 (SIRT1) activity and protein expression through the cAMP-CREB pathway was observed after ghrelin and ghrelin potentiator treatment in ghrelin receptor 1a-expressing cells and human umbilical vein endothelial cells. Furthermore, rikkunshito increased hypothalamic SIRT1 activity and SIRT1 protein expression of the heart in the all three mouse models of aging. Pericarditis, myocardial calcification and atrophy of myocardial and muscle fiber were improved by treatment with rikkunshito. Ghrelin signaling may represent one of the mechanisms activated by CR, and potentiating ghrelin signaling may be useful to extend health and lifespan.

Pain-releasing action of platelet-activating factor (PAF) antagonists in neuropathic pain animal models and the mechanisms of action.

BACKGROUND: Platelet-activating factor (PAF) has been implicated in the pathology of neuropathic pain. Previous studies reported that PAF receptor (PAF-R) antagonists have varied anti-allodynia effects by route of administration and nerve injury models in rats. METHODS: The present study elucidated the effectiveness of PAF antagonists against neuropathic pain in four different models of peripheral nerve injury and provided insights into the mode of anti-allodynia action. RESULTS: PAF antagonists, TCV-309, BN 50739 and WEB 2086 by intravenous (i.v.) and oral administration have potent and long-lasting anti-allodynia action in mice neuropathic pain models. Treatment with PAF antagonists before surgery delayed the initiation of allodynia until the effects of these treatments were abolished. Intrathecal (i.t.) injection of the PAF antagonists and siRNA against PAF receptor ameliorated allodynia. I.t. injection of the glycine receptor (GlyR)α3 siRNA reduced the anti-allodynia effect of PAF antagonists. This evidence suggests that the anti-allodynia effect of PAF antagonists is at least in part mediated by spinal relief of PAF-induced dysfunction of GlyRα3. An analysis of the mode of anti-allodynia action of TCV-309 in vivo revealed a competitive action against PAF shortly after the injection of TCV-309, converting to a non-competitive action later. CONCLUSIONS: The present results revealed the effectiveness in anti-allodynia of PAF antagonists in different nerve injury models, and the unique mode of action; long-lasting anti-allodynia effects mediated by spinal GlyRα3 with a competitive manner at the initial stage and the following non-competitive manner of inhibition.

Potentiation of ghrelin signaling attenuates cancer anorexia-cachexia and prolongs survival.

Cancer anorexia-cachexia syndrome is characterized by decreased food intake, weight loss, muscle tissue wasting and psychological distress, and this syndrome is a major source of increased morbidity and mortality in cancer patients. This study aimed to clarify the gut-brain peptides involved in the pathogenesis of the syndrome and determine effective treatment for cancer anorexia-cachexia. We show that both ghrelin insufficiency and resistance were observed in tumor-bearing rats. Corticotropin-releasing factor (CRF) decreased the plasma level of acyl ghrelin, and its receptor antagonist, α-helical CRF, increased food intake of these rats. The serotonin 2c receptor (5-HT2cR) antagonist SB242084 decreased hypothalamic CRF level and improved anorexia, gastrointestinal (GI) dysmotility and body weight loss. The ghrelin receptor antagonist (D-Lys3)-GHRP-6 worsened anorexia and hastened death in tumor-bearing rats. Ghrelin attenuated anorexia-cachexia in the short term, but failed to prolong survival, as did SB242084 administration. In addition, the herbal medicine rikkunshito improved anorexia, GI dysmotility, muscle wasting, and anxiety-related behavior and prolonged survival in animals and patients with cancer. The appetite-stimulating effect of rikkunshito was blocked by (D-Lys3)-GHRP-6. Active components of rikkunshito, hesperidin and atractylodin, potentiated ghrelin secretion and receptor signaling, respectively, and atractylodin prolonged survival in tumor-bearing rats. Our study demonstrates that the integrated mechanism underlying cancer anorexia-cachexia involves lowered ghrelin signaling due to excessive hypothalamic interactions of 5-HT with CRF through the 5-HT2cR. Potentiation of ghrelin receptor signaling may be an attractive treatment for anorexia, muscle wasting and prolong survival in patients with cancer anorexia-cachexia.

