We have previously reported that the basal activity of L-type Ca²⁺ channel is suppressed by the inhibitors of calmodulin (CaM)-dependent protein kinase II (CaMKII), and that CaMKII activated by CaM recovers Ca²⁺ channel activity after run-down. In this study, we have investigated how CaMKII and CaM play their roles in the maintenance of the basal activity of Ca²⁺ channel. [Method] Single Ca²⁺ channel activities were recorded with patch clamp technique in guinea-pig ventricular myocytes. CaMKIIα-T286D, a mutant recombinant CaMKIIα (presented by Dr. Miyamoto E. & Dr. Takeuchi Y.), was produced by site-directed mutagenesis and GST fusion protein purification. CaMKIIα-T286D had partial enzymatic activity in the absence of CaM and Ca²⁺, and was used as an activated CaMKII. [Results & Discussion] After the patches were excised into basic internal solution, the Ca²⁺ channel activity disappeared (run-down). Application of CaMKIIα-T286D (1, 3, 10, 30 μM) recovered the Ca²⁺ channel activity to only 0.5, 3, 5 and 4% of that recorded in the cell-attached mode. The effect of CaM to reverse run-down was time-dependent, but in the presence of CaMKIIα-T286D, it became time-independent. These results suggest that both of CaMKII and CaM are involved in the maintenance of the basal activity of Ca²⁺ channel. CaMKII may keep the channel in a state that can be reactivated by CaM. [Jpn J Physiol 54 Suppl:S67 (2004)]

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Chloride channels have significant functions in glial astrocytes. However, only GABA_A receptor, glycine receptor and ClC-2 chloride channels have been identified in astrocytes. Thus, we here examined the expression of the ClC-1 chloride channel in rat astrocytic glioma C6 cells and rat astrocytes in primary culture. In C6 cells, five isoforms of rat ClC-1, but not rat skeletal muscle ClC-1 (SM ClC-1), were detected at the mRNA expression level. Comparison with SM ClC-1 showed that these isoforms share a short 3' end with a deletion of the nucleotides from 3115 to 3197 and a substitution of T by C at nucleotides 480 and 1733 as common features. Three of the five isoforms, M1, M2 and M3, were produced by deletion of ClC-1 exon 7, insertion of ClC-1 exon 7a and insertion of a trinucleotide, TAG, at nucleotide 858, respectively. Another isoform, M4, was produced by deletion of ClC-1 exon 8 at nucleotide 937, and the other, M5, was the same as SM ClC-1 except for the short 3' end and substitutions at the two positions. In Xenopus oocytes, only the M5 isoform could be expressed as a functional anion-selective channel. This glial ClC-1 isoform showed less dependence on voltage and extracellular Cl⁻ than SM ClC-1, whereas the anion selectivity sequence and the pharmacological sensitivity of the glial isoform were the same as those of SM ClC-1. In rat astrocytes, two additional distinct isoforms were found to be expressed in addition to the M2 isoform. [Jpn J Physiol 54 Suppl:S68 (2004)]

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The properties of VSOR Cl⁻ currents in single ventricular myocytes isolated from ClC-3 knockout mice were compared with those from ClC-3-expressing wild-type or heterozygous mice. Basal whole-cell currents recorded under isotonic conditions were indistinguishable between ClC-3-deficient and -expressing cells. ClC-3-deficient cardiomyocytes responded to osmotic swelling with activation of whole-cell anion currents that exhibited moderate outward rectification, time- and voltage-dependent inactivation, low-field anion selectivity and sensitivity to a Cl⁻ channel blocker. These biophysical and pharmacological properties of VSOR Cl⁻ currents in ClC-3-deficient cells were identical to those in ClC-3-expressing cells. Moreover, the VSOR Cl⁻ current density, which is an indicator of plasmalemmal expression of the functional channels, was essentially the same in cells isolated from these 3 types of mice. Activation of protein kinase C (PKC) was found to upregulate VSOR Cl⁻ currents in ClC-3-deficient and -expressing cells. This PKC effect is opposite to the reported downregulating effect of PKC activators on ClC-3-associated Cl⁻ currents. Thus, it is concluded that functional expression of VSOR Cl⁻ channels on the plasma membrane in mouse cardiomyocytes is independent of molecular expression of ClC-3. [Jpn J Physiol 54 Suppl:S68 (2004)]

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To clarify the role of the intracellular Ca²⁺ and Cl⁻ in the activation process of the Na⁺-K⁺-2Cl⁻ cotransporter in rat parotid acinar cells, we made simultaneous recording of the membrane conductance and the reversal potential of the Cl⁻ current at each peak of the oscillatory Cl⁻ current by using the gramicidin-perforated patch recording method. The conductance reflects the intracellular Ca²⁺ level. The intracellular Cl⁻ concentration was calculated from the reversal potential. We blocked the K⁺ channel by charybdotoxin and obtained the data in the second carbachol (CCh, 0.25 μM) application, changing the membrane potential from -80 to -85 mV. The intracellular Cl⁻ concentration was 30-60 mM just after the start of CCh application. It decreased to 10-30 mM in 2 min and then increased to a steady-state level of 20-40 mM. The increase was blocked by the Na⁺-K⁺-2Cl⁻ cotransporter blocker, bumetanide, suggesting that it reflects the cotransporter activity. The membrane conductance began to increase just after the start of CCh application and became almost steady-state in 0.5 min. These suggest that the Cl⁻ channel is activated by the intracellular Ca²⁺ increase before the Na⁺-K⁺-2Cl⁻ cotransporter activation, and that the decrease in the intracellular Cl⁻ concentration may play an important role in the activation of the cotransporter under the condition of the high intracellular Ca²⁺ level. [Jpn J Physiol 54 Suppl:S68 (2004)]

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In potassium channels, it has been proposed that two different gates are arranged in series along the pore. To investigate the mechanism of gating, KcsA channel was studied using a modified tip-dip method and single-channel currents were analyzed using the hidden Markov model. Effect of various concentration of sucrose (10%-40%) on the gating kinetics was tested. As the concentration of sucrose increased, the single-channel conductance was decreased and the open time was remarkably prolonged. In asymmetric concentrations of sucrose, sucrose in the intracellular side was effective to control the gating. Contrarily to what has been believed, in which high concentration of non-electrolyte forced the channel closed through collapsing effect of osmotic pressure, we found that the open probability was increased. Various non-electrolytes, such as sorbitol and glycerol, were applied and similar effects were observed. Since the viscosity of these solutions varied significantly, kinetic viscous effect on gating was less likely. A gating model built from the single-channel data identified that the fast gating mode was strongly affected by non-electrolytes. Thus, it is suggested that the effect of non-electrolytes is medicated through altered hydration state of the channel upon gating. [Jpn J Physiol 54 Suppl:S68 (2004)]

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The vacuolar type H⁺-ATPase (V-ATPase) expressed in the plasma membrane is the the primary proton secreting mechanism responsible for osteoclastic bone resorption. The V-ATPase is a multi-subunit complex composed of a membrane sector (V₀) and a catalytic sector (V₁). Since V₀ acts as a passive H⁺ conductor, voltage may be a key controller for the H⁺ secretion. We examined voltage-dependency of the H⁺ transport via H⁺-translocating component of V₀, subunit c: the cDNA was obtained from murine osteoclasts, cloned and expressed in COS7 cells. The pH recovery from cell acidosis in transformed cells were recorded under a variety of pH gradients. Cations and anions were replaced by NMDG⁺ and aspartate⁻. At the pH gradient of 1.8, outward currents were increased by depolarization. The I-V curve shifted to more positive voltages by acidifying the extracellular solution. N,N^,-Dicyclohexylcarbodiimide (DCCD), an inhibitor of subunit c, blocked the depolarization-dependent pH recovery and the outward currents. These data suggest that depolarization potentiates H⁺ efflux via subunit c localized at the plasma membrane. The membrane potential of osteoclasts, changing over a wide range (-120-0 mV), may regulate the H⁺ secretion in the absence of ATP-hydrolysis. [Jpn J Physiol 54 Suppl:S69 (2004)]

