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From postnatl-day-0 to postnatal-day-2, a few acetylcholinesterase (AChE)-active and choline acetytransferase (ChAT)-immunoreactive nerve fibers and relatively numerous vesicular acetylcholine transporter (VAChT)-immunoreactive puncta were observed in the rat adrenal medulla. Despite relatively numerous clear vesicles in the nerve fibers, the synthesis and hydrolysis of acetylcholine may not be fully activated until postnatal-day-2. The number of AChE-active and ChAT-immunoreactive nerve fibers dramatically increased and that of VAChT-immunoreactive puncta gradually increased from postnatal-day-3 to postnatal-week-1. The synthesis and hydrolysis of acetylcholine may be dramatically activated in the nerve fibers of the medulla until postnatal-week-1. From postnatal-week-2 to postnatal-week-3, the number of AChE-active and the ChAT-immunoreactive nerve fibers gradually increased and reached the adult levels. The VAChT-immunoreactive puncta per unit area was maximum number at postnatal-week-2. The synthesis and hydrolysis of acetylcholine in the nerve fibers of the medulla may be completed between postnatal-week-2 to postnatal-week-3. The diameter of the VAChT-immunoreactive puncta gradually increased from postnatal-day-0 with aging. However, the number of the VAChT-immunoreactive puncta gradually decreased from postnatal-week-2 onwards. In electron-microscopy, the VAChT-immunoreactive deposits were seen in clusters of clear vesicles, and the diameter of the nerve fibers and the number of clear vesicles at postnatal-week-8 increased compared with those at postnatal-week-2. The AChE-active, ChAT-immunoreactive, and VAChT-immunoreactive nerve fibers observed around noradrenaline (NA) cells were denser than those around adrenaline (A) cells in the medulla at postnatal-week-8. These suggest that the preferential innervation of NA and A cells may cause the differential secretion NA and A. Anat Rec, 292:371–380, 2009. © 2009 Wiley-Liss, Inc.

Substance P (SP)-immunoreactive nerve fibers in the adrenal medulla was very few during postnatal day 0-5, indicating that its synthesis in the neurons and the transport to nerve endings was incomplete by the end of this period. Since the number of SP-immunoreactive nerve fibers gradually increased during postnatal week 1-2, it was hypothesized that SP was not fully transported to nerve endings until postnatal week 1-2. At postnatal week 3, numerous SP-immunoreactive nerve fibers contacted some noradrenaline (NA) cells but not adrenaline (A) cells in the medulla. From postnatal week 3 onward, the abundance and expression patterns of SP-immunoreactive nerve fibers in the medulla were similar to those in adults. At postnatal week 3, the innervation with SP-immunoreactive nerve fibers was completed indicating the possibility that SP affected on the secretory activity of NA cells but not of A cells in the medulla. The medullary SP-immunoreactive nerve fibers contacting the chromaffin cells possessed a few dense-cored vesicles in their endings at postnatal week 8. Very few SP-immunoreactive chromaffin cells were found in the medulla