Characterization of functional effects of Z-338, a novel gastroprokinetic agent, on the muscarinic M1, M2, and M3 receptors expressed in Xenopus oocytes.

This study characterized the functional effects of a novel gastroprokinetic agent, N-[2-(diisopropylamino)ethyl]-2-[(2-hydroxy-4,5-dimethoxybenzoyl)amino]-1, 3-thiazole-4-carboxyamide monohydrochloride trihydrate (Z338), on the muscarinic M1, M2, and M3 receptors expressed in Xenopus oocytes using the two-electrode voltage clamp method. Z-338 did not produce by itself any currents in oocytes expressing muscarinic M1, M3 receptors or muscarinic M2 receptors/G protein-gated inward rectifying K+ channels (Kir3.1 channels). In oocytes expressing muscarinic M1 receptors, Z-338 inhibited the acetylcholine-induced Ca2+ -activated Cl- current with an IC50 of 1.8 microM. In oocytes expressing muscarinic M2 receptors/Kir3.1 channels, Z-338 inhibited the acetylcholine-induced K+ currents with an IC50 of 10.1 microM, whereas in oocytes expressing muscarinic M3 receptors, Z-338 did not inhibit the acetylcholine-induced Ca2+ -activated Cl- current in a concentration-dependent manner. These results indicate that Z-338 is a potent antagonist not for muscarinic M3 receptor but for both muscarinic M1 and M2 receptors. Thus, Z-338 is a gastrokinetic agent with a unique profile.

Mechanisms of cytosolic Ca2+ suppression by prostaglandin E2 receptors in rat melanotrophs.

We have previously reported that voltage-dependent Ca2+ (VDC) channels of rat melanotrophs are inhibited by prostaglandin E2 (PGE2). In this study, mechanisms involved in the inhibitory actions of PGE2 receptors of rat melanotrophs were analysed using reverse transcriptase-polymerase chain reaction (RT-PCR), Ca2+-imaging and whole-cell, patch-clamp techniques with recently developed EP agonists, each of which is selective for the known four subclasses of EP receptors (EP1-4). PGE2 reversibly suppressed the cytosolic Ca2+ concentration ([Ca2+]i). The maximum reduction in [Ca2+]i by PGE2 was comparable to that by dopamine or to that by extracellular Ca2+ removal. RT-PCR analysis of all four EP receptors revealed that EP3 and EP4 receptor mRNAs were expressed in the intermediate lobe. The effects of PGE2 to suppress [Ca2+]i were mimicked by the selective EP3 agonist, ONO-AE-248, whereas three other EP agonists, ONO-DI-004 (EP1), ONO-AE1-259 (EP2) and ONO-AE1-329 (EP4), had little or no effect on [Ca2+]i. All four G-protein activated inward rectifying K+ (GIRK) channel mRNAs were identified in intermediate lobe tissues by RT-PCR. Dopamine concentration-dependently activated GIRK currents, whereas PGE2 did not activate GIRK currents, even at the concentration causing maximal inhibition of VDC channels. These results suggest that PGE2 acts on EP3 receptors to suppress Ca2+ entry of rat melanotrophs by selectively inhibiting VDC channels of these cells. We have compared the possible cellular and molecular mechanisms of inhibition by dopamine and PGE2.

Production of cAMP by adrenomedullin in human oligodendroglial cell line KG1C: comparison with calcitonin gene-related peptide and amylin.