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We reported that genistein stimulates Cl- transport by activating apical Cl- channels and basolateral Na+/K+/2Cl- cotransporter (NKCC) in renal epithelial A6 cells. In this study, we found that insulin enhanced the genistein action on Cl- transport. Therefore, we determined whether insulin shows the enhancement by affecting translocation of Cl- channel and/or NKCC in genistein-treated cells. Brefeldin A (BFA) diminished the Cl- transport by decreasing the NPPB-sensitive conductance (Gt) and the action was less effective in insulin-treated cells. On the other hand, pretreatment with MG132 caused further increases in the Cl- transport without affecting the NPPB-sensitive Gt in both insulin-treated and -untreated cells, however MG132 was much more effective in insulin-treated cells than in insulin-untreated cells. These results suggest that: 1) the enhancement of genistein action by insulin occurs through translocation of the Cl- channel to the apical membrane, 2) insulin shows the enhancement by increasing both rates of insertion and endocytosis of NKCC, 3) the insulin action is larger in the insertion of NKCC than the endocytosis. Supported by JSPS 15659052 (YM), 15590189 (NN), 15790120 (HM). [Jpn J Physiol 54 Suppl:S69 (2004)]

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The kidney strictly controls the body Na+ content that is one of important factors determining the body fluid content and blood pressure by regulating the Na+ reabsorption in the distal nephron. The transepithelial Na+ reabsorption is mediated by epithelial Na+ channel (ENaC), which is controlled by aldosterone (ALDO), ADH and osmolality. A flavonoid, quercetin, which is contained in natural foods such as onions and soy beans is clinically reported to reduce high blood pressure. We previously reported that quercetin decreased the Na+ reabsorbtion in the distal nephron via suppression of _lpha-ENaC expression by activating the basolateral Na+/K+/2Cl- cotransporter. On the other hand, the ENaC expression is well known regulated by ALDO, however the interaction between ALDO and quercetin on the ENaC expression is not clear. To study the effect of quercetin on the ALDO-induced Na+ reabsorption through ENaC, we measured the benzamil-sensitive short-circuit current (Isc) as Na+ reabsorption in renal epithelial A6 cells. Although quercetin did not affect the basal Na+ reabsorption in ALDO-untreated A6 cells, quercetin reduced the ALDO-induced Na+ reabsorption dose-dependently by decreasing the benzamil-sensitive conductance. We suggest here that quercetin suppresses the ALDO action on ENaC. Supported by JSPS 15659053 (YM), 15590189 (NN), 15790120 (HM). [Jpn J Physiol 54 Suppl:S69 (2004)]

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The fluorescent Mg²⁺ indicator furaptra (mag-fura-2) was introduced into single ventricular myocytes by incubation with acetoxy-methyl ester of the indicator. Intracellular free Mg²⁺ concentration ([Mg²⁺]_i) was estimated from the fluorescence intensity ratio at 382 nm and 350 nm excitation in Ca²⁺-free conditions (0.1 mM EGTA) at 25°C. After the cells were loaded with Mg²⁺ by incubation in high extracellular Mg²⁺ concentration ([Mg²⁺]_o), reduction of [Mg²⁺]_o to 1 mM induced a decrease in [Mg²⁺]_i. The decrease in [Mg²⁺]_i was accelerated by initial [Mg²⁺]_i above the basal level (~0.8 mM) with a half maximum [Mg²⁺]_i of ~1.5 mM. On the other hand, raising [Mg²⁺]_o slowed the decrease in [Mg²⁺]_i with 50% reduction of the rate at ~10 mM [Mg²⁺]_o. Since it was likely that a part of furaptra molecules were distributed in intracellular organelles, we assessed the fluorescence signal emitted from the organelles. The cell membrane of the indicator-loaded myocytes were permeabilized with saponin (50 μg/ml) in "the internal solution" that contained 0.8 mM free Mg²⁺ and 5 mM ATP (pH 7.1). While the indicator fluorescence intensity at 350 nm excitation was quickly dissipated by ~80% after saponin treatment, the intensity ratio was essentially unchanged. Raising free Mg²⁺ concentration of the internal solution to 4 mM only caused a small change in the fluorescence ratio that corresponded to an increase in [Mg²⁺]_i of ~0.15 mM. We conclude that the decrease in [Mg²⁺]_i observed in the present study primarily reflects Mg²⁺ efflux, which is modulated by Mg²⁺ gradient across the cell membrane. [Jpn J Physiol 54 Suppl:S69 (2004)]

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Intrathecal administration of lidocaine primarily causes degeneration of the dorsal root in rats. To determine membrane mechanisms of neurotoxicity of lidocaine, the lidocaine-induced impairment of axonal transport in cultured mouse dorsal root ganglion neurons was investigated. Lidocaine (1-100 mM) inhibited axonal transport in a time- and dose-dependent manner. Higher concentrations (50-100 mM) of lidocaine caused membrane rupture and cell death. Ca²⁺-free solution and the Ca²⁺-calmodulin kinase II (CAM kinase II) inhibitor KN-62 reduced the inhibition of axonal transport but not membrane rupture. Lysophosphatidic acid, a bioactive phospholipid, blocked both the inhibition of axonal transport and membrane rupture. Thus, lidocaine at lower concentrations (<10 mM) may form pores in the membrane, resulting in Ca²⁺ influx and activation of CAM kinase II to inhibit axonal transport. Neurotoxic effect of higher concentrations of lidocaine seems to be a direct disruption of cell membrane. Lysophosphatidic acid may be useful to prevent neurotoxocity of lidocaine even at higher concentrations. [Jpn J Physiol 54 Suppl:S70 (2004)]

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Integrins are transmembrane receptors that mediate cell to substlate adhesion by forming links between the extracellular matrix and cytoskeleton. They participate in organizing cell adhesion sites and the actin-containing cytoskeleton, but also mediate signal transduction such as gene expression. Consequently integrins provide an intersection where mechanical forces, cytoskeletal organizations, biochemical signals and adhesions can meet. We wonder how a single receptor can fulfill the mechano-chemical signal transduction. To address this question, we first analyzed the distribution of individual integrin heterodimers in the ventral membrane of endothelial cells at ultrastructural level. To this end, immuno-freeze-etching-replica method (IFER) was applied to the exposed intracellular surface of the basal membrane prepared by a brief sonication. EM images stained with immuno-gold of integrins showed that there were many small sized (9.7 ± 9.3 ×10⁻³ μm²; mean ± SD) integrin aggregates in the region out of focal adhesion but near stress fibers. It should be noted that integrins in an aggregate seemed to be connected each other with fine filamentous structures. Moreover time-laps imaging of the integrin aggregates showed that some of them were very stable like as fixed points. This result suggests that the integrin aggregate may form an unknown type of fixed point at the basal membrane different from that at Focal adhesion. [Jpn J Physiol 54 Suppl:S70 (2004)]

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We studied an increase in the internal Ca concentration evoked the depletion of internal Ca stores (Ca-entry) with a SERCA inhibitor thapsigargin and/or a salivary gland secretagogue ACh in CD38 knockout (CD38 KO) mouse submandibular acinar cells. CD38 can generate cyclic ADP-ribose in the acinar cells. Thapsigargin evoked the Ca-entry divided into two phases, a rapid and a slow phase, equally in wild and CD38 KO cells. The elimination of CD38 greatly enhanced ACh induced Ca-entry. The presence of external Ni or excess of external K (90 mM) reduced the thapsigargin- and ACh-evoked Ca-entry. A phospholipase A₂ inhibitor 4-BPB (4-bromophenacyl bromide) enhanced the ACh-induced Ca-entry. The results suggest that the submandibular Ca-entry depends on the membrane potential, arachidonate formation , and the elimination of cyclic ADP-ribose. [Jpn J Physiol 54 Suppl:S70 (2004)]

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Sperm-specific phospholipase C-zeta (PLCζ) is known to induce intracellular Ca²⁺ oscillations and subsequent early embryonic development, when expressed in mouse eggs by injection of RNA encoding PLCζ. We addressed characteristics of recombinant mouse PLCζ protein generated by baculovirus/Sf9-cell expression system. PLCζ protein injected into mouse eggs induced repetitive Ca²⁺ spikes quite similar to those produced by injection of sperm extract, probably via produced InsP₃ and Ca²⁺ release from the ER. Recombinant PLCδ1 also induced Ca²⁺ oscillations, but 20-fold higher concentration was required. In the enzymatic assay, PLCζ exhibited a significant PtdInsP₂-hydrolyzing activity at [Ca²⁺] as low as 10 nM and had 70% maximal activity at 100 nM [Ca²⁺] that is usually the basal [Ca²⁺]i level of cells. On the other hand, the activity of PLCδ1 increased at [Ca²⁺] between 1 and 30 μM. EC₅₀ was 52 nM for PLCζ and 5.7 μM for PLCδ1: PLCζ has approximately 100-fold higher Ca²⁺-sensitivity than PLCδ1. Such high Ca²⁺-sensitivity of PLC activity as PLCζ that can be active in cells at the resting state is thought to be an appropriate characteristic as the sperm factor which is introduced into the egg cytoplasm upon sperm-egg fusion, first triggers Ca²⁺ release, and maintains Ca²⁺ oscillations. [Jpn J Physiol 54 Suppl:S70 (2004)]

