We have previously shown that presenilin 1 and 2 (PS1 and PS2) bind to syntaxin 5 (Syx5). In this study, we examined the interaction of PSs with Syx isoforms, the effects of Syx overexpression on β-amyloid precursor protein (βAPP), and the Aβ peptide production in NG108-15 cells. The data showed that Syx5 which localizes from the endoplasmic reticulum to the Golgi binds to PS holoproteins, while other Syxs, so far studied did not. Among familial Alzheimer's disease (FAD)-linked PS mutants, PS1deltaE9, which lacks the region containing the endoproteolytic cleavage site, showed markedly decreased binding to Syx5. The interaction domains in Syx5 were mapped to the transmembrane region and to the cytoplasmic region containing the α-helical domains, which are distinct from the H3 (SNARE motif). Furthermore, among the Syxs examined, the overexpression of Syx5 resulted in the accumulation of βAPP and reduced the secretion of Aβ peptides (Aβ40 and Aβ42) in NG108-15 cells. Taken together, these results indicate that Syx5 may play a specific role in the modulation of processing and/or trafficking of FAD-related proteins in neuronal cells by interaction with PS holoproteins in the early secretory compartment. [Jpn J Physiol 55 Suppl:S83 (2005)]

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The A subclass of the ATP-binding cassette (ABC) transporter superfamily possesses a structural feature that distinguishes it from other ABC transporters, and is proposed to be involved in transmembrane transport of endogenous lipids. We previously cloned the full-length cDNA of mouse ABCA17, a novel ABC transporter belonging to the A subclass, and found that the protein is localized in the anterior head of sperm and elongated spermatids. However, the functional significance of ABCA17 is unknown. To determine if ABCA17 is involved in regulation of lipid metabolism, we performed metabolic labeling analysis in HEK293 cells using [C¹⁴]-labeled acetate. When expressed in HEK293 cells, ABCA17 protein was localized to the ER. Analysis revealed that intracellular esterified lipids including cholesteryl esters, fatty acid esters, and triacylglycerols were significantly decreased in HEK293 cells stably expressing ABCA17 compared to those in untransfected cells. These results suggest that ABCA17 may be involved in sorting esterified neutral lipids from the ER to other intracellular compartments. Since a decrease in cholesterol is critical during capacitation, ABCA17 may well play an important role in sperm capacitation. [Jpn J Physiol 55 Suppl:S83 (2005)]

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Organic Anion Transporter 4 (OAT4) is expressed in renal proximal brush border and mediates the transports of sulfate conjugates. The C-terminal domain of OAT4 is exposed to the cytoplasmic compartment and contains PDZ motif, one of the famous protein-protein interaction modules, suggesting that it may interact with PDZ proteins. In the present study, using the C-terminus of OAT4 as bait, we performed yeast two-hybrid assay against prey vectors containing PDZ domain containing proteins such as PDZK1, NHERF1, and IKEPP. We found that OAT4 C-terminus interacted with two PDZ proteins, PDZK1 and NHERF1. Such an interaction requires the PDZ motif of OAT4 in its extreme intracellular C-terminal region as identified by both yeast two-hybrid and in vitro binding assays. In addition, the first and fourth PDZ domains of PDZK1 and the first domain of NHERF1 associate with the OAT4 C-terminus. Immunohistochemical study revealed that OAT4 and two PDZ proteins are expressed at the apical membrane of renal proximal tubules. The association of OAT4 with PDZK1 or NHERF1 enhanced estrone sulfate transport activities in HEK293 cells (1.4 to 1.6-folds), and the deletion of the URAT1 C-terminal PDZ motif abolished this effect. The augmentation of the transport activity was accompanied by a significant increase in the V_max of estrone sulfate transport via OAT4. The elucidation of these interactions will lead to the further understanding of the function and regulation of renal apical organic anion transports in the proximal tubules. [Jpn J Physiol 55 Suppl:S84 (2005)]

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Salivary glucometry was assessed as a non-invasive monitor for blood glucose. Glucose concentration in the saliva and perfusate were compared in the isolated perfused submandibular glands of rats and rabbits. The ratio of saliva glucose to perfusate glucose (S/P-G) was in reciprocal relationship to the rate of fluid secretion. The saliva glucose concentration was higher as the saliva was secreted at lower rate. As the fluid secretion was higher, the value of S/P-G decreased and reached to the constant value. The present findings support that blood glucose can be estimated from the salivary glucometery when the fluid secretion rate is enough high. For practical application, the saliva was sampled from ductal openings of the sublingual/submandibular glands and the parotid gland of 42 adult human volunteers (20-28 years old) under resting and salivary stimulation with oral application of a sweet tablet. The ratio of salivary glucose to blood glucose (S/B-G) was examined among different individuals. The salivary flow rates increased by stimulation with a sweat tablet. It was shown that salivary glucose concentration showed well correlation with blood glucose under this stimulatory condition. [Jpn J Physiol 55 Suppl:S84 (2005)]

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In renal epithelial A6 cells, hypotonicity induces Na⁺ reabsorption by translocation of epithelial Na⁺ channel (ENaC) in a protein tyrosine kinase (PTK)-dependent manner. However, it is unknown what type of PTK is involved in hypotonic action. We hypothesized that receptor tyrosine kinases (RTKs) existing in cell membrane act on ENaC translocation by sensing the hypotonicity-induced change of cell volume. To clarify the hypothesis, we examined a role of epidermal growth factor receptor (EGFR), one of the RTKs, in hypotonicity-induced ENaC translocation. AG1478, an inhibitor of EGFR, blocked the stimulatory action of hypotonicity on ENaC activity that was measured as benzamil-sensitive short-circuit current and conductance 120 min after exposure to hypotonicity. Next, we investigated the influences of ERK1/2 and PI3-K, representing two main downstream signaling pathways of EGFR. PD98059, an inhibitor of ERK1/2, did not affect the hypotonicity action on ENaC. The hypotonicity action on ENaC was inhibited by LY294002, an inhibitor of PI3-K. These findings indicate that the signal of hypotonicity is converted into EGFR activation, which causes ENaC translocation through a PI3-K-dependent pathway. [Jpn J Physiol 55 Suppl:S84 (2005)]

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The interlobular duct segments isolated from guinea-pig pancreas secrete HCO₃⁻ at ∼0.5 nmol sec⁻¹ cm⁻² into a HCO₃⁻-rich (125 mM) luminal fluid following secretin stimulation. Under these conditions intracellular HCO₃⁻ is ∼20 mM and intracellular potential is ∼-60 mV. If apical HCO₃⁻ transport is mediated by an anion conductance, apical membrane HCO₃⁻ permeability of 0.25 μm sec⁻¹ is required. To estimate the apical HCO₃⁻ conductance in pancreatic duct cells, we measured changes in intracellular pH (pH_i) when the cells were depolarized by raising extracellular K⁺ ([K⁺]_B). The isolated ducts were superfused with HCO₃⁻/CO₂-free HEPES-buffered solution containing H₂DIDS (dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and methyl-isobutyl amiloride to inhibit basolateral HCO₃⁻/H⁺ transport. The lumen was microperfused with a solution containing 125 mM HCO₃⁻, 24 mM Cl⁻, and 5% CO₂. pH_i was measured with BCECF in ducts stimulated with dibutyryl cAMP. With 1 mM [K⁺]_B, pH_i was ∼6.5. When [K⁺]_B was raised to 70 mM, pH_i started to increase. HCO₃⁻ influx calculated from the rate of pH_i increase and intracellular buffering capacity (at pH_i of 6.6) was 0.063 ± 0.009 mM sec⁻¹ (mean ± SEM, n = 5). From luminal HCO₃⁻ (125 mM), intracellular HCO₃⁻ (4 mM) at pH_i of 6.6, and membrane potential (∼-40 mV), the HCO₃⁻ permeability coefficient of the apical membrane was estimated to be ∼0.15 μm sec⁻¹. [Jpn J Physiol 55 Suppl:S84 (2005)]