The actions and the presence of adrenomedullin (AM) were investigated in cultured human oligodendroglial cell line KG1C. AM and AM mRNA were detected in KG1C cells by immunohistochemistry and RT-PCR. mRNAs for calcitonin receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMPs) 1, 2 and 3 but not for calcitonin receptors were detected in the cells, while mRNAs for CRLR, calcitonin receptors and all RAMPs were detected in the human cerebellum. Application of AM resulted in time- and concentration-dependent increases in the cAMP level of KG1C cells. Calcitonin gene-related peptide (CGRP) and amylin, peptides structurally related to AM, also increased cAMP. The potencies for the cAMP production of the three peptides were CGRP > or =AM >> amylin with EC(50) of 8, 18, 90 nM, respectively. The responses induced by AM were strongly inhibited by the CGRP(1) receptor antagonist human CGRP(8-37), and inhibited also by the AM receptor antagonist human AM(22-52). In contrast, the responses induced by CGRP or amylin were inhibited only by CGRP(8-37) and not by AM(22-52). The responses induced by all three peptides were unaffected by the amylin receptor antagonist human amylin(8-37). The CGRP(2) receptor agonist human [Cys(Acm)(2,7)]CGRP significantly increased the cAMP level but the increase was smaller than that caused by CGRP. This increase in cAMP was unaffected by CGRP(8-37), AM(22-52) or by amylin(8-37). These results suggest that in KG1C cells, AM increases cAMP through AM and CGRP(1) receptors, whereas CGRP does so through CGRP(1) and CGRP(2) receptors, and amylin exerts its effects through CGRP(1) receptors. Collectively, these findings imply that AM released from oligodendroglial cells may play a role in the regulation of oligodendrocytes via autocrine/paracrine through AM receptors and CGRP(1) receptors.

Inhibition by tramadol of muscarinic receptor-induced responses in cultured adrenal medullary cells and in Xenopus laevis oocytes expressing cloned M1 receptors.

Tramadol is a widely used, centrally acting analgesic, but its mechanisms of action are not completely understood. Muscarinic receptors are known to be involved in neuronal function in the brain and autonomic nervous system, and much attention has been paid to these receptors as targets of analgesic drugs in the central nervous system. This study investigated the effects of tramadol on muscarinic receptors by using two different systems, i.e., a Xenopus laevis oocyte expression system and cultured bovine adrenal medullary cells. Tramadol (10 nM-100 microM) inhibited acetylcholine-induced currents in oocytes expressing the M1 receptor. Although GF109203X, a protein kinase C inhibitor, increased the basal current, it had little effect on the inhibition of acetylcholine-induced currents by tramadol. On the other hand, tramadol did not inhibit the current induced by AlF4-, a direct activator of GTP-binding protein. In cultured bovine adrenal medullary cells, tramadol (100 nM-100 microM) suppressed muscarine-induced cyclic GMP accumulation. Moreover, tramadol inhibited the specific binding of [3H]quinuclidinyl benzilate (QNB). Scatchard analysis showed that tramadol increases the apparent dissociation constant (Kd) value without changing the maximal binding (Bmax), indicating competitive inhibition. These findings suggest that tramadol at clinically relevant concentrations inhibits muscarinic receptor function via QNB-binding sites. This may explain the neuronal function and anticholinergic effect of tramadol.

Selective expression of aquaporin 1, 4 and 5 in the rat middle ear.

The middle ear cavity is an air-filled space that must be maintained for effective sound transmission to the inner ear. To examine the mechanisms of water homeostasis in the middle ear, we investigated whether aquaporins (AQPs), a family of water-permeable channels, were expressed in the middle ear. Reverse transcription-polymerase chain reaction and immunoblot analyses revealed that mRNAs encoding AQP1, 4 and 5 (but not 2 or 3) subtypes were expressed in rat middle ear epithelium; AQP1, 4 and 5 were detected as 28-, 30- and 30-kDa proteins, respectively. Immunohistochemical analysis showed that AQP1 was localized at capillary endothelial cells and fibroblasts in lamina propria mucosae; AQP4 was present solely at the basolateral membrane of ciliated cells, whereas AQP5 was on the apical surface of ciliated cells as well as of flat and columnar epithelial cells. The characteristic different localizations of AQP1, 4 and 5 subtypes in the middle ear suggest that middle ear water homeostasis requires the coordinated operation of these AQPs.