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A transient increase of the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) (Ca²⁺-transient) in the early stage of fertilization is requisite for the activation of sea urchin eggs. We have found that immediately after insemination, cGMP begins to increase, followed by cADP-ribose and IP₃, all preceding the explosive increase of [Ca²⁺]_i (Kuroda et al., Development 128, 2001). Kuo et al. (Nature 406, 2000) have claimed that nitric oxide synthase (NOS) or nitric oxide (NO) is the primary egg activator. NO could activate the formation of cGMP/cADP-ribose, and S-nitrosylation of src kinase could accelerate the IP₃ production. On the contrary, Leckie et al. (J. Biol. Chem. 278, 2003) have argued that NO production was not the cause but the effect of [Ca²⁺]_i increase. We examined two inconsistent reports by simultaneous measurements of the NO level and [Ca²⁺]_i and those of cGMP and IP₃ contents of eggs during fertilization in the presence or absence of inhibitors against NOS and NO-dependent soluble guanylyl cyclase (sGC), and NO scavenger. We obtained the following results: (1) NOS inhibitors could not block the Ca²⁺-transients and NO production without lethal effects on sperm; (2) freshly prepared oxyhemoglobin could not block Ca²⁺-transients; (3) on the other hand, an inhibitor of NO-sensitive sGC elongated the latent period of Ca²⁺-transients, abolished the cGMP production immediately after insemination, and significantly delayed the IP₃ production. Even now the situation is confused. [Jpn J Physiol 54 Suppl:S71 (2004)]

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Adrenal chromaffin cells of adult guinea-pig rapidly secrete catecholamine in response to hypoxia, whereas those of adult rat fail to do. Thus, we explored what was responsible for this difference. Rat chromaffin cells showed a little, if any, secretory response to various mitochondrial inhibitors, whereas guinea-pig chromaffin cells responded markedly to them. The order of rank of the stimulating potency was oligomycin A<CN⁻<CCCP. This result suggests that the inhibitor-induced secretion may not be due to a change in reactive oxygen species or NADH concentrations. Alternatively, it may be ascribed to a decrease in cellular ATP contents. Thus, this possibility was examined indirectly using the Mg²⁺ indicator MgGreen. Since the majority of cellular ATP is present as MgATP, a decrease in ATP can be measured as an increase in Mg²⁺ ions. Application of CCCP resulted in about 25% and 12% increases in MgGreen fluorescence in guinea-pig and rat chromaffin cells, respectively. Similarly, the extent of CN⁻-induced increase in the fluorescence in the latter was about half of that in the former, suggesting that the extent of ATP hydrolysis in response to mitochondrial inhibitors was larger in guinea-pig than that in rat chromaffin cells. Consistent with this result, a relative expression level of ATP synthase, which may operate in the reverse mode with consumption of ATP in hypoxia, was larger in guinea-pig than that in rat adrenal medulla. The present results raise the possibility that ATP synthase in mitochondria may play an important role for hypoxia detection in adrenal chromaffin cells. [Jpn J Physiol 54 Suppl:S71 (2004)]

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A chromaffin cell model to analyze Ca²⁺ dynamics and secretory responses was constructed by assembling cell functions including nicotinic excitatory synapse, voltage-gated Na⁺, K⁺ and Ca²⁺ channels, Ca²⁺-activated K+ channel, buffered Ca²⁺ diffusion, Ca²⁺-handling by ER, Ca²⁺ extrusion, fluorescent Ca2+ indicator and exocytotic secretion. Standard values of parameters were determined so as to fit basic data reported in the literature. The model was applied to simulate experimental results obtained from chromaffin cells in the perfused rat adrenal medullary tissue. Observed profiles of Ca²⁺ responses to repetitive electric stimulation (1 to 50 Hz) of splanchnic nerve terminals were adequately simulated with minor adjustments of the standard parameters. Simultaneously observed secretory responses were also approximated by simulation, provided that the calculated responses were processed with time constants to detect catecholamine in the experiments. Furthermore, the inclusion of Ca²⁺ store that comprises InsP₃ receptor/channel and Ca²⁺ pump into the model enabled us to simulate various characteristic modes of muscarine-induced Ca²⁺ transients in rat chromaffin cells in a Ca²⁺-deficient medium. The model provides a useful tool for analyzing and predicting quantitative relations in various events occurring in stimulation-secretion coupling in chromaffin cell. [Jpn J Physiol 54 Suppl:S71 (2004)]

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Iindomethacin effects on Ca²⁺-regulated exocytosis activated by acetylcholine (ACh, 10 μM) were studied in guinea pig antral mucous cells using video optical microscopy. Indomethacin, which inhibits prostaglandin E₂ (PGE₂) synthesis, decreased the frequency of ACh-stimulated exocytotic events by 30% in a dose-dependent manner, and an inihibitor of PKA (10 μM H-89) also decreased the frequency of ACh-stimulated exocytotic events by 30%. However, the addition of PGE₂ (1 μM), which accumulates cAMP via the EP4 receptor, prevented the indomethacin-induced decrease in the frequency of ACh-stimulated exocytotic events. A similar inhibition was induced by aspirin. Thus, indomethacin decreased the frequency of ACh-stimulated exocytosis by reducing PGE₂ synthesis. SC560 (a COX1 inhibitor) decreased the frequency of ACh-stimulated exocytotic events by 30%, but NS389 (a COX2 inhibitor) did not. Moreover, indomethacin also decreased the frequency of exocytotic events stimulated by ionomycin. Thus, indomethacin inhibited COX1, which was stimulated by an increase in intracellular Ca²⁺ concentration ([ Ca²⁺]_i). In conclusion, in antral mucous cells, ACh increases [ Ca²⁺]_i, which stimulates exocytotic events and PGE₂ release via COX1. The released PGE₂ enhances the Ca²⁺-regulated exocytosis mediated by cAMP accumulation. The autocrine mechanism mediated by PGE₂, which is inhibited by indomethacin, maintains the exocytotic events in ACh-stimulated antral mucous cells. [Jpn J Physiol 54 Suppl:S71 (2004)]

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Effects of mechanical stress on integrin clusters at focal contacts (FCs) were examined in cultured human umbilical vein endothelial cells (HUVECs). HUVECs were stained with an Alexa-antibody against an extracellular domain of the β1 integrin for live imaging of β1 integrins. Localized mechanical stimulus was applied to the FCs at the basal membrane by displacing a fibronectin coated glass bead that was attached on the apical cell surface and connected to the basal FCs via stress fibres. When the bead was displaced to stretch the connected stress fibers, integrin clusters at the basal membrane started to disappear within 1 min. Many clathrin positive integrin-containing vesicles were observed in the cytosol after the integrin disappearance, suggesting facilitated endocytosis of integrins. The mechano-induced integrin endocytosis was inhibited by external Ca²⁺ depletion or PAO (100 nM, for 5 min), a blocker of tyrosine phosphatase. These results suggest that mechanical stress on FCs facilitates the clathrin-dependent endocytosis of integrins via increased intracellular Ca²⁺ probably by activated SA channels and tyrosine dephosphorylation of focal proteins. We also observed FCs disappearance when an entire HUVEC was cyclically stretched. [Jpn J Physiol 54 Suppl:S72 (2004)]

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Human albumin has one free sulfhydryl group which has been reported to be highly oxidized in various renal and hepatic dysfunctions. However, the oxidation mechanism still is not clear. The isolated rat salivary gland allow us to sample intercellular fluids, venous effluent and saliva, thus to understand the site of oxidation through capillary, across vascular wall or through paracellular route. The rat submandibular gland was isolated and perfused with the perfusate containing commercial human albumin. The collected samples were analyzed by our special HPLC system.
The findings indicated that: 1) despite the evidence that the salivary gland has no intrinsic secretion system of human albumin, small amount of the albumin could be detected in the saliva at 1 μM carbachol stimulation and its concentration was approximately 0.1-0.5% of that in the perfusate; 2) a distinguishing feature was existence of S-nitrosoalbumin fraction only in saliva, and its concentration was approximately 10-20% of that in the salivary albumin; 3) in the salivary gland, the intercellular passage tended to increase an irreversible albumin fraction which was directly oxidized by reactive oxygen species. These facts suggested that the human albumin in saliva might be passed through the paracellular pathway such as tight junction in rat submandibular gland and modification of the albumin might be occurred during the pathway. [Jpn J Physiol 54 Suppl:S72 (2004)]