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NaCl absorption in the gastrointestinal tract is mediated by coupled Na⁺/H⁺ and Cl⁻/HCO₃⁻ exchangers. Recent studies have suggested that SLC26A3 (also called DRA) is a major Cl⁻/HCO₃⁻ exchanger in the colon, yet little is known about its functional properties. Since multiple isoforms of the Cl⁻/HCO₃⁻ exchanger are co-expressed in an intact colonic cell, complicating the functional analysis of an individual isoform, we generated an N-terminal hemagglutinin (HA) epitope-tagged human SLC26A3 construct and expressed transiently in CHO cells by using inducible gene expression systems. Cl⁻/HCO₃⁻ exchange activity was assessed by measuring intracellular pH using fluorescent dye BCECF. When the cells were induced DRA, the cells were conferred 100μM DIDS insensitive Cl⁻/HCO₃⁻ exchange activity. Pharmacological analyses revealed that SLC26A3 was inhibited by 50 μM niflumic acid (90%), by 50 μM tenidap (30%). Ion selectivity experiments showed that SLC26A3 can also transport Br⁻ and I⁻ but not SO₄²⁻. Increased intracellular cAMP and/or Ca²⁺ elicited by enterotoxins and some neurotransmitters is known to result in inhibition of electroneutral NaCl absorption in intestinal epithelium. In SLC26A3-expressing cells, elevating [Ca²⁺ ]i with ionomycin rapidly inhibited SLC26A3 activity, while did not affect increasing cAMP. Immunoblotting and fluorescence imaging analysis of cells before and after treatment of ionomycin revealed no visible differences in the density or distribution of SLC26A3. [Jpn J Physiol 55 Suppl:S85 (2005)]

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We have previously shown that ileal villi exhibit Cl⁻/HCO₃⁻ exchange activity by microfluorometry and express at least three Cl⁻/HCO₃⁻ exchangers, i.e., Slc26a3, Slc26a6 and AE2, by RT-PCR. This study further characterized Cl⁻/HCO₃⁻ exchange activity. [Methods] Isolated villous tips from mouse ileum were loaded with BCECF and intracellular pH (pH_i) was determined by microfluorometry on the stage of inverted microscopy. The samples were illuminated alternately at 440 and 500 nm, and the fluorosence was measured at 520∼570 nm through interference filter. HCO₃⁻/CO₂-buffered solution was used for superfusing the samples. [Results] The pH_i was increased by removing Cl⁻ from superfusing solution and recovered upon re-addition of Cl⁻. The pHi recovery upon re-addition of Cl was hardly suppressed by 300 μM DIDS, while it was inhibited by tenidap (10 μM, 40% inhibition). The rates of pH_i increase and decrease upon Cl⁻ removal and readdition, respectively, were not affected by the treatment of the sample with forskolin (10 μM). [Conclusion] These results suggest that Slc26a3 is mainly responsible for pHi change induced by Cl⁻ removal protocol in villous tip cells. [Jpn J Physiol 55 Suppl:S85 (2005)]

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FOK rats are resistant to heat-exposure and cold-exposure, and great amount of salivation is found under the condition of heat-exposure (HE). To investigate how the enhanced salivation in FOK rats is related to the resistance to HE, we investigate immunohistochemical expression of aquaporin5 (AQP5) in submandibular gland in FOK rats and WKAH rats. AQP5 was detected in apical membrane (AM) and lateral membrane (LM) of acinar cells in both strains, although AQP5 expression was also found in basal membrane (BM) in FOK rats. Although expression pattern of AQP5 did not changed in both strains under HE (40°C), immunoreactivity to AQP5 was significantly decreased in WKAH rats. However, it did not changed in FOK rats. Blockage of parasympathetic effect by atropine (1 mg/kg) under HE caused inhibition of salivation, while decrease of AQP5 was found in not acinar cells but granular duct in FOK rats. On the other hand, injection of parasympathetic agonist policarpine (1mg/kg) induced salivation in both strains. Localization of AQP5 after pilocarpine injection was similar to that of HE. Expression of AQP5 in acinar cells was significantly only in WKAH rats. However, decrease of AQP5 expression in granular cells was found in both strains, and it was stronger in FOK rats. Data suggest that mechanism of enhanced salivation under HE is different from that by pilocarpine injection in FOK rats. [Jpn J Physiol 55 Suppl:S85 (2005)]

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Retinal rod and cone photoreceptors express two distinct Na⁺/Ca²⁺-K⁺ exchanger (NCKX) gene products. Both the rod NCKX1 and cone NCKX2 are polytopic membrane proteins that use both the inward Na⁺ gradient and the outward K⁺ gradient to drive Ca²⁺ extrusion across the plasma membrane. Here, we have identified a single residue (out of >100 tested) for which substitution removed the K⁺ dependence of NCKX-mediated Ca²⁺ transport. Charge-removing substitution of Asp⁵⁷⁵ by either asparagine or cysteine rendered the mutant NCKX2 proteins independent of K⁺, while the charge-conservative replacement of Asp⁵⁷⁵ to glutamate resulted in a nonfunctional mutant NCKX2 protein, accentuating the critical role for the K⁺ dependence of NCKX2. Asp⁵⁷⁵ is conserved in the NCKX1-5 genes, while an asparagine is found in this position in the three NCX genes, coding for the K⁺-independent Na⁺/Ca²⁺ exchanger. [Jpn J Physiol 55 Suppl:S85 (2005)]

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The aims of this study are to analyze arterial pressure variability (APV) and heart rate variavility (HRV) in conscious rats using power spectral techniques, and to examine the hemodynamic effects of nitric oxide (NO) and ATP-sensitive K⁺ (K_ATP) channels on sympathetic activity in arterial pressure regulation. Experiments were performed on male normotensive Whistar rats (n = 32), 10–13 wks of age. A polyethylene catheter for arterial pressure measurement was inserted from the right femoral artery into the terminal abdominal aorta under anesthesia. Another catheter for injection of drugs was inserted into the right external jugular vein. Three days post-operation, AP and HR were measured in the conscious resting state. Hexamethonium C6 (25 mg/kg), L-NAME (5 mg/kg), L-arginine (150–200 mg/kg), glibenclamide (3 mg/kg), cromakalim (0.067 mg/kg) and prazosin (200 μg/kg) were infused. The effects of the drugs on APV and HRV were examined using power spectral analysis with a sampling interval of 0.05 seconds. Findings indicate that: (1) sympathetic activity (< 0.6 Hz) in arterial pressure regulation was buffered by NO. NO-buffering capacity was modulated by activation of K_ATP channels, (2) very low oscillations (0.002–0.2 Hz) were mediated by sympathetic nerve activity, vasomotion, and neurohumoral factors, (3) low oscillations (0.2–0.6 Hz) were mediated by the baroreflex, and (4) high oscillations (0.6–2.5 Hz) were mediated vagally. We concluded that the NO-buffering effects on sympathetic activity may be backed up by activation of K_ATP channels. [Jpn J Physiol 55 Suppl:S86 (2005)]

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Multiple-site optical recording methods, using voltage-sensitive dyes and a multi-element (16 X 16) photodiode array, were employed to simultaneously monitor action potentials from many sites in the isolated rat atrium preparation. The wavelength of the incident light was changed within the range of 500–750 nm, and the action spectra were measured for several voltage-sensitive dyes. These dyes belonged to merocyanine-oxazolone, merocyanine-rhodanine and oxonol groups. Most of the dyes had the action spectra which were similar to those reported in the previous report. However, some dyes exhibited different spectra from those of other preparations. Species-to-species difference of the action spectrum of the voltage-sensitive dye would be discussed. [Jpn J Physiol 55 Suppl:S86 (2005)]

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Although erythrocyte filterability is a determinant of microcirculation, this notion has not been verified quantitatively so far. Therefore, we investigated the correlation of erythrocyte filterability and microcirculation in 10 males with life style-related diseases. For these subjects, we investigated the effects of oligomeric procyanidins (OPCs) on erythrocyte filterability using a nickel mesh filtration technique and on skin microcirculation using laser Doppler flowmetry. The correlation of erythrocyte filterability and microcirculation was analyzed. The erythrocyte filterability before the intake of OPCs (64.6±14.5%) became 69.8±7.7% 1 week after starting intake, and was significantly improved 2 weeks after intake (77.3±5.6%, p<0.02). Correspondingly, laser Doppler flowmeter signals were significantly augmented 1 week (1515±138 mV, p<0.001) and 2 weeks (1570±128 mV, p<0.001) after intake compared with that before intake (1335±126 mV). Furthermore, we found a good correlation (R=0.92) between the improvement of filterability and augmentation of microcirculation. The present study shows that the combined studies involving the nickel mesh filtration technique and laser Doppler flowmetry are relevant for evaluating the rheologic functions of nutritional foods from in vitro and in vivo standpoints. [Jpn J Physiol 55 Suppl:S86 (2005)]