Adrenomedullin inhibits the pressor effects and decrease in renal blood flow induced by norepinephrine or angiotensin II in anesthetized rats.

Adrenomedullin (AM), a hypotensive peptide originally isolated from human pheochromocytoma, has been reported to regulate renal functions. In patients with glomerulonephritis, the serum levels of AM are elevated as well as hypertensive agents norepinephrine (NE) and angiotensin II (AII). The effects of AM on the NE- or AII-induced pressor effects and renal blood flow responses, however, are not well clarified. We examined the effects of AM on blood pressure and renal blood flow induced by NE or AII in anesthetized rats. Arterial blood pressure and renal blood flow were measured using a calibrated pressure transducer and a laser Doppler flowmeter, respectively. Drugs were injected into the tail vein with a syringe. Intravenous administration of AM (1-3 nmol/kg) decreased the arterial blood pressure in anesthetized rats in a dose-dependent manner, whereas it did not affect the renal blood flow. NE or AII administration in anesthetized rats caused both increases in blood pressure and decreases in renal blood flow. Simultaneous administration of AM with NE or All prevented the increasing effects of blood pressure and inhibited the decreases in renal blood flow caused by NE or AII. These findings suggest that AM may have a protective role against the pressor effects and decrease in renal blood flow caused by NE or AII.

GTP gamma S-induced Ca2+ activated Cl- currents: its stable induction by Gq alpha overexpression in Xenopus oocytes.

In native Xenopus oocytes, injection of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) (30 mM, 5 nl) did not induce Cl- current in 11 out of 22 oocytes. Injection of increased concentration of GTPgammaS (100 mM, 5 nl) into the oocytes induced Cl- currents in 16 out of 17 oocytes; however, the size of the induced currents was extremely varied. In oocytes overexpressing Gq alpha, GTPgammaS (30 mM, 5 nl) faithfully evoked Ca2+-activated Cl- currents. These results indicate that heterogeneous expression of Gq alpha in Xenopus oocytes provides a useful system for studying the functional roles of Gq alpha in regulating cellular events.

Sequential changes in transforming growth factor (TGF)-beta1 concentration in synovial fluid and mRNA expression of TGF-beta1 receptors in chondrocytes after immobilization of rabbit knees.

We have previously reported that a combination of transforming growth factor (TGF)-beta1 and basic fibroblast growth factor (bFGF) synergistically increases the proliferation of chondrocytes obtained from knee joint immobilized for 7-14 days in male Japanese white rabbits. In the present study, we performed experiments with chondrocytes and syn ovial fluid obtained from rabbit knees immobilized for 0-42 days, to clarify the sequential changes in TGF-beta1 and bFGF concentrations in synovial fluid and the mRNA expression of TGF-beta1 receptor type I (RI) and II (RII) in chondrocytes after immobilization. The combination of TGF-beta1 and bFGF had a synergistic effect on the proliferation of chondrocytes obtained from knee joints immobilized for 7-14 days. The concentration of TGF-beta1 in synovial fluid was significantly higher (up to 3.6-fold) at 7-28 days after immobilization compared with that at 2 days. The mRNA for RI and RII was expressed during the whole immobilization period. The con centration of bFGF was kept at the same level at 2-7 days after immobilization, and gradually decreased thereafter. In the early stages of degenerated cartilage, up to 14 days after immobilization, the concentrations of both TGF-beta1 and bFGF were higher in the synovial fluid and mRNA expression of TGF-beta1 receptors in chondrocytes was kept.

Down-regulation of cell surface insulin receptors by sarco(endo)plasmic reticulum Ca2+-ATPase inhibitor in adrenal chromaffin cells.