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We already reported that glutamic acid was released from C6 glioma cells by the hypotonic challenge via the pathway distinct from Cl⁻ channels or glutamate transporters. Recently we found that glutamic acid was released from C6 glioma cells by the application of extracellular glucose. We measured the extracellular concentration of glutamic acid using the system we established before. The concentration of glutamic acid increased from ~4μM at the control condition in the absence of glucose to ~15μM by the application of the 5mM glucose. Application of mannitol, sucrose, lactose, or galactose did not show any increase of extracellular glutamic acid. Blockers of volume-sensitive Cl⁻ channel, including phloretin and arachidonic acid, did not block glucose-induced glutamic acid release from C6 cells. Gd³⁺, the blocker for ATP release from swollen cells, or inhibitors of glutamate transporters also failed to block glutamic acid release from C6 cells by extracellular glucose. These characteristics are quite similar with those of swelling-induced glutamic acid release. We conclude that glutamic acid is released from C6 cells by extracellular glucose possibly via the common membrane permeation pathway of swelling-induced glutamic acid release. [Jpn J Physiol 54 Suppl:S72 (2004)]

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It has been reported that ClC-2 is an apical Cl⁻ channel which is responsible for gastric acid secretion in rabbit parietal cells (Sherry et al. 2001). However, it seems to be difficult to secrete Cl⁻ via the apical ClC-2 channel, because the potential difference across the apical membrane is about 10 mV. Herein, we checked whether ClC-2 is really expressed in rabbit and rat parietal cells. Animals were humanely killed by overdose of urethane. Northern blot analysis showed that ClC-2 mRNA is highly expressed in rabbit parietal cells. However, Western blot analysis using two kinds of anti-ClC-2 antibodies (ACL-002 and CLC21-A) showed that ClC-2 protein is not significantly expressed in rabbit and rat parietal cells, while these antibodies clearly reacted with cloned ClC-2 expressed in HEK293 cells. Control antigen peptides for the antibodies almost completely abolished the single band of cloned ClC-2 (95 kDa), but the peptide did not affect any of non-specific bands of the membrane fraction (rich in gastric H⁺,K⁺-ATPase) from the parietal cells. Our present results suggest that ClC-2 may not directly contribute to gastric acid secretion. [Jpn J Physiol 54 Suppl:S72 (2004)]

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[Objective] The oral cavity is a first barrier toward the entry of bacteria and viruses into the body. In this study, we investigated the role of toll-like receptors (TLRs) in the submandibular gland (SMG) during the host defense against LPS, as the saliva is mostly secreted from the SMG. [Materials and Methods] TLR4-mutant and it's wild type mice (C3H/HeJ and C3H/HeN, respectively) were used; RT-PCR, immunohistochemistry, and Western blotting were employed to investigate the expression of inflammation cytokines. [Results and Discussion] In the SMG of C3H/HeN mice, mRNAs for TNF-α and IL-1β were strongly induced by LPS injection (i.p). Induction of these cytokines was transient, being maximum at around 6 h after injection. These inflammation cytokines were not induced in C3H/HeJ mice. Denervation of the superior cervical trunk and chorda tympani nerve did not diminish the LPS-induced elevation of IL-1β mRNA in submandibular gland, indicating the irrelevance of the central nervous system in this induction. In addition, TLR4 mRNA was shown to be strongly expressed in the SMG. These facts suggest that TLR4 is involved in the induction. The IL-1β protein was detected in the SMG of C3H/HeN mice, and it localized in the secretary granules present in the granular convoluted tubular cells. The IL-1β protein was secreted into saliva and its level was increased by LPS injection. The present study suggest that one of inflammation cytokines, IL-1β is induced and secreted into saliva in response to the inflammation happens in the local sites including the oral cavity. [Jpn J Physiol 54 Suppl:S73 (2004)]

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To investigate the effect of cell to cell interaction on depolarization-evok ed exocytosis of β-cells, we evaluated changes of capacitance of intact β-cells within the mouse islet in non-stimulatory(5mM) and stimulatory(10mM) glucose concentrations. Capacitance was measured using the software-based lock-in amplifier in standard whole cell configuration with pipette solution containing 3mM ATP, which allows maximum exocytosis and minimum metabolic effe ct by glucose in the patched cells. The value of exocytosis in 10mM glucose(21±6fF, Mean±SEM, n=7) evoked by depolarizing pulse(-70 to -0mV, 500msec) was significantly smaller than that in 5mM glucose(72±11 fF, n=13). Application of wortmannin(100nM), phosphatidylinositol 3-kinase inhibitor, reversed the suppression of exocytosis in 10mM glucose(83±21 fF, n=7). On the other hand, wortmannin did not change the value in 5mM glucose. Since phosphatidy linositol 3-kinase dependent pathway is a key signal transduction of insulin in β-cells, these results indicate that the suppression of exocytosis of β-cells in 10mM glucose may be induced by insulin locally released by autoc rine and paracrine mechanisms. [Jpn J Physiol 54 Suppl:S73 (2004)]

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A role of pertussis toxin (PTX)-sensitive pathway in regulation of glucose-stimulated Ca²⁺ signaling in rat islet β-cells was investigated by using clonidine. In isolated β-cells, clonidine reversibly reduced the levels of intracellular Ca²⁺ concentration ([Ca²⁺]_i) elevated by glucose stimulation (elevation of extracellular glucose concentration from 5.5 to 22.2 mM). This clonidine effect was antagonized by yohimbine, and abolished in β-cells pre-treated with PTX. Clonidine showed little effect on the peak magnitude of whole-cell currents through L-type Ca²⁺ channels (I_Ca(L)), but increased the inactivation process of the currents. Clonidine increased the magnitude of the voltage-dependent K⁺ currents (I_VK) in a concentration-dependent manner. These clonidine effects on I_Ca(L) and I_VK were abolished in β-cells treated with PTX. On the other hand, clonidine was ineffective to ATP-sensitive K⁺ channels. These results suggest that the PTX-sensitive pathway increases I_VK activity and decreases I_Ca(L) activity of islet β-cells, resulting in a decrease in the levels of [Ca²⁺]_i elevated by depolarization-induced Ca²⁺ entry. This mechanism seems responsible at least in part for well-known inhibitory action of PTX-sensitive pathway on glucose-stimulated insulin secretion from islet β-cells. [Jpn J Physiol 54 Suppl:S73 (2004)]

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In order to explore a role of lipid metabolism in islet β-cell functions, we examined the effect of oleic acid (OA) treatment on insulin secretion and electrical properties of islet β-cells. Islets isolated from rat pancreas were exposed with 0.1 mM OA for 3 days. The amount of protein in OA-treated islets was similar to that of OA-untreated (control) islets. On the other hand, the amount of insulin in OA-treated islets was lower than that in control islets. Insulin secretion from OA-treated islets exposed to a low (2.8 mM) glucose was slightly higher than that from control islets. On the other hand, in OA-treated islets an elevation of glucose concentration (16.7 mM) did not increase insulin secretion, whilst it significantly increased in control islets. In the low glucose condition, β-cells isolated from control islets were silent with ATP-sensitive K⁺ channels (K_ATP) being open, whereas OA-treated β-cells showed spontaneous action potential firing with K_ATP mostly closed. Under a condition of null ATP inside the membrane, K_ATP activities of excised patches from control and OA-treated β-cells were similar. ATP sensitivity to K_ATP for OA-treated cells, however, was higher than that for control cells. These results suggest that treatment of islets with OA interferes the glucose responsiveness of β-cells by evoking electrical excitation even in a low glucose condition. [Jpn J Physiol 54 Suppl:S73 (2004)]

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Insulin secretion from pancreatic β cells is modulated by numerous extracellular signaling molecules. Glucagon-like peptide 1 (GLP-1) among them is a powerful insulinotropic peptide. Upon binding of its receptor, GLP-I increases cyclic adenosine monophosphate (cAMP) levels via a G-protein-coupled activation of adenylate cyclase (AC), leading to activation of protein kinase A (PKA). GLP-1 through cAMP-PKA signaling pathway modulates Ca²⁺ signals. Our recent work has demonstrated that depolarization-evoked Ca²⁺ influx via voltage dependent Ca²⁺ channels in insulin secreting cells (INS-1 cells) induces activation of conventional PKC (cPKC) as well as structurally Ca²⁺-independent novel PKC (nPKC). We investigated whether GLP-1 as a Ca²⁺ modulator can activate PKCα and PKCε as representatives of cPKC and nPKC in INS-1 cells. GLP-1 and 8-Br-cAMP (Br) evoked-Ca²⁺ signal induced GFP-tagged PKCα and PKCε. This result indicated that GLP-1-evoked Ca²⁺ signals can activate PKCs. We verified this by showing in three deferent ways: firstly, GLP-1-induced translocation of GFP-tagged myristoylated alanine-rich C kinase substrate, secondly, GLP-1-induced translocation of GFP-tagged C1 domain of PKCγ as a diacylglycerol (DAG) sensor, thirdly GLP-1-induced translocation of GFP-tagged pleckstrin homology domain as a marker of phospholipase C (PLC) activation. Thus, we concluded from these observations that GLP-1 -evoked Ca²⁺ signal generates DAG through PLC activation thereby activating both PKCα and PKCε. [Jpn J Physiol 54 Suppl:S74 (2004)]