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Chronic hypoxia causes sustained pulmonary hypertension and, although impairment of the pulmonary endothelial nitric oxide (NO) pathway has been implicated, no study has described the central role of NO in modulating pulmonary vascular tone or acute hypoxic pulmonary vasoconstriction (HPV). Centrally, NO inhibits sympathetic tone, so we hypothesised that acute central NO would modulate pulmonary vascular tone and acute HPV, especially in the hypertensive state. Male adult Sprague-Dawley rats were exposed to normoxia (N) or hypoxia (CH=12% O₂) for 14 days. Mean pulmonary (MPAP) and systemic arterial pressure (MABP), cardiac output (CO) and heart rate (HR) were measured in anesthetized, artificially-ventilated rats. N- and CH-rats were exposed to acute hypoxia (10% O₂ for 4 min) after the intracerebroventricular (icv) administration of aCSF (control) and again after either icv L-NAME (150μg in 10μl) or SIN-1 (100μg/10μl). Chronic hypoxia caused pulmonary hypertension (MPAP of 20±1 mmHg in N-rats, cf. 30±1 mmHg in CH-rats) and attenuated HPV. Central inhibition of NO synthesis (L-NAME) did not alter baseline MPAP or the acute HPV in either N-rats or CH-rats, but it did elevate MABP. The NO-donor (SIN-1) did not alter baseline MPAP, but it enhanced (N-rats) or restored (CH-rats) the HPV and decreased MABP. This study indicates that, although central NO has a limited role in the tonic modulation of MPAP, exogenous NO enhances the acute HPV. [Jpn J Physiol 55 Suppl:S87 (2005)]

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We have previously shown that the defense response was attenuated in prepro-orexin gene knockout mice (Am J Physiol, 2003, 285:R581). Because the defense response was not completely abolished in the knockout mice, we have speculated that the co-transmitters contained in the orexinergic neurons may also participate in the defense response. To test the hypothesis, here we used orexin neuron-ablated mice that were generated by introducing a transgene to express a neurotoxin under regulation of the orexin-promoter. In urethane-anesthetized mice, a GABA-A receptor antagonist, bicuculline, was microinjected into the perifornical area in the posterior hypothalamus, so called defense area. Defense response was defined as simultaneous elevation of blood pressure, heart rate, and minute ventilation and shift of blood flow from the visceral to the muscular vasculature. Excitation of the perifornical area by disinhibition with bicuculline resulted in an attenuated defense response in orexin neuron-ablated mice. However, the defense response was still remained about a half magnitude as compared to the wild-type controls as was the case in prepro-orexin knockout mice. We conclude that orexin but not co-transmitters in the orexinergic neurons plays a role as a master switch to elicit multifaceted autonomic and respiratory changes in the defense response. [Jpn J Physiol 55 Suppl:S87 (2005)]

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We investigated the properties of the cross-bridge-dependent change in the Ca²⁺ affinity of troponin C (TnC) (downstream mechanism) during relaxation because it has not been understood due to the difficulty of mechanical perturbation during relaxation. The aequorin method was used to intact ferret papillary muscles and a tetanic contraction was induced by a repetitive electrical stimulation in the presence of ryanodine. The cross-bridge-dependent change in the Ca²⁺ affinity of TnC was estimated by measuring the extra-Ca²⁺, the transient increase in the intracellular Ca²⁺ concentration in response to a rapid reduction in muscle length, and was estimated by the change in the slope of the extra-Ca²⁺-tension relation. The extra-Ca²⁺-tension relation measured during relaxation became steep compared to that during contraction. 20 mM Caffeine, by which the maximal activated tension was significantly reduced, made the extra-Ca²⁺-tension relation steeper during contraction. 3 mM 2,3-butanedione monoxime, an inhibitor of the cross-bridges, made the extra-Ca²⁺-tension relation steeper during contraction. These results indicate that the downstream-dependent change in the Ca²⁺ affinity of troponin-C was reduced during relaxation compared to that during contraction due to the decreased number of attached cross-bridge. [Jpn J Physiol 55 Suppl:S87 (2005)]

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[Aim] Pulmonary veins (PVs) are major arrhythmogenic foci to initiate and perpetuate atrial fibrillation. Previous electrophysiological studies demonstrated myocardial cells in PVs next to the left atrium (myocardial sleeve) show spontaneous pacemaker activities, but its molecular basis is still unclear. We investigated mRNA and protein level of the PV in comparison with the SA node (SAN) and the right atrium (RA). [Methods] Tissue samples from PV, SAN and RA were dissected from rabbit hearts (n=7). Quantative real-time PCR was used to quantify mRNA expression for connexin (Cx)45, Cx43, Cx40, hyperpolarization-activated nucleotide-gated channels (HCN1, 4), L-type calcium channel alpha-subunits (Cav1.2, 1.3), cardiac-type sodium channel alpha-subunit (Nav1.5), atrial natreutic peptide (ANP) and middle neurofilament (NF-M). Protein expression of HCN1,4 and Cxs was estimated by immunohistochemistry. [Results] In comparison with RA, SAN was characterized by higher mRNA levels of Cx 45, HCN1, HCN4, Cav1.3 and NF-M and a lower mRNA levels of Cx43, Cx40, Cav1.2, Nav1.5 and ANP. PV, like SAN, had a lower mRNAs of ANP and Nav1.5, but mRNAs of Cx43, Cx45, Cx40, HCN1, HCN4, Cav1.2, Cav1.3 and NF-M were comparable to RA. Unlike SAN, proteins of Cx45, HCN1 and HCN4 were not recognized in PV. [Conclusions] The PV exhibited different mRNA and protein expression profiles than SAN in terms of ion channels and connexins. The spontaneous pacemaker activity of PV cannot be attributed to HCN1,4 and Cav1.3 channels. [Jpn J Physiol 55 Suppl:S87 (2005)]

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Purpose: Although the sympathetic nervous system regulates the heart via both humoral and neural factors, interaction between the two factors remains unknown. The purpose of the present study was to elucidate the effects of plasma NE on dynamic heart rate (HR) regulation by the cardiac sympathetic nerve. Methods: In 7 anethetized rabbits with vagotomy, we stimulated the right cardiac sympathetic nerve using a binary white noise (0-1 Hz, 2-s switching interval) while measuring the HR response. Effects of continuous intravenous infusion of NE at 1 and 10 μg/kg/h on the transfer function from sympathetic stimulation to HR were examined. Results: NE infusions at 1 and 10 μg/kg/h increased plasma NE concentration from 547±128 to 965±118 and 7120±1080 pg/ml (means±SE), respectively. The transfer function from sympathetic stimulation to HR approximated a second-order low-pass filter with lag time. Dynamic gain did not differ among conditions of control and NE infusions (5.4±1.4, 5.2±0.9, 5.3±1.1). Other parameters of the transfer functions including natural frequency, damping ratio, and lag time did not differ among the three conditions. Conclusion: Plasma NE concentration up to approximately 10 times a resting level hardly affected dynamic HR response to sympathetic stimulation. The neural regulation appears predominant over the humoral regulation in the sympathetic control of HR. [Jpn J Physiol 55 Suppl:S88 (2005)]

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Experiments have demonstrated that hypothermia prolongs action potential duration (APD) of myocytes. However, the effect of regional cooling on tissue responses to electrical stimulation is unknown. Methods: To develop a membrane model that reproduces the effects of cooling, equations representing temperature quotient Q₁₀ dependence of membrane and gap-junctional currents were incorporated in the LRd model. Responses to both pacing and shocks at various temperatures were simulated in a 3×3-cm bidomain sheet with a 1-cm in diameter central region of cooling. Results: Lowering regional temperature from 37 to 32°C during pacing at 500 ms resulted in Q₁₀ value of 2.0 for APD (166 vs. 235 ms) and of 1.3 for conduction velocity (CV) along fibers (58 vs. 50 cm/s). Further decrease in temperature to 27°C brought about higher Q₁₀ (2.2 and 2.6 for APD and CV, respectively) and led to lower safety factor for propagation. At a temperature of 22°C, triggered activity was induced. Intriguingly, for regional cooling at below 32°C, a weak electric shock (6 V/cm) formed strong virtual electrodes (VEs; regions of positive and negative membrane polarization far from shock electrodes) around the cooled region border. Conclusion: While significant myocardial cooling causes arrhythmogenesis, shock-induced VEs formed in a weakly-cooled region may suggest a novel approach to low-energy defibrillation since the VEs underlie success of defibrillation and fibrillation control. [Jpn J Physiol 55 Suppl:S88 (2005)]