Long-term (> or =12 h) treatment of cultured bovine adrenal chromaffin cells with thapsigargin (TG), an inhibitor of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), caused a time (t(1/2)=16.3 h)- and concentration (IC50=37.8 nM)-dependent decrease of cell surface 125I-insulin binding by 35%, but did not change the Kd value. TG caused a sustained increase of cytoplasmic concentration of Ca2+ ([Ca2+]c) in a biphasic manner, and the effect of TG on 125I-insulin binding was abolished by BAPTA-AM. Western blot analysis showed that TG lowered insulin receptor (IR) beta-subunit level in membrane, but did not alter total cellular levels of IR precursor and IR beta-subunit. Internalization of cell surface IR, as measured by using brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network (TGN), was not changed by TG. These results suggest that inhibition of SERCA by TG and the subsequent increase of [Ca2+]c down-regulates cell surface IR by retarding externalization of IR from the TGN.

Up-regulation of cell surface sodium channels by cyclosporin A, FK506, and rapamycin in adrenal chromaffin cells.

Treatment of cultured bovine adrenal chromaffin cells with cyclosporin A (CsA) increased cell surface [(3)H]saxitoxin ([(3)H]STX) binding by 56% in a time (t(1/2) = 15.2 h)- and concentration (EC(50) = 2.9 microM)-dependent manner but did not change the K(d) value. In CsA-treated cells, veratridine-induced (22)Na(+) influx was augmented with no change in the EC(50) of veratridine; also, alpha- and beta-scorpion venom and Ptychodiscus brevis toxin-3 enhanced veratridine-induced (22)Na(+) influx in a more than additive manner, as in nontreated cells. CsA treatment for 1 to 24 h inhibited calcineurin activity, measured by the in vitro assay, with the IC(50) of 0.6 microM but did not alter cellular level of calcineurin. FK506 or rapamycin elevated [(3)H]STX binding by 36 or 25%, whereas GPI-1046, an immunophilin ligand incapable to inhibit calcineurin, or okadaic acid, an inhibitor of protein phosphatases 1 and 2A, had no increasing effect. The rise of [(3)H]STX binding by CsA was attenuated by the coincident treatment with brefeldin A (BFA), an inhibitor of vesicular exit from the trans-Golgi network. The internalization rate of cell surface Na(+) channels, as determined in the presence of BFA, was decreased in CsA (but not rapamycin)-treated cells (t(1/2) = 20.3 h), compared with nontreated cells (t(1/2) = 13.7 h). CsA treatment, however, did not elevate cellular levels of Na(+) channel alpha-subunit and Na(+) channel alpha- and beta(1)-subunit mRNAs. In CsA-treated cells, veratridine-induced (45)Ca(2+) influx via voltage-dependent Ca(2+) channels and catecholamine secretion were enhanced, whereas high K(+)-induced (45)Ca(+) influx was not. Thus, the inhibition of calcineurin or rapamycin-binding protein causes up-regulation of cell surface functional Na(+) channels via modulating externalization and internalization of Na(+) channels, thus enhancing Ca(2+) channel gating and catecholamine secretion.

Heterogeneous increases of cytoplasmic calcium: distinct effects on down-regulation of cell surface sodium channels and sodium channel subunit mRNA levels.