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Ghrelin is a new peptide, purified from the stomach as an endogenous ligand for growth-hormone secretagogue receptor (GHS-R). Ghrelin stimulates growth-hormone release and food intake and is thought to play a central role in the energy metabolism. Ghrelin and GHS-R are present in the pancreatic islets. This study was aimed at examining the effects and action mechanisms of ghrelin on Ca²⁺ signaling and insulin release in islet β-cells. Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was measured in rat β-cells with fura-2 microfluorometry. Single β-cells were isolated from the rat pancreas and incubated with 1 mM fura-2/AM for 30 min at 37°C in KRB containing 2.8 mM glucose. Fluorescent images due to 340 and 380 nm were detected every 5 sec, and the ratio image was produced by an Argus-50 system. Ratio value was converted to [Ca²⁺]_i with calibration curves. Insulin release experiments were performed in islets under static incubation conditions and insulin was determined using EIA kit. Ghrelin at 10⁻⁸ M suppressed increases in [Ca²⁺]_i induced by 8.3 mM glucose and those by pituitary adenylate cyclase-activating polypeptide (PACAP) (10⁻⁹ M) at 8.3 mM glucose in single β-cells. Ghrelin also suppressed the [Ca²⁺]_i increases by forskolin, a cAMP increasing agent, but not those by 10⁻⁵ M Ach in single β-cells. Insulin release in response to 8.3 mM glucose was inhibited by ghrelin. The results indicate that ghrelin counteracts Ca²⁺ signaling evoked by glucose and PACAP-cAMP pathway in islet β-cells, which is linked to inhibition of insulin release. [Jpn J Physiol 54 Suppl:S74 (2004)]

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Ghrelin, isolated from the human and rat stomach, is the endogenous ligand for the growth hormone (GH) secretagogue-receptor (GHS-R). Both ghrelin and GHS-R are also present in the pancreas. This present study explored a possible physiological function of ghrelin in the regulation of insulin and glucose metabolism. Systemic administration of ghrelin increased blood glucose concentrations in a GH-independent manner in fasted mice. In the glucose tolerance test (GTT), ghrelin increased glucose levels further and decreased insulin levels. Conversely, administration of GHS-R antagonists lowered fasting glucose concentrations, markedly attenuated increases in glucose levels and enhanced increases in insulin levels during GTT. In isolated rat pancreatic islets, glucose-induced insulin release was reduced by ghrelin, while it was enhanced by GHS-R antagonists and anti-ghrelin antiserum. Cytosolic Ca²⁺ responses to glucose were enhanced by GHS-R antagonist in islets and suppressed by ghrelin in single β-cells. These findings suggest that endogenous ghrelin in islets reacts with GHS-Rs on β-cells and inhibits glucose-induced insulin release, thereby upwardly regulating blood glucose levels both in the basal state and upon glucose challenge. [Jpn J Physiol 54 Suppl:S74 (2004)]

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The carcinoma cells in tissues break through the vessel wall into the blood stream and conversely pass through the vessel wall into distant tissues or organs. Thus, the cell-to-cell interaction between VSMCs and tumor cells is critical to the pathophysiological process of angiogenesis, vascular remodeling and metastasis. In this study, the growth rate of vascular smooth muscle cells (VSMCs), which were derived from aorta of mice deficient in the fibrinolytic factors; tissue-type plasminogen activator (t-PA^−/-), urokinase (u-PA^−/-), u-PA receptor (u-PAR^−/-) and type 1 plasminogen activator inhibitor (PAI-1^−/-), as well as wild-type (WT) mice, was investigated in the presence of mouse melanoma cells (B16). In the VSMCs cultured with a basal medium, there was no difference in the growth rate among the gene-lacking VSMCs and WT VSMCs. On the other hand, when these VSMCs were cultured with B16 cells in either the mixed culture or a double-chamber, only u-PAR^−/-VSMCs showed a significantly lower growth rate. In addition, these suppressive effects on u-PAR^−/-VSMCs were also observed in the presence of B16-derived conditioned medium (B16/CM). These effects on growth activity were partially associated with the levels of tyrosine phosphorylation of 77-kDa protein and mitogen-activated protein kinase (MAPK, p42/p44) activity. The findings suggest that u-PAR plays an important role in the proliferative response of VSMCs and that without u-PAR, there is no intracellular signaling for cell proliferation. [Jpn J Physiol 54 Suppl:S74 (2004)]

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The immunosuppressant drug FK506 is knowm to modulate the ryanodine receptor (RyR) by dissociating FK506-binding protein (FKBP) from the RyR-FKBP complex in skeletal muscle. Cyclic ADP-ribose (cADPR) is also known to dissociate FKBP12.6, an isoform of FKBP, from cardiac-type RyRs. In the present study, the effects of FK506 and cADPR on Ca²⁺ release through FKBP-stripped RyRs were investigated in rat pancreatic acinar cells. After a steady state of ATP-dependent ⁴⁵Ca²⁺ uptake into the microsomal vesicles had been reached, cADPR- or FK506-induced ⁴⁵Ca²⁺ release from the vesicles was measured. Pretreatment of the vesicles with FK506 (3 μM) significantly increased cADPR (0.5-10 μM)-induced ⁴⁵Ca²⁺ release from the vesicles. The cADPR-induced ⁴⁵Ca²⁺ release in the presence of FK506 may not be due to the dissociation of FKBP, since it is thought that FKBP had been removed from the RyR by FK506 before the addition of cADPR. Pretreatment with cADPR (0.5 μM) significantly increased FK506 (0.1-10 μM)-induced ⁴⁵Ca²⁺ release from the vesicles. If the FKBP had been removed from the RyR by cADPR, the FK506-induced ⁴⁵Ca²⁺ release in the presence of cADPR may not be due to the dissociation of FKBP. The results suggest that an FKBP-independent mechanism is involved in FK506- or cADPR-induced Ca²⁺ release through RyRs in rat pancreatic acinar cells. [Jpn J Physiol 54 Suppl:S75 (2004)]

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Cardiac ATP-sensitive K⁺ (K_ATP) channels are composed of a K⁺ channel subunit, Kir6.2 and sulfonylurea receptor (SUR) 2A, whereas vascular nucleotide-dependent K⁺ channels are formed from Kir6.1 and SUR2B. SUR is an ABC protein possessing two cytoplasmic nucleotide-binding domains (NBD1 and NBD2). Intracellular ATP and ADP inhibit K_ATP channels by binding to Kir6.2 while activating them through NBDs. It was reported that the E171Q mutant of MJ0796, a prokaryotic ABC protein which is entirely an NBD, forms an ATP sandwich dimer in the presence of Mg-free, Na-bound ATP. SUR2A and SUR2B bearing the corresponding mutation in both NBDs (SUR2A(D834N, E1471Q) and SUR2B(D834N, E1471Q)) were coexpressed with Kir6.2 in HEK293T cells. Both SUR2A(D834N, E1471Q)/Kir6.2 and SUR2B(D834N, E1471Q)/Kir6.2 channels were spontaneously activated by intracellular Na- but not K- or Mg-ATP as measured with the inside-out configuration of the patch-clamp method. This reaction was never observed with wild-type SUR2A/Kir6.2 and SUR2B/Kir6.2 channels. Na-ATP increased the mutant channel activity up to ~40% of the maximum in a concentration-dependent manner. Nicorandil, a K_ATP channel opener, increased the activity of the mutant than the wild-type K_ATPchannels with higher potency in the presence Na-ATP. Therefore, dimerization of NBD1 and NBD2 seems to be sufficient to induce opening of the SUR2A- or SUR2B-containing K_ATP channels. [Jpn J Physiol 54 Suppl:S75 (2004)]