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[Porpose] It is reported that in neonatal period, the pulmonary vagal reflex may promote the oscillations of PP interval from after 1 month postnatal. However, the modulability of the atrioventricular (AV) conduction (PR interval) by respiratory vagal input is unclear in the low-birth-weight infants (LBWI). We attempted to evaluate what correlation and change of PP interval and PR interval variabilities in 0 day and 1month of age. [Methods] Sixteen preterm, LBWI (mean gestational age 33±1 weeks, mean birth weight 1380±110g) were evaluated. ECG, respiratory wave (RW) were recorded using the polygraph system. The frequency domain analysis of PP, PR interval and RW were carried out by FFT method. Power spectrum density was obtained from four frequency regions; low (LF:0.036∼0.146 Hz), high (HF:0.146∼0.390 Hz), total (TF:0.036∼2.000 Hz) and respiratory frequency (RSA). We compared the ratio (LF/HF, RSA/TF) of each intervals between 0 day and 1 month of age. Statistical analysis was performed. [Results] LF/HF of PP decreased in 1 month from day-old 0. Contrary, RSA/HF of PP significantly increased. LF/HF of PR also significantly increased in 1 month from day-old 0, whereas RSA/HF of PR remained statistically. [Conclusion] It is suggested that AV nodal conduction was dominated by non-respiratory related vagus nerve activity, and controlled by an autonomic regulation independent of sinus node after postnatal 1 month. [Jpn J Physiol 55 Suppl:S88 (2005)]

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The effects of a herbal medicine, fuscoporia, a kind of fungi, which had been used for traditional medicine by Ainu race in Hokkaido, were studied in SHRSP. Five 7-week-old HRSPwere supplied with cooled hot-water extracts of fuscoporia ad libitum instead of water (test group). Other four 7-week old specimens of SHRSP were supplied with cooled boiled water ad libitum. Both groups were fed with the normal rat chow for 60 days (control group). On the 30th and 60th days the blood pressure and heart rate were measured on the tail. On the end of the final measurements thd venous blood was sampled from abdominal vein under ethyl-ether anesthesia and leparatomy. Hearts werre removed, weighed and used for the capillary analysis by the use of the double staining method for enzyme expressions of alkaline phosphatase and dipeptidylpeptidase IV. The mean arterial blood pressure was 196±14 mmHg in the control group and 154±31 mmHg in the test group on an average and ±S.D. (p=0.020). The number in leukocytes was 4.55±0.92 x10³/μl vs.5.98±0.50 x10³ /μl, respectively (p=0.032). The content in HbA1c showed a trend of decrease (0.20±0.14% vs. 0.082± 0.04%, p=0.078). No clear changes were found in heart weight, heart rate, red blood cell number, total cholesterol, triglyceride and HDL. Also in the capillary network no significant differnce could be seen by the double staining method. Although the improved variables are limmited, hot water soluble fraction of fuscoporia seems to improve several physiological functions in SHRSP. [Jpn J Physiol 55 Suppl:S88 (2005)]

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It is well known that norepinephrine causes an increase in hepatic oxygen consumption, as well as hepatic venoconstriction. However, it is not known whether oxygen consumption increases during anaphylactic hepatic venoconstriction. We determined oxygen consumption during hepatic venoconstriction induced by norepinephrine or anaphylaxis in isolated rat livers perfused portally and recirculatingly at constant flow with diluted blood (Hct 12.2%). Oxygen consumption was measured by monitoring continuously the differences in portal and hepatic venous O2 content using the absorption spectrophotometer (ODM-1, Biomedical Science, Kanazawa), the sensors of which were equipped in the inflow and outflow perfusion lines. The hepatic anaphylaxis was induced by an injection of ovalbumin (0.1mg) into the perfusate of the isolated liver derived from the rat sensitized with ovalbumin (1 mg). Hepatic venoconstriction and liver weight loss were similarly observed in response to norepinephrine (0.01∼10 μM) and anaphylaxis. However, the response of oxygen consumption was different between anaphylaxis and norepinephrine; hepatic anaphylaxis caused a decrease in oxygen consumption, while norepinephrine increased oxygen consumption, as reported previously. We conclude that anaphylactic hepatic venoconstriction is accompanied by a decrease in hepatic oxygen consumption in rats. [Jpn J Physiol 55 Suppl:S89 (2005)]

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Measurement of cytosolic Ca²⁺ ([Ca²⁺]_i) is one of the important methods for investigating various cellular functions. Aequorin is a Ca²⁺-sensitive photoprotein extracted from jellyfish (native aequorin; N-AQ) and has been widely used for measurement of [Ca²⁺]_i in living cells. Aequorin has been employed for the simultaneous measurement of [Ca²⁺]_i and tension in muscle, particularly in mammalian myocardium. Recently, aequorin can be produced using a genetic engineering method (recombinant aequorin; R-AQ). However, the property of R-AQ and the applicability of R-AQ to the measurement of [Ca²⁺]_i in living cells were not fully investigated. We prepared R-AQ using apoaequorin expressed in Escherichia coli and coelenterazine. R-AQ showed higher sensitivity for Ca²⁺ in the middle range of pCa-light relation. Decay of the aequorin light signal in the solution with higher Ca²⁺ concentration, expressing the consumption rate of aequorin, was almost the same in both N-RQ and R-AQ. The pCa-light relation of N-AQ and R-AQ was similarly shifted to the right by Mg²⁺. The pCa-light relation of N-RQ and R-AQ was hardly influenced by pH change in the range tested (pH 6.5, 7.0 and 7.5). We tried to measure the intracellular Ca²⁺ transient (CaT) with tension in mouse papillary muscle using R-AQ. The measured CaT did not significantly differ form that with N-RQ. These results indicate that R-AQ could be used for the measurement of [Ca²⁺]_i in living cells as in the case of N-AQ. [Jpn J Physiol 55 Suppl:S89 (2005)]

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In cardiac myocyte, binding of catecholamine to β₁-adrenergic receptor activates protein kinase A (PKA) via G protein (Gs) and adenylyl cyclase that synthesizes cyclic AMP (cAMP). PKA, when activated by cAMP, phosphorylates a wide spectrum of target proteins including L-type Calcium channel, phospholamban, contracting proteins, and ryanodine receptor. Thus, the β-adrenergic signaling pathway dynamically regulates excitation-contraction (E-C) coupling through a combination of feedforward and feedback mechanisms. To understand mechanisms of β₁-adrenergic signaling pathway in cardiac E-C coupling quantitatively, we have modeled the β₁-adrenergic signaling pathway based on Saucerman et al. (J. Biol. Chem. 2003; 278 47997-48003, and Prog. Biophys. Mol. Biol. 2004; 85 261-78) and incorporated in the Kyoto model which includes cardiac E-C coupling and ATP metabolism (Prog. Biophys. Mol. Biol. 2004; 85 279-299). The parameters of the β₁-adrenergic signaling pathway model were modified to fit a wide range of experimental data, and new kinetic models of target protein phosphorylation by PKA, such as phospholamban and L-type Calcium channel were constructed. The β agonist, isoproterenol, dependence of E-C coupling was simulated using this model. [Jpn J Physiol 55 Suppl:S89 (2005)]

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The intracellular signaling pathways involved in the activation of P2Y receptor-mediated Cl⁻ currents (I_Cl,P2Y) were studied in mouse ventricular myocytes with the whole-cell clamp method. With high concentrations of EGTA in the pipette, extracellular application of UTP (0.1-100 μM) elicited a Cl⁻ current which showed a time-independent activation during step voltages and a linear I-V relationship in symmetrical Cl⁻ solutions. Its anion selectivity was Cl⁻ > I⁻ > Asp⁻. The potency of UTP (EC50 = ∼1 μM) for the current activation was almost equal to that of ATP, while ADP and UDP were less potent. Suramin, but not PPADS, inhibited the activation of I_Cl,P2Y. These data suggest that the P2Y-receptor subtype involved is P2Y₂. The activation of I_Cl,P2Y by UTP was suppressed by extracellular application of PLC inhibitors or intracellular dialysis of anti-G_αq/11 antibody, while the suppression was relieved by application of PKC activators. These results suggest that the UTP activates I_Cl,P2Y through Gq/11-coupled P2Y₂ receptor-PLC-PKC signaling. When the cells were dialyzed with non-hydrolyzable ATP analogues (ATPγS or AMP-PNP) without ATP, UTP failed to activate I_Cl,P2Y, whereas I_Cl,P2Y was persistently activated by UTP in the cells dialyzed with both ATP and ATPγS, suggesting that phosphorylation and ATP hydrolysis are involved in the activation of I_Cl,P2Y. The G_αq/11-PLC-PKC signaling and intracellular ATP hydrolysis may be the essential mechanisms for the activation of I_Cl,P2Y by UTP in cardiac cells. [Jpn J Physiol 55 Suppl:S89 (2005)]