1. Long-term (> or = 12 h) treatment of cultured bovine adrenal chromaffin cells with A23187 (a Ca(2+) ionophore) or thapsigargin (TG) [an inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)] caused a time- and concentration-dependent reduction of cell surface [(3)H]-saxitoxin (STX) binding capacity, but did not change the K:(D:) value. In A23187- or TG-treated cells, veratridine-induced (22)Na(+) influx was reduced (with no change in veratridine EC(50) value) while it was enhanced by alpha-scorpion venom, beta-scorpion venom, or Ptychodiscus brevis toxin-3, like in nontreated cells. 2. The A23187- or TG-induced decrease of [(3)H]-STX binding was diminished by BAPTA-AM. EGTA also inhibited the decreasing effect of A23187. A23187 caused a rapid, monophasic and persistent increase in intracellular concentration of Ca(2+) ([Ca(2+)](i)) to a greater extent than that observed with TG. 2,5-Di-(t-butyl)-1,4-benzohydroquinone (DBHQ) (an inhibitor of SERCA) produced only a rapid monophasic increase in [Ca(2+)](i), without any effect on [(3)H]-STX binding. 3. Reduction in [(3)H]-STX binding capacity induced by A23187 or TG was attenuated by Gö6976 (an inhibitor of conventional protein kinase C) or calpastatin peptide (an inhibitor of calpain). When the internalization rate of cell surface Na(+) channels was measured in the presence of brefeldin A (an inhibitor of vesicular exit from the trans-Golgi network), A23187 or TG accelerated the reduction of [(3)H]-STX binding capacity. 4. Six hours treatment with A23187 lowered Na(+) channel alpha- and beta(1)-subunit mRNA levels, whereas TG had no effect. 5. These results suggest that elevation of [Ca(2+)](i) caused by A23187, TG or DBHQ exerted differential effects on down-regulation of cell surface functional Na(+) channels and Na(+) channel subunit mRNA levels.

Aquaporin subtypes in rat cerebral microvessels.

We investigated the expression of aquaporin (AQP) subtypes in the rat cerebral microvessels by reverse transcription-polymerase chain reaction, immunoblotting and immunohistochemistry. mRNA for AQP4, but not for AQP1, 2, 3 or 5, was detected in the microvessels. Immunoblot analysis showed that AQP4 protein was detected as a 30 kDa band with higher molecular weight bands. Immunohistochemical staining showed that AQP4 was located on cell surface of the cerebral microvessels. These results suggest that AQP4 in the cerebral microvessels is involved in the regulation of water transport between blood and brain.

Post-translational reduction of cell surface expression of insulin receptors by cyclosporin A, FK506 and rapamycin in bovine adrenal chromaffin cells.

Long-term (>/=3 h) treatment of cultured bovine adrenal chromaffin cells with cyclosporin A (CsA) decreased cell surface (125)I-insulin binding by 62% in a concentration (IC(50)=18 microM)- and time (t(1/2)=16 h)-dependent manner, but did not change the K(d) value. FK506 (1 microM) or rapamycin (3 microM) treatment reduced (125)I-insulin binding. Western blot analysis showed that CsA treatment decreased insulin receptor (IR) beta-subunit level (t(1/2)=15 h) in membrane fraction, but did not alter total cellular levels of IR precursor and IR beta-subunit. Internalization rate of cell surface IR measured by using brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network, was comparable between non-treated and CsA-treated cells. Thus, CsA, FK506 and rapamycin inhibit peptidyl prolyl cis-trans isomerase activities of cyclophilin and FK506-binding protein, and down-regulate IR presumably by reducing cell surface externalization of IR.

Adrenomedullin receptors in rat cerebral microvessels.

To characterize the sites of action of adrenomedullin (AM) in the cerebral microvasculature, we studied the effect of AM on cyclic AMP (cAMP) level as well as expression of AM and its receptor in the rat cerebral microvessels. The microvessels were prepared from rat cerebral cortex by albumin flotation and glass bead filtration technique. AM and calcitonin gene-related peptide (CGRP) increased cAMP level in the microvessels in a concentration-dependent manner. The effect of AM was more than 100 times more potent than that of CGRP. The accumulation of cAMP by AM was inhibited by AM[22-52], an AM receptor antagonist, but not by CGRP[8-37], a CGRP receptor antagonist, suggesting that AM increased cAMP accumulation by acting on receptors specific to AM. [125I]AM binding to the microvessels was displaced by AM and less potently by AM[22-52]. The displacing potencies of CGRP and CGRP[8-37] were very weak. mRNAs for AM as well as calcitonin-receptor-like receptor and receptor-activity-modifying protein 2 which form a receptor specific to AM, were highly expressed in the microvessels. These results provide biochemical and pharmacological evidence that AM is produced in and acts on the cerebral microvessels in an autocrine/paracrine manner and is involved in regulation of cerebral microcirculation.