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In brown adipocytes, the β-action of noradrenaline is known to cause thermogenesis, which seems to be facilitated by the action of thyroid hormone. On the other hand, the α-action of noradrenaline causes a large increase in Ca²⁺ via intracellular Ca²⁺ release and subsequent store-operated Ca²⁺ entry, albeit its physiological function is unknown. We have studied by fluorometric Ca²⁺-imaging techniques how thyroxin modifies the actions of noradrenaline to increase intracellular Ca²⁺ in cultured rat brown adipocytes. Noradrenaline (1-10 μM) caused initially sharp and then plateaued rises in Ca²⁺, which was blocked by phentorlamine (10 μM: α-antagonist), but not by propranolol (1 μM: β-antagonist). Phenylephrine (10 μM: α-agonist) caused a rise in Ca²⁺ similar to that by noradrenaline. When rat adipocytes were pretereated with thyroxin (1-4 μM) for more than 4 min, which produced no or slight decrease in Ca²⁺, noradrenaline induced a transient rise in Ca²⁺ in short duration, which was followed by a marked, sustained reduction in Ca²⁺. This decrease in Ca²⁺ was blocked by propranolol, but not by phentolamine. Isoprenaline (1 μM: β-agonist) produced a similar decrease in Ca²⁺ under this condition, but not if no pretreatment with thyroxin was made. These results suggest that thyroxin primes the mechanism of the β-action of noradrenaline to decrease intracellular Ca²⁺ in brown adipocytes presumably via the enhancement of Ca²⁺ uptake and/or extrusion, overcoming the α-action to raise Ca²⁺. [Jpn J Physiol 54 Suppl:S75 (2004)]

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The subcellular localization and adjacent pH of the α₁-adrenoceptor (α₁-AR) subtypes conjugated with pH sensor GFP (α₁-AR/pH) were assessed using real-time imaging of living, stably expressed CHO-K1 cells. The α_1B-AR/pH fluorescence was detected predominantly on the cell surface and intracellular pH distribution showed homogeneous pattern. Stimulation of the α_1B-AR with norepinephrine (NE, 10⁻⁷M) led to an increase in pH, promoted rapid internalization and caused Ca²⁺ oscillations. α_1A-AR/pH fluorescence was detected not only on the cell surface but also inside the cell and this localization was unaffected by exposure to NE during observation period. Intracellular pH on the cell surface was higher than intracellular compartment before NE. Stimulation of the α_1A-AR with NE led to a decrease in pH and caused only a Ca²⁺ transient. The α_1D-AR/pH fluorescence was detected mainly intracellularly, and this localization was unaffected by exposure to NE. Intracellular pH distribution of α_1D-AR/pH was homogeneous as in α_1B-AR/pH. Stimulation of the α_1D-AR/pH with NE led to an increase in pH and caused Ca²⁺ oscillations. These data show that transfected α₁-AR/pH fusion proteins are functional and that there are differences in the cellular distribution of α₁-AR, agonist-mediated internalization, Ca²⁺ response and neighbor pH among α₁-AR subtypes. These differences could contribute to the diversity in physiologic responses regulated by the α₁-ARs. [Jpn J Physiol 54 Suppl:S75 (2004)]

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In our previous study [M. Fujinoki et al. (2001) Biomed. Res. 22, 45-58], we reported that 66k-Da protein, 58k-Da protein, two types of 36k-Da protein, which were designated as 36K-A protein and 36K-B protein, obtained from hamster sperm flagella were phosphorylated at serine residues associated with motility. In the present experiments, we identified and characterized these four proteins. Two 36k-Da proteins were phosphorylated in a cAMP dependent manner. In order to identify those four phosphoproteins, they were analyzed by peptide mass finger printing and amino acid sequencing. From the results, the 66k-Da protein was identified as beta tubulin. Protein of 58kDa was identified as ATP synthase F1 beta. Both 36k-Da protein were identified as pyruvate dehydrogenase E1 beta lacked N-terminal 30 amino acids. Moreover, we examined localization of 58k-Da protein and two 36k-Da proteins. The 58k-Da protein was assumed to be existed in the equatorial segment of acrosomal region of sperm head and the middle piece and the principal piece of sperm flagellum. The 36K-A protein was localized in the principal piece. On the other hand, the 36K-B protein was localized in the middle piece. [Jpn J Physiol 54 Suppl:S76 (2004)]

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We studied the molecular mechanism of cyclic stretch dependent stress fiber disassembly using a semi-intact cell system. Human umbilical vein endothelial cells were cultured on an elastic substratum, permeabilized by digitonin, termed semi-intact cells here, and mechanically stretched (20%, 1 Hz) in standard buffer or in brain extract solution. Stress fibers disassembled when the cells were cyclically stretched in the presence of brain extract. The disassembly was also observed when tension in stress fibers was decreased with compression (30%) of the substratum, implying that loss of mechanical tension caused stress fiber disassembly in a cytoplasmic factor dependent way. We examined two candidate molecules for the stress fiber disassembly: cofilin and gelsolin, actin severing proteins. Purified cofilin (2μg/ml) disassembled stress fibers in semi-intact cells during cyclic stretch, but gelsolin (2μg/ml) did not even in the presence of 1 mM Ca²⁺. Cofilin alone disassembled stress fiber without cyclic stretch when actomyosin ATPase was inhibited or ATP was removed from the buffer; these treatments decline the tension in stress fibers. By contrast continuous stretch of the cell inhibited the cofilin dependent stress fiber disassembly with or without ATP. These results indicate that mechanical tension in actin cytoskeleton prevents actin cytoskeleton from ADF/cofilin dependent disassembly. [Jpn J Physiol 54 Suppl:S76 (2004)]

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Subepithelial fibroblasts form a cellular network with gap junctions under the epithelium of gastrointestinal tract. The network keeps close contact with epithelium, capillaries and nerve terminals, and may play important roles in various signaling in intestinal villi. We found that they change cell-shape, depending on intracellular cAMP level and several bioactive substances such as endothelins (ETs), and that they release ATP by mechanical stress and transmit Ca²⁺ signal via P2Y activation in surrounding cells. These dynamical changes of their property may control mechanical and sieve properties of the network. Here, we have confirmed the sub-types of ET receptors using wild-phenotype and sl/sl rats, a deletion mutant of ET_B gene, and also sub-types of ATP receptors. We have also clarified the cell-shape dependence of intercellular communication via mechanically released ATP and gap junctions. It is known that nerve terminals of mucosal sensory neurons distributes under the epithelium of villi and possess P2X receptors. The source of ATP was thought to be a release from enterochromaffin cells. However, the characteristics of subepithelial fibroblasts we made clear suggests that ATP is released from subepithelial fibroblasts by mechnical deformation of the villi. So, we propose that subepithelial fibroblasts work as a mechano-sensor in the intestine. A finding that neurons (NG108-15) co-cultured with subepithelial fibroblasts were activated by mechanically induced Ca²⁺-waves on subepithelial fibroblasts supports the idea. [Jpn J Physiol 54 Suppl:S76 (2004)]

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Bronchiolar ciliary cells were isolated from rat lungs. The β2-adrenergic regulation of ciliary beat frequency (CBF) was studied using video-optical microscopy. Terbutaline (an β2-agonist) increased CBF, and it also decreased the volume of the ciliary cells. These terbutaline actions were mediated by cAMP. Amiloride (1 μM) and bumetanide (20 μM) potentiated cell shrinkage and the CBF increase, and they shifted the terbutaline dose-response curve to the lower-concentration side. Quinidine (500 μM), in contrast, increased cell volume and suppressed the CBF increase. Moreover, a KCl solution containing amiloride (1 μM) and strophanthidin (100 μM) increased cell volume and suppressed the CBF increase, and then the subsequent removal of either amiloride or strophanthidin decreased cell volume and further increased CBF. NPPB (10 μM) or glybenclamide (200 μM) had no effects on the terbutaline actions. Thus, in terbutaline-stimulated ciliary cells, cell shrinkage enhances the CBF increase, in contrast, cell swelling suppresses it. However, hypo-osmotic cell swelling enhanced the CBF increase, while isosmotic swelling suppressed it. These suggest that isosmotic and non-isosmotic volume changes of terbutaline-stimulated bronchiolar ciliary cells may trigger different signaling pathways. In conclusion, terbutaline increases CBF and decreases the volume of the rat bronchiolar ciliary cells via cAMP accumulation under an isosmotic condition, and the isosmotic cell shrinkage enhances the CBF increase by increasing cAMP sensitivity. [Jpn J Physiol 54 Suppl:S76 (2004)]