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The pathophysiology of the hepatic vascular response to anaphylaxis in rabbit is not known. We studied effects of anaphylaxis on hepatic vascular resistances and liver weight in isolated perfused livers derived from sensitized New Zealand white rabbits. Sensitization was performed by a subcutaneous injection of ovalbumin (1 mg) four times at one week intervals. The livers were perfused portally and recirculatingly at constant flow with diluted blood. With the use of the double-occlusion technique to estimate the hepatic sinusoidal pressure, pre-sinusoidal resistance (Rpre) and post-sinusoidal resistance (Rpost) were calculated. An injection of the antigen (2 mg) into the perfusate caused hepatic venoconstriction, reflected by an increase in portal vein pressure. This anaphylactic hepatic venoconstriction was characterized by an increase in Rpre greater than Rpost, and was accompanied by a liver weight loss. In conclusion, anaphylaxis causes predominantly pre-sinusoidal constriction and a decrease in liver blood volume in isolated sensitized-rabbit livers. [Jpn J Physiol 55 Suppl:S90 (2005)]

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Myocardial gap junction is prerequisite for normal rhythmic cardiac contraction through providing an electrical pathway between neighboring cells. The conductance is voltage- and intracellular [Ca²⁺]-dependent. The aim of this study is to model the voltage- and Ca²⁺- dependent gating properties of gap junction, and to investigate the relationship between cardiac function and gap junction conductance through computer simulation. A new Ca²⁺ dependent gate was added to the voltage-dependent gate model developed by Vogel and Weingart (1998). The Ca²⁺ gating kinetics was determined based on two experimental findings: 1) Gap junction closes at the physiological concentration range of intracellular Ca²⁺ (Noma and Tsuboi, 1987), 2) Changes in the gap junction conductance is less than 10% during a twitch contraction (Weidmann, 1970). This gap junction model was used to connect 50 cell models with membrane dynamics as well as Ca²⁺-transient defined by Kyoto Model (Matsuoka et al., 2003) in a linear strand, and effects of altering gap junction conductance on cardiac function were evaluated. Lowering the gap junction conductance caused a decrease in E_max and energy efficiency along with the decrease in the conduction velocity. The results suggested that decreased gap junction conductance, under pathological conditions such as ischemia, enhances contractile dysfunction. [Jpn J Physiol 55 Suppl:S90 (2005)]

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Erythrocyte flow behavior in microvessels affects not only flow resistance, but also oxygen transfer to peripheral tissues. Erythrocytes flowing through a microvessel tend to accumulate in the central flow axis, and thus cell-free layer appears along inner wall of the vessel. The oxygen release from flowing erythrocytes was examined using an oxygen-permeable fluorinated ethylenepropylene copolymer tube (inner diameter, 0.027 mm). After exposing the narrow tube to deoxygenated environment, the oxygen saturation was measured with a spectrophotometer under a microscope. We modified the flow behavior of erythrocytes by applying accelerational force perpendicularly to the flow direction using a centrifuge or by perfusing erythrocytes reduced in sialic acid content of the membrane proteins with neuraminidase to decrease electrostatic interaction among erythrocytes.(1) With increasing the centifugal force, the oxygen release from flowing cells was suppressed accompanying the shift of the cell flow column to centrifugal side and the compression of the flow column.(2) With decreasing sialic acids of erythrocytes, oxygen release from cells was decreased, especially at low pH condition (pH=6.5). In conclusion, this research suggests that accumulation of flowing erythrocytes affects the oxygen diffusion from erythrocytes to tissue in microcirculation. [Jpn J Physiol 55 Suppl:S90 (2005)]

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Acute intoxication due to alcohol consumption has been known to elicit reversible cardiac muscle dysfunction. Acetaldehyde (AcA), the major oxidized product of alcohol, may be a primary factor underlying alcohol-induced muscle dysfunction. We examined whether clinically relevant concentrations of AcA function as a primary toxin underlying muscle dysfunction, by measuring channel activity of rabbit purified ryanodine receptors (RyR2) with a lipid bilayer method and Ca²⁺ transient of rat single cardiac myocytes with a confocal imaging system. When applied to the RyR2 channel, AcA at one μM enhanced channel activity. AcA-induced channel activation was dose-dependent and was kept high even in presence of 300 μM AcA. Exposure of single myocytes to one μM AcA did not significantly affect parameters (resting intracellular Ca²⁺ level, time to peak, decay time) of Ca²⁺ transient elicited by single electrical field stimulation. However, AcA slowed decay time of Ca²⁺ transient on application of 2nd to 4th field stimulation and significantly retarded exponential decrement in amplitude of Ca²⁺ transient in respond to 2nd, 3rd and 4th field stimlation, in constant with a fast decrease in intact myocytes. These results suggest that AcA at clinical concentrations plays a role as the primary factor for alcohol-induced cardiac muscle dysfunction by modifing SR function. [Jpn J Physiol 55 Suppl:S90 (2005)]

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In the myocardium, dysfunction of the gap junction impairs intercellular electrical coupling and can be one of arrhythmogenic factors. The function of the gap junction depends essentially on characteristics of the connexin which composes the gap junction channel. In this study, relation between changes of expression of the connexin43 (Cx43) and susceptibility of myocardium to fibrillation was examined on the aconitine (AC)-induced cardiac fibrillation model in the isolated hearts of adult guinea-pig and rat. In the normal hearts, ventricular flutter induced by AC (0.1μM) (on Langendorff perfusion) shifted spontaneously to fibrillation about 10 min later. Low concentration of heptanol shifted the flutter to the fibrillation within several seconds showing asynchronous intercellular electrical interactions. At the beginning of the fibrillation, expression of Cx43 at the intercalated disk (ID) was sporadic coupled with a reduction of PKA-mediated and an augmentation of PKCε-mediated phosphorylation of Cx43. These events were enhanced as the fibrillation was advanced. During fibrillation, level of cardiac tissue Angiotensin II (Ang.II) was raised. In Ang.II analog- or PMA-pretreated hearts and diabetic hearts in which PKCε was activated, expression of Cx43 at ID was sparse. In these pathological hearts, time of shift from the flutter to the fibrillation was remarkably shortened within 3 min. These results suggest that remodeling of Cx43 due to an activation of PKCε and an acceleration of proteolytic degradation makes the heart susceptible to the fibrillation. [Jpn J Physiol 55 Suppl:S91 (2005)]

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We investigated the dynamical mechanisms of biological pacemaker (BP) generation in human ventricular myocytes (HVMs) by bifurcation analyses of a mathematical model. Equilibrium points (EPs), periodic orbits, stability of EPs, and bifurcation points were calculated as functions of conductance or amplitude of inward-rectifier K⁺ current (I_K1), L-type Ca²⁺ current (I_Ca,L), delayed-rectifier K⁺ current (I_K), Na⁺-Ca²⁺ exchanger current (I_NaCa) or constant bias current (I_bias) for constructing bifurcation diagrams. Pacemaker activity (stable limit cycle) abruptly appeared around an unstable EP via a saddle-node bifurcation, when I_K1 was suppressed by 84.6%. After the bifurcation, the I_K1-downregulated HVM has an unstable EP only, which is essentially important for stable BP generation. To elucidate how individual sarcolemmal currents contribute to EP instability and BP generation, we further explored the bifurcation structures of the system during decreases in each current. Our results suggest that 1) I_K1 suppression is necessary for creation of the BP cell system, 2) I_Ca,L is, but I_K or I_NaCa is not, responsible for EP instability as a requisite for stable BP generation, 3) I_K is indispensable for robust pacemaking with large amplitude, high upstroke velocity and stable frequency, and 4) I_NaCa is the dominant pacemaker current, but not necessarily required for BP oscillations. [Jpn J Physiol 55 Suppl:S91 (2005)]