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Cell volume regulation is a fundamental function for living cells. In hypertonic situations, the intracellular osmolality is increased by accumulation of osmolytes and water is driven into the cell for recovery of original cell volume. In some cases, cells with chronic exposure to hypertonic media ultimately swell over their initial volume (rebound cell swelling: RCS). The RCS was considered to cause edema, however mechanisms of this phenomenon were not well known. In the present study, to elucidate molecular mechanisms of RCS, we observed cell volume and morphological change of C6 glia cells incubated in hypertonic media. Cells were cultured up to 50% confluence in normal media and exposed to the hypertonic media for 48 h, which were made by adding sucrose and urea directly to normal media. When cells were acclimated to the hypertonic sucrose and urea media, the cell volume exceeded its initial volume. The RCS in the sucrose medium was accompanied by intracellular vacuolation, whereas cells in the urea medium did not form vacuoles in the cytoplasm. In hypertonic sucrose media, cells had a rounded shape, while cells in the urea medium exhibited a flatter shape and an irregular surface with flutter edge. These results suggest that a feature of RCS is dependent on kinds of osmolytes, and that a disruption of cellular metabolism and formation of cytoskeletons are caused by extracellular hyperosmolality. Supported by JSPS 15659052 (YM), 15590189 (NN), 15790120 (HM), MHLW 15A-1. [Jpn J Physiol 54 Suppl:S77 (2004)]

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Sphingosine-1-phosphate (S1P) is a bioactive lysophospholipid that induces a variety of biological responses in diverse cell types. Many of these responses are mediated by S1P receptors, S1P₁/EDG1, S1P₃/EDG3, and S1P₂/EDG5. We previously showed that S1P₂/EDG5, via G₁₂ and G₁₃ proteins, mediated inhibiton of insulin-like growth factor-I (IGF-I)-induced Rac activation and cell migration, whereas S1P₁/EDG1 and S1P₃/EDG3 each alone, via G_i protein, mediated Rac activation and stimulation of cell migration. However, it is not known how G₁₂and G₁₃ proteins mediate inhibition of Rac and cell migration in S1P₂/EDG5 signaling. We found that the pretreatment with C3 toxin, which inactivates Rho, and the expresson of dominat negative Rho A prevented S1P₂/EDG5-mediated inhibition of Rac and cell migration. On other hand, the expression of constitutively active Rho inhibited IGF-I induced Rac activation and cell migration. However, Rho kinase inhibitors did not affect S1P₂/EDG5-mediated inhibition of Rac or cell migration. These results indicate that Rho, but not Rho kinase, mediates inhibition of Rac activity and cell mobility on stimulation of S1P₂/EDG5. [Jpn J Physiol 54 Suppl:S77 (2004)]

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The lysophospholipid mediator sphingosine-1-phosphate (S1P) exerts pleiotropic actions on diverse cell types through Edg family G protein-coupled receptors. Among them, of particular interest is an inhibitory action of S1P on cell migration. We have previously shown that S1P induces activation of Rho via Edg-5, which in turn mediates inhibition of PDGF-induced Rac activation and chemotaxis in VSMCs. In non-muscle cells, it was previously demonstrated that G_αq as well as G_α12 and G_α13 mediate Rho activation. However, it is not fully understood in VSMCs which heterotrimeric G protein mediates Rho activation and how the G protein regulates the activity of Rho. The C-terminal peptides of G_α subunits (G_α-CT) act as specific inhibitors for respective G proteins. Adenovirus-mediated expression of either G_αq-CT, G_α12-CT, or G_α13-CT, but not G_αs-CT, abolished S1P stimulation of RhoA and S1P inhibition of PDGF-induced Rac activation and chemotaxis. Interestingly, the PLC inhibitor U-73122 and the Ca²⁺-chelator BAPTA-AM did not affect S1P-induced RhoA activation. These results suggest in VSMCs that both G_αq and G_α12/13 mediate Rho activation, which in turn mediates inhibition of cellular Rac and chemotaxis, and that the G_αq-mediated Rho activation is independent of calcium mobilization. [Jpn J Physiol 54 Suppl:S77 (2004)]

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Sphingomyelinase (SMase) is a key enzyme which produces lipid mediator, ceramide. SMase induced endothelium-dependent vasorelaxation, which was inhibited not only by LNMMA but also by high K⁺-depolarization. Fluorometric measurement of NO and [Ca²⁺]i revealed that SMase produces NO without [Ca²⁺]i elevation in endothelial cells in situ, in which only eNOS, but not iNOS, was expressed. Confocal images of immunostaining showed SMase induced dissociation of eNOS from plasma membrane caveolae and translocation to cytosol in endothelial cells in situ. Both microelectrical and fluorometric measurements of the membrane potential demonstrated that SMase induced membrane hyperpolarization in smooth muscle cells in situ of arterial strips with endothelium, but not in smooth muscle cells without endothelium and endothelial cells per se. SMase and bradykinin increased ceramide level of endothelial cells in situ. These results suggest that SMase induces endothelium-dependent vasorelaxation, as mediated by both endothelial NO production and EDH of smooth muscle, and is accompanied with ceramide production but not [Ca²⁺]i elevation. [Jpn J Physiol 54 Suppl:S77 (2004)]

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Using video-enhanced microscopy, we found that 4-aminopyridine (4-AP), known as a membrane permeable blocker of K⁺ channels, induced Brownian movement of organelles within 5 min in cultured dorsal root ganglion (DRG) neurons and non-neuronal cells. The Brownian movement gradually became rapid, and then organelles excavated the surrounding cytoplasm and formed vacuoles. Intracellular actin filaments were pushed away from the vacuoles but microtubules were not significantly affected. These phenomena were reversed by washout after 1-hour treatment of 4-AP, but longer treatment (4-24 hours) resulted in cell death. Other aminopyridines, 2-AP, 3-AP and 3,4-diamynopyridine, also evoked Brownian movement of organelles and formed vacuoles, but other K⁺ channel blockers tetraethylammonium (TEA), Cs⁺ and apamin had no effect. The observed effects of 4-AP were prevented by elimination of intracellular Cl⁻ from extracellular medium and by the vacuolar H⁺ ATPase inhibitor bafilomycin A1. These results suggest that intracellular 4-AP and Cl⁻ may be coupled to activation of vacuolar H⁺ ATPase to cause Brownian movement of organelles. Aminopyridines therefore possess a unique effect on organelle movement and cytoplasm, which are associated with changes in the cytoskeleton organization and cell death. [Jpn J Physiol 54 Suppl:S78 (2004)]

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Microglia are resident macrophages in the central nervous system. In response to injuries or inflammation, microglia are stimulated to exhibit drastic changes in morphology and functions, and called as "activated microglia". Activated microglia proliferate vigorously, migrate and accumulate to the site of the inflammation, and phagocytose pathogens and cellular debris. Further, activate microglia produce various bioactive molecules to assist repair and regeneration of the nervous system. In this report, to elucidate molecular mechanisms of microglia activation, we focused on motility and phagocytosis of microglia. Motility and phagocytosis are underlain by dynamic remodeling of the action cytoskeleton. Microglia exhibit extremely active motility and phagocytosis, suggesting existence of microglia-specific regulatory mechanisms. We therefore analyzed signaling molecules involved in regulation of the actin cytoskeleton of activated microglia. Iba1 is a small calcium-binding protein expressed specifically in microglia. We previously reported that expression of Iba1 is upregulated during microglia activation, and that Iba1 is involved in motility and phagocytosis of microglia. Furthermore, Iba1 was shown to be cooperating with a small G protein, Rac, in regulation of the actin cytoskeleton. Herein, we demonstrated intracellular behavior of these signaling molecules by using visualizing system such as green fluorescent protein (GFP). [Jpn J Physiol 54 Suppl:S78 (2004)]

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The docking/fusion of transport vesicles mediated by the SNARE proteins is thought to be regulated by Sec1/Munc-18 (SM) proteins, but their precise roles are still unclear. There are 6 SM protein encoding genes on the C. elegans genome, however, no systematic functional studies have yet been performed.
In this work, we focused on the Ce-vps45 whose orthologous protein in yeast plays an important role in vacuolar protein sorting. The putative null mutation, tm246, showed a maternal-effect ts larval lethal phenotype. The ts lethal phenotype of tm246 mutant was rescued when Ce-vps45 cDNA was speficically expressed in the intestine. This result suggest that Ce-vps45 gene products may function in membrane trafficking from Golgi's apparatus to lysosome in mainly intestinal cells where lysosome is highly developed. To visualize specific membrane trafficking pathway, transgenic analyses using fluorescent reporters in the mutant background are in progress.
In addition to disfunction of intracellular membrane trafficking, the mutant tm246 shows abnormal endocytotic uptake of proteins from pseudocoelom to coelomocytes. Both RME (receptor mediated endocytosis) and non-RME appeared to be impaired. We infer that the CE-VPS45 protein is necessary for both biosynthetic and endocytic membrane trafficking pathways. [Jpn J Physiol 54 Suppl:S78 (2004)]