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It is well known that Mg²⁺ inhibits Ca²⁺-induced Ca²⁺ release (CICR) (physiological Ca²⁺ release mechanism) in sarcoplasmic reticulum (SR) of cardiac muscles. Previously, we suggested that intracellular Na⁺ enhances Ca²⁺ release from SR in aequorin-injected ferret ventricular muscles. In the present study, we directly demonstrate the enhancement of CICR by Na⁺ using skinned myocardium. We measured the relative amount of Ca²⁺ released by Ca²⁺ (2 min application of solutions at pCa7-4) from SR using a fluorescent Ca²⁺ indicator (fluo-3) in saponin-treated ferret thin ventricular muscle bundles. Ca²⁺ was released in the solutions containing 1.5 mM free-Mg²⁺ with or without Na⁺. Na⁺ was replaced with three monovalent cations (K⁺, Li⁺, N-methyl-D-glucamine⁺). We also measured Ca²⁺ release in Mg²⁺-free solutions with or without Na⁺. The amount of released Ca²⁺ was normalized to that by 50 mM caffeine which releases all Ca²⁺ in the SR. The amount of Ca²⁺ released by Ca²⁺ in the presence of 100% Na⁺ was higher than that in Na⁺-free solutions containing other monovalent cations. There were no significant differences in the relative amount of Ca²⁺ released by Ca²⁺ (pCa6) among these three different cations. CICR was inhibited by Mg²⁺. However, the inhibitory effect of Mg²⁺ on CICR disappeared in the solutions with 100% Na⁺, i.e., in the absence of Mg²⁺, the enhancement of CICR by Na⁺ was not observed. We conclude that Na⁺ might act on the same site where Mg²⁺ binds to inhibit CICR. [Jpn J Physiol 55 Suppl:S91 (2005)]

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Cilostazol, an inhibitor of cyclic nucleotide phosphodiesterase 3 (PDE3), has been used clinically for the treatment of chronic arterial occlusive diseases and secondary prevention of cerebral infarction. The beneficial effect of cilostazol is attributed to both anti-platelet aggregation and vasodilation. Effects of cilostazol on resistance-sized cerebral arteries, however, have not been well elucidated. In this study, we investigated the mode of action of cilostazol on pressurized rabbit cerebral arterioles. Cerebral arterioles were cannulated and monitored with an inverted microscope. The vessels developed myogenic tone by constricting approximately 50% from the maximum passive diameter. Cilostazol (10⁻⁹–10⁻⁴ M) produced concentration-dependent vasodilation. The agent also produced a similar relaxation in the arterioles precontracted by 5-hydroxytryptamine or U46619, a stable analog of thromboxane A₂. Cilostazol (10⁻⁶ M) augmented the adenosine-induced vasodilation of the arterioles. Adenosine (10⁻⁷ M) also caused a potentiation of the cilostazol-mediated vasodilation. Nω-nitro-L-arginine methyl ester (L-NAME) (10⁻⁴ M), a nitric oxide synthase inhibitor, or aspirin (10⁻⁵ M), a cyclooxygenase inhibitor, did not significantly alter the cilostazol-induced arteriolar dilation. Furthermore, vasodilation by cilostazol was observed with endothelial-denudated arterioles. These results suggest that cilostazol produces the vasodilation of rabbit cerebral arterioles independent on the functional endothelium. [Jpn J Physiol 55 Suppl:S91 (2005)]

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The cardiovascular adaptation to voluntary static exercise is controlled by the autonomic nervous system. Two neural mechanisms are responsible for the cardiovascular adaptation at the onset of exercise. One is central command descending from higher brain centers, and the other is muscle mechanosensitive reflex from activation of mechanoreceptors in the contracting muscles. In this study we examined which mechanism played a major role in the initial cardiovascular adaptation during static exercise. To solve this, we studied the effect of intravenous injection of gadolinium (Gd, 55μM), a blocker of stretch-activated ion channel, on the increases in heart rate (HR) and mean arterial blood pressure (MAP) at the onset of static exercise (pressing a bar with a forelimb) in conscious cats. Gd did not blunt the initial increases in HR and MAP at the onset of exercise, although the peak force applied to the bar was gradually decreased following Gd. Next, we examined the effect of Gd on the reflex changes in HR and MAP in response to passive mechanical stretch of a forelimb or hindlimb in anesthetized cats. Gd significantly blunted their increases during passive stretch, indicating that Gd may block the stretch-activated ion channel and thereby attenuate the reflex responses. In conclusion, the present finding that Gd did not affect the initial cardiovascular responses at the onset of voluntary static exercise suggests that central command predominantly contributes to the initial cardiovascular adaptation. [Jpn J Physiol 55 Suppl:S92 (2005)]

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We hypothesized that calpain inhibitor-1 (CI) protected left ventricular (LV) function from ischemic-reperfusion (IR) injury by inhibiting the proteolysis of α-fodrin. To test this hypothesis, we investigated the effect of CI on LV mechanical work and energetics in the cross-circulated rat hearts that underwent 15-min global ischemia and 60-min reperfusion (n=9). After IR with CI, mean end-systolic pressure at midrange LV volume (ESP_mLVV) and systolic pressure-volume area (PVA) at mLVV (PVA_mLVV) were hardly changed, although they were significantly (P<0.01) decreased after IR without CI. Mean myocardial oxygen consumption per beat (VO₂) intercepts of VO₂-PVA linear relations were also unchanged after IR with CI, although they were significantly (P<0.01) decreased after IR without CI. VO₂ for the Ca²⁺ handling was significantly higher than pre IR from 10 to 15 min after IR. CI did not block this energy-wasting process. There were no significant differences in O₂ costs of LV PVA and contractility among the hearts in control (or normal), post IR and post IR with CI. Western blotting of α-fodrin and the immunostaining of 150-kD products of α-fodrin confirmed that CI almost completely protected proteolysis of α-fodrin. Our results indicate that CI prevents the heart from IR injury associated with the impairment of total calcium handling by directly inhibiting the proteolysis of α-fodrin. [Jpn J Physiol 55 Suppl:S92 (2005)]

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We hypothesized that after ischemic-reperfusion (IR) contractile failure similar to that after high Ca²⁺ infusion occurs. To test this hypothesis, we investigated left ventricle (LV) mechanical work and energetics in the cross-circulated rat hearts that underwent 15-min global ischemia and 60-min reperfusion. Mean systolic pressure-volume area (PVA; total mechanical energy per beat) at midrange LV volume (PVA_mLVV) was significantly decreased. Mean myocardial oxygen consumption per beat (VO₂) intercepts (VO₂ for the total Ca²⁺ handling in excitation-contraction coupling and basal metabolism) of VO₂-PVA relation was significantly decreased without change in its slope. After 30-min infusion of a reverse-mode Na⁺/Ca²⁺ exchanger (NCX) inhibitor, KB-R7943 (KBR, 10 µM) during IR, the decrease in mean PVA_mLVV and VO₂ intercept was significantly but partially reduced (n=6). Although VO₂ for the Ca²⁺ handling was finally decreased after 60 min of IR, it was significantly higher than pre IR from 10 to 15 min after IR. This energy-wasting process was completely blocked by KBR. α-fodrin proteolysis significantly increased after IR was significantly reduced by KBR. We clarified the contribution of reverse-mode NCX to the contractile failure after IR due to the impairment of the total Ca²⁺ handling in the excitation-contraction coupling and that NCX inhibitor antagonized this failure by preventing the heart from Ca²⁺ overload. [Jpn J Physiol 55 Suppl:S92 (2005)]

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Biological and physico-chemical mechanisms of lymphatic metastasis of tumor cells and tumor-mediated lymphangiogenesis remain still unclear. And while many angiogenic molecules are widely studied, only few lymphangiogenic molecules except for VEGF-C/D are examined. To address molecular biology of lymphatic endothelial cells (LECs), firstly we should establish the cell lines isolated from small-sized lymph vessels. Thus we have attempted to develop a novel method for the isolation and culture of anatomically-defined LECs of rat thoracic ducts and afferent lymph vessels of iliac lymph nodes. Lymph vessels were dissected using a dissecting microscope. Then LECs were isolated enzymatically by intraluminal perfusion of trypsin. The perfusates were centrifuged and the cells were collected and harvested in hypoxic environment. The colonies were characterized with immunohistochemistry. Proliferation assay was also done for 96 hours. Using this technique, the LECs were able to isolate successfully and show VEGFR3. The LECs from the afferent vessels demonstrated higher proliferation activity compared with the LECs isolated from the thoracic ducts. Wound assay activity with VEGF-C stimulation is observed clearly both in the LECs from thoracic ducts and afferent vessels. Unlike casual techniques with the antigenic selection, the advantages of this method has enabled us to define directly and easily lymphatic endothelial cells, and then to offer suitable cultured lymphatic endothelial cells to study the mechanisms of lymphangiogenesis and lymphatic metastasis of tumor cells. [Jpn J Physiol 55 Suppl:S92 (2005)]

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Oxidized LDL has been reported to activate vascular endothelial cells and promote monocyte transmigration through endothelium, but the direct evidence for the effect of oxidized LDL on the process has not been fully addressed. Using HUVECs (Human Umbilical Vein Endothelial Cells) and freshly isolated human monocytes, we developed an in vitro proatherogenic model with oxidized LDL embedded in very thin (30-50 μm) subendothelial collagen gels, which resembled the accumulation of oxidized LDL in subendothelium of atherosclerotic plaques in vivo, and enabled to trace 3D-kinetics of individual living monocytes. Using this model, we showed that significantly greater number of adherent monocytes crawled on endothelium to paracellular junctions, and started invasion in the presence of oxidized LDL, but once they started invasion, number and kinetics (migration distance, speed, etc. during and after invasion) of monocytes were not significantly different in the presence or absence of oxidized LDL. These results give the first direct evidence that oxidized LDL specifically promotes monocyte post-adhesion dynamics on endothelium (probably crawling), but not invasion itself at junctions, or subendothelial migration after invasion. [Jpn J Physiol 55 Suppl:S93 (2005)]

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The mitochondrial NADH fluorescence firstly decreased and then increased when the positive staircase of contraction was induced by increasing the stimulation rate in guinea-pig ventricular cells. We studied mechanisms by incorporating the well-established model of oxidative phosphorylation (Korzeniewski & Zoladz, 2001) in our membrane excitation-contraction Kyoto model. When the stimulation rate was switched to 3.3 Hz after establishing a steady-state at 0.1 Hz, the mitochondrial NADH concentration declined and reached a new steady state, and cytoplasmic ATP concentration significantly decreased. Assuming a delayed activation of NADH production during 3.3 Hz stimulation well reconstructed the biphasic response of mitochondrial NADH and remarkably attenuated the decrease in cytoplasmic ATP. Thus, the model supported our hypothesis that the initial decrease in mitochondrial NADH is due to feedback control by ADP (Chance and Williams, 1956), while the delayed increase in mitochondrial NADH is caused by the activation of NADH production. The model predicted that this activation of NADH production attenuates the contribution of feedback control by about 25% when compared at the same rate of ATP synthesis during 3.3 Hz stimulation. [Jpn J Physiol 55 Suppl:S93 (2005)]

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Reactive oxygen species (ROS) have been implicated in hypertensive organ damage. However the precise mechanism of ROS-mediated organ damage is not fully understood. We recently demonstrated that up-regulation of NADPH oxidase, which is a superoxide-producing enzyme, was associated with renal injury in hypertensive Dahl salt-sensitive (DS) rats. In the present study we investigated the role of monocyte chemoattractant protein-1 (MCP-1) in renal injury in hypertensive DS rats and the effect of antioxidant treatment on MCP-1 expression. High salt diet (8% NaCl) significantly increased urinary MCP-1 excretion and MCP-1 mRNA expression in kidney in DS rats, associating with urinary hydrogen peroxide excretion and expression of p47phox, which is a subunit of NADPH oxidase. Immunostaining showed strong expression of MCP-1 in damaged renal tubules and the expression of MCP-1 was correlated with macrophage infiltration. Antioxidant N-acetyl-L-cysteine supplement with high salt diet abrogated the increase in MCP-1 expression, macrophage infiltration, and tubular and glomerular lesion in the kidney. These data suggest that ROS-associated MCP-1 expression plays an important role in renal injury in hypertensive DS rats. [Jpn J Physiol 55 Suppl:S93 (2005)]

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In ventricular myocytes, α₁-adrenoceptor stimulation (α₁ARS) increases L-type Ca²⁺ current (I_Ca,L) observed with perforated patch clamp recording. We previously showed that Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) inhibition with KN-93 significantly blocked the potentiation of I_Ca,L by α₁ARS. However CaMKII involvement in α₁-adrenoceptor mediated modulation of I_Ca,L in mammalian ventricular myocytes and the mechanism of I_Ca,L potentiation during α₁ARS are not been fully investigated. The purpose of this study is to clarify the role of CaMKII in the regulation of I_Ca,L during α₁ARS. In Western immunoblotting analysis, phenylephrine significantly increased the amount of autophosphorylated CaMKII (p-CaMKII) and this increase was completely blocked by KN-93. We also investigated the changes in subcellular localization of p-CaMKII using immunofluorescence microscopy and immunoelectron microscopy. Before α₁ARS phospho-CaMKII was exclusively located just beneath the surface plasma membrane (plasmalemma). But after α₁ARS, it was localized close to sarcolemmal invaginations called transverse-tubules (T-tubules) where L-type Ca²⁺ channels highly exist. These findings strongly support the results of our electrophysiological experiments and we conclude that 1) CaMKII which exists near T-tubules was activated by α₁ARS and 2) CaMKII activation directly potentiates I_Ca,L in rat ventricular myocytes. [Jpn J Physiol 55 Suppl:S93 (2005)]

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Lymphocytes re-circulate between vascular and lymphatic systems. Lymphocytes enter to lymph nodes via high endothelium venules (HEV) and then escape from the nodes via efferent lymph vessels. There is, however, little information how lymphocytes pass through the lymph nodes via lymph dynamics including afferent and efferent lymph vessels. The present study thus investigated lymphatic traffic of lymphocytes in isolated lymph nodes of rats by an intravital-microscope system. Seven-week-old male Wistar rats were anesthetized with pentobarbital-Na (50 mg/kg). The mesenteric and iliac lymph nodes were isolated and then used in the present study. Lymphocytes were collected from mesenteric lymph nodes and labeled with CSFE. Afferent and efferent lymph vessels of iliac lymph nodes were cannulated in an organ chamber, while the arteries and veins were ligated. Isolated lymph nodes were circulated with Krebs-bicarbonate solution at a constant perfusion pressure. CSFE-labeled lymphocytes were selectively circulated into the lymph nodes via afferent lymph vessel. Lymphocytes lymphatic dynamics were measured through a fluorescent microscope system and a color chilled CCD-camera. CSFE-labeled lymphocytes circulated through afferent lymph vessel homed at the lymph node and did not appear to efferent lymph vessel for 6 hrs after the circulation. These results suggest that lymphocytes from afferent lymph vessels did not run through the lymph nodes for 6 hrs in vitro experiments. [Jpn J Physiol 55 Suppl:S94 (2005)]

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The interaction between cardiomyocyte contraction and organ level dynamics was investigated to assess the physiological significance of the length-dependent force generation. We developed a simple hypothetical circulation model consisting of 3 components, a single ventricle, a preload and an afterload. The Kyoto model of a single cardiomyocyte was combined with the circulation model and we implemented the contraction process using the NL model (Negroni & Lascano, 1996). A thin-walled spheric geometry is used as a rough estimate of the left ventricle. We employed the Laplace law to convert the wall tension to the intraventricular pressure assuming uniform wall tension in all direction. Using this model, the relationship between the contraction of cardiomyocyte and the ventricular ejection-fraction (EF) was investigated. Computational results suggested that the EF value obtained by using the original NL model was less than 30% because the force generation during systole was strongly inhibited by the cell shortening. An improved result of the EF > 60% was achieved by a slight modification in the length-dependency on force generation. It was also shown that the EF could be increased assuming the dead space occupied by the papillary muscles within a ventricular cavity. [Jpn J Physiol 55 Suppl:S94 (2005)]

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It is known that dysfunction of sciatic nerves in diabetes is related to disturbance of blood microcirculation in the axon and sheath of peripheral nerves. Oxygen free radicals also affect vascular reactivity and produce dysfunction of blood microcirculation in organs. There is, however, little information that high oxygen tension regulates mechanical activity of arterioles in sciatic nerves. The present study thus investigated effects of high oxygen tension on the activity of arterioles in rat sciatic nerves by an intra-vital videomicroscope system. Seven-week-old male Wistar rats were anesthetized by subcutaneous injection of 2% α-chloralose and 10% urethane. The left femoral artery was cannulated to measure arterial blood pressure. The arterioles of right sciatic nerves were observed by using of the intra-vital videomicroscope. Krebs-bicarbonate solution (KBS) was superfused onto the sciatic nerve and changes in the diameter of the arterioles were investigated in response to normal (PO₂; ∼5 mmHg) or high oxygenated (PO₂; ∼140 mmHg) KBS. The superfusion of normal KBS onto the sciatic nerves did not affect diameter of the arterioles, while the superfusion of high oxygenated KBS significantly and time-dependently constricted the arterioles for 10 min (from 18.0 ± 2.0 μm to 9.7 ± 2.0 μm). These results suggest that high oxygen tension constricts rat sciatic nervous arterioles and affect microcirculation of the peripheral nervous system in vivo. [Jpn J Physiol 55 Suppl:S94 (2005)]