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Acinar cells of salivary parotid gland produce saliva by two types of secretion; water fluid and exocytosis of proteins like amylase. Exocytosis of amylase is mainly regulated by intracellular cAMP, and VAMP2, one of the SNARE proteins, is essential for cAMP-dependent amylase secretion. However, it is difficult to investigate the molecular mechanism of parotid acinar cells since there is no stable cell line that has the ability to release amylase upon stimulation. To examine the functions of parotid acinar cells, we tried to establish a transfection system of exogenous genes to primary culture. Acinar cells were dispersed from rat parotid glands by enzyme digestion, and cultured in Waymouth's medium that contained rat serum. Cells that cultured in dishes coated with various extracellular matrix kept amylase activity, and have the ability to release amylase in response to isoproterenol at least for 48 h. The cells spread on the dish bases and formed filopodia although they have granules that contain amylase, suggesting they are derived from acinar cells. VAMP2 gene fused with enhanced green fluorescence protein (EGFP-VAMP2) was transfected to dispersed acinar cells and cultured for 48 h. EGFP-VAMP2 protein was observed to be correctly localized at granules that contained amylase. Therefore, the cells cultured by this method can be used to examine the effects of exogenous genes on functions of parotid acinar cells like regulated exocytosis and maturation of secretory granules. [Jpn J Physiol 54 Suppl:S78 (2004)]

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Viral vectors have been mainly used for the various studies of gene and cell therapies because of their inherent property of high transfection. However, the clinical applications of viral vectors are limited by their cytotoxicity and immunogenicity. Hemagglutinating virus of Japan envelope (HVJ-E) vector we used in this study is a novel non-viral vector which is inactivated and lost the ability to replicate.
We demonstrated the transduction of β-galactosidase (β-gal) as a delivery protein incorporated with HVJ-E into the rat brainstem in vivo. We microinjected HVJ-E/β-gal (n=16)(100nl/site) and control HVJ-E vector (n=13) into the nucleus tractus solitarius of anesthertized rats. The rats were fixed by intracardic perfusion with 2% paraformaldehyde-0.2% glutaraldehyde in phosphate-buffered saline (pH7.4) at 3, 6, 12, 24 hours after microinjection. Brainstems were removed and cryostat sections(40μm) were obtained from the region of the injection and assayed by X-gal staining. Sections from HVJ-E/β-gal-injected rats showed β-gal activities in all cases. Sections from HVJ-E-injected rats showed no staining with X-gal. Histological stainings were assessed by measuring the colored strength in a defined area using the Mac Scope software. It is suggested that the delivery of proteins using HVJ-E vector may introduce a new system of transduction to study a therapeutic neurobiology. [Jpn J Physiol 54 Suppl:S79 (2004)]

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The catalytic subunit of Na⁺, K⁺-ATPase shows at least four isoforms, α1, α2, α3 and α4, each derived from a distinct gene. Here, we investigated expression levels of mRNA and protein of Na⁺, K⁺-ATPase α-isoforms in the human colorectal cancer mucosa and the normal mucosa. The specimens were obtained from surgical resection of patients in accordance with the recommendations of the Declaration of Helsinki. Informed consents were obtained from the patients at Toyama Medical and Pharmaceutical University Hospital. First, we examined the protein expression level of α1-isoform by using specific antibodies. Interestingly, decrease in the expression of α1-isoform was observed in all the cancer mucosae compared with the normal mucosae. But ouabain (5 μM)-sensitive Na⁺, K⁺-ATPase activities in both cancer and normal musosae were not significantly different. Real-time PCR (TaqMan) assay showed that the mRNA expression of α2- and α4-isoforms decreased in almost all the cancer mucosae. Interestingly, the expression level of the α3-isoform in the cancer mucosa was higher than that of the normal mucosa. These results suggest that decrease in protein expression of α1-isoform and increase in that of α3-isoform may be associated with colorectal cancers. [Jpn J Physiol 54 Suppl:S79 (2004)]

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Allograft (e.g., skin and Meth A tumor)-induced macrophage (AIM) exhibits a unique (cell-to-cell contact-dependent and TNF-α- and NO-independent) cytotoxic activity against the allograft. In the last meeting, we reported that two cDNA clones (#15-4-4 and #12-4-5) were isolated by the expression cloning method using neutralizing monoclonal antibodies to AIM cytolysis, that the #15-4-4 cDNA (261bp fragment) had a region homologous to potential phospholipid-transporting ATPase IIb, and that the protein encoded by #12-4-5 cDNA (551bp fragment) was homologous to a functionally as yet unknown protein. Here, we partially characterized the nature of protein encoded by #12-4-5 cDNA. Although RT-PCR showed that the cDNA was expressed strongly in AIM and weakly in Mac-1⁺ cells (peripheral blood) and F4/80⁺ cells (transplantation site) with a size of 2.3kb, Northern blot analyses revealed the AIM-specific expression of #12-4-5 cDNA with smaller sizes of alternatively spliced valiants. Of particular interest, the protein expressed as a capsid fusion protein on T7 phages did bind to H-2K^d (a mouse MHC of allograft) tetramer, suggesting that the protein encoded by #12-4-5 cDNA might be a recognition molecule for allogeneic MHC. [Jpn J Physiol 54 Suppl:S79 (2004)]

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To evaluate the relationship between IgE production of IgE-producing cells and expression of EBV latent infection membrane protein 1 (LMP1), one of the proteins encoded by the virus in latent infection, IgE mRNA levels were examined upon transient transfection of LMP 1 in the spleen cells derived from Brown-Norway (BN) rats with or without immunization with DNP-Ascaris suum (As), as well as in the rat IgE-producing hybridoma cells FE-3. The spleen cells were collected from spleens of Brown-Norway rats 7 days after immunization with DNP-As. IgE mRNA expression was examined using RT-PCR or Northern blot analysis. Transfection of LMP1 expression vector, pSG5-LMP1, into cells was performed by lipofection method. In the non-immunized BN rats, IgE mRNA expressions were not altered by IL-4 treatment in the spleen cells transfected with pSG5 control vector as well as in those with pSG5-LMP1. IgE mRNA levels were considerably higher in the spleen cells transfected with pSG5-LMP1 than in those with control vector. In contrast, IgE mRNA levels were reduced upon transfection with pSG5-LMP1 in the spleen cells derived from Brown-Norway rats immunized with DNP-As. IgE mRNA levels were not affected by transfection with pSG5-LMP1 in the FE-3 cells. These results suggest that IgE mRNA expressions in IgE-producing cells are modified by LMP1 gene expression and that this modification leading to changes in expression level of IgE mRNA might be dependent on the differentiation stage of immunocompetent cells upon exposure to allergen. [Jpn J Physiol 54 Suppl:S79 (2004)]

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We have investigated a role of SMemb gene expression in the phenotype conversion of vascular smooth muscle (VSM) cells. In the present study, we employed SMemb specific short interfering RNAs (siRNAs) to inhibit SMemb mRNA expression. We designed three different siRNAs targeting SMemb mRNA. The two siRNAs were designed to target open reading frame (named as ORF-1 and ORF-2), and the one siRNA was designed to target 3' untranslated region (named as 3' UTR). The siRNAs at different concentrations (1, 10 and 100 nM) were transfected into rabbit cultured VSM cells by the lipofection method. After 6 hours transfection, 10% fetal bovine serum (FBS)-culture medium was added and incubated for another 42 hours. We extracted total RNA to determine the level of SMemb mRNA expression by RT-PCR. Consequently, the 3' UTR and ORF-2 siRNAs dose-dependently decreased the SMemb mRNA expression. The SMemb protein expression in the cells was evaluated by immunohistochemistry. The number of FITC-positive cells in siRNA transfected group was less than that in the control group. We also extracted crude myosin extract to determine the level of SMemb protein expression by dot blot analysis. All the SMemb protein expressions were inhibited to about half by 100 nM of the three siRNAs. Other myosin heavy chain (MHC) isoform mRNA expressions such as SM1 and SM2 were not changed by treatment with the three siRNAs. Thus, the three different siRNAs specific for SMemb could inhibit SMemb mRNA and protein expressions. [Jpn J Physiol 54 Suppl:S80 (2004)]

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