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The lymph nodes are known to trap with foreign particles, such as perfluorocarbon emulsions, liposomes, carbon particles and tin colloid. For this reason, these particles are used as tracer and vehicle of drugs which target the lymph nodes. Little information, however, exists regarding barrier function of the lymph nodes. Thus, in this study, we have attempted to develop new animal model for evaluating barrier mechanisms through the lymph node. Male Japan White rabbits were anesthetized with ketamine chloride (20 mg/kg iv.) and pentobarbital sodium (20 mg/kg iv.). Centripetal and retrograde cannulations were performed in one of the popliteal afferent and efferent lymph vessels, respectively. All other efferent lymphatics were ligated completely. Fluorescently labeled latex microspheres (0.2–2.0 μm in diameter) were administered into the afferent lymph vessel. Then, changes in concentration of the microspheres in the efferent lymph vessel were determined by measuring the number of the particles under a fluorescent microscope. Several percent of the microspheres with 0.2 μm in diameter was appeared in the efferent lymph by 30 min after the administration. No microsphere with 2.0 μm in diameter was observed in the efferent lymph by 120 min after the administration. These results strongly suggest that size-dependent barrier mechanisms exist in the lymph node. [Jpn J Physiol 55 Suppl:S94 (2005)]

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Bloodless method for measuring human cardiac stroke volume (SV) was proposed by Kubicek et al. in 1966 (Aerosp. Med.). If it is assumed that the variation of the recorded thoracic impedance (Z_t) is attributable to that of the blood volume, SV is given by the equation, SV=A|dZ_t/dt|_maxVET (A; constant, VET; ventricular ejection time). The Z_t, however, usually involves a slow respiration-synchronized component. This slow component might be due to the volume-change in the air or in the blood or both, which obscures quantitative assessment of the beat-to-beat SV change. It should be noted that the respiration itself affects SV. Without an autonomic regulation of the heart rate, both SV and |dZ_t/dt|_max may fluctuate with respiratory variation of the venous return. Thus it seems essential to hold breath during recording and then calculate an average of several |dZ_t/dt|_maxs to exclude the erroneous component. In the present study we tried to specify the origin of the slow component of Z_t under various recording conditions with different placements of electrodes. The beat-to-beat VET was measured by comparing dZ_t/dt with heart sound, which was not significantly changed by breathing. Thus it was suggested that the errors in measuring SV might be due mainly to the respiratory variation of |dZ_t/dt|_max. It will be shown that the erroneous component is minimized in some recording conditions. Limitations of the non-invasive SV measurement will be discussed. [Jpn J Physiol 55 Suppl:S95 (2005)]

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We have previously observed the functional differences in myocardial contraction between normal control (F1B strain) and cardiomyopathic hamsters (Bio TO-2 strain). To understand the mechanisms underlying myocardial contractile dysfunction in TO-2 hamster, we compared the composition of myosin isoforms and the concentration of myosin heavy chain (MHC) and actin in ventricular muscles of F1B and TO-2 hamsters. Myosin isoforms were separated with pyrophosphate / polyacrylamide gel electrophoresis and their relative proportions were densitometrically quantified. Ventricular myosin from TO-2 consisted of three isoforms, V1 (∼50%), V2 (∼30%) and V3 (∼20%), while F1B myosin contained mostly V1 isoform, accounting for our previous observation that under unloaded conditions, the maximum shortening velocity was lower in TO-2 than in F1B cardiomyocytes. The concentrations of MHC and actin relative to those of total ventricular protein were lower in TO-2 than in F1B hamster, being consistent with our previous finding, the lower isometric peak force in TO-2 than in F1B cardiomyocytes. These results suggest that the shift of myosin isoform from V1 to V2 and V3 and the decrease in contractile protein concentration may contribute to myocardial contractile dysfunction in dilated cardiomyopathic hamster (TO-2). [Jpn J Physiol 55 Suppl:S95 (2005)]

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[Background] Much attention has been devoted to the role of apoptosis in the pathogenesis of drug-induced cardiac toxicity. However, the pro-apoptotic effects of antiarrhythmic drugs are poorly understood. Therefore, we examined the effects of several antiarrhythmic drugs, i.e., pilsicainide, propranolol, nifekalant, verapamil, and amiodarone, on apoptotic parameters in H9c2 cardiac cells. [Methods] The cultured H9c2 cells were incubated for 24 hours with 1, 2, 4, and 8 times the highest clinically attainable concentration of each drug. To determine apoptosis in H9c2 cells, mitochondrial transmembrane potential (Δψ_m) and the percentage of the cells with hypodiploid DNA were measured quantitatively by flow cytometric assay with DiOC₆(3) and propidium iodide, respectively. The activity of caspase-3 was determined by colorimetric protease assay. [Results] Amiodarone at 4 and 8 times but not at 2 times or less the highest clinically attainable concentration induced loss of Δψ_m, the occurrence of hypodiploid cells, and activation of caspase-3. Other drugs did not produce these pro-apoptoic effects even at 8 times the concentration. [Conclusion] All the antiarrhythmic drugs tested except amiodarone did not induce apoptosis in the H9c2 cells up to the level of 8 times the greatest therapeutic range. Na⁺, K⁺ and Ca²⁺ channels blockers, or β adrenergic receptor antagonist did not induce apoptosis, suggesting that the pro-apoptotic effect of amiodarone is independent of its multichannel blocking actions. [Jpn J Physiol 55 Suppl:S95 (2005)]

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Venous pooling/stasis caused by immobility is an important risk factor for deep vein thrombosis during seat position. To assess venous stasis in the lower limbs, popliteal venous flow velocity and cross sectional area, oxy- and deoxy-hemoglobin (Hb) concentration of the calf muscle, and O₂ saturation were measured during 60 min seat position. Oxy-Hb concentration was gradually decreased and deoxy-Hb concentration was gradually increased during seat position, and O₂ saturation in the calf muscle was significantly depressed from 70 ± 7 to 60 ± 7%. Popliteal venous flow velocity was significantly decreased from 16 ± 4 to 5 ± 0.5 cm/s, however cross sectional area was increased 15 ± 5 to 64 ± 10 mm², and then popliteal venous blood flow was not altered during seat position. Thus, during seat position, venous flow velocity of the lower limbs was depressed but total blood flow volume was kept by increasing cross sectional area. Effects of electrical muscle stimulation on lower limbs hemodymnamics were further assessed. [Jpn J Physiol 55 Suppl:S95 (2005)]

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Background: To maintain intracellular environment, pH is regulated by several acid-extruding membrane mechanisms. HCO₃⁻ plays important role in regulating intracellular pH and therefore simultaneous monitoring of extracellular pH and pCO₂ leads to understand how the homeostasis are kept. Here, we developed pH microsensor system based on ion-sensitive field effect transistor (ISFET) which can detect pH and pCO₂ changes in less than 1 microL solution and evaluated the metabolism in the immediate vicinity of endothelial cells of excised rabbit aorta. Materials and Methods: Two sensors were placed at the center of excised rabbit aorta, one was for pH monitoring and the other covered with CO₂ permeable silicon tube for pCO₂ monitoring. We used Tyrode solution buffered with NaHCO₃ which was much more sensitive to pH change than blood. After circulation of this solution, we stopped the solution and monitored the time course of pH and pCO₂ changes for 10 minutes. Results: pH and pCO₂ were changed monotonically and saturated at 10 minutes. Changes of pCO₂ at 10 minutes were significantly larger than that of pH (-0.76 ± 0.10 and -0.93 ± 0.09 respectively, n = 5, p < 0.05). Conclusion: Larger change in pCO₂ compared to pH suggested secretion of alkaline, which should be HCO₃⁻. Using chemical equilibrium of CO₂, lactate, and bicarbonate, we found that the amount of bicarbonate ion secretion was ∼one-third of CO₂ production. [Jpn J Physiol 55 Suppl:S96 (2005)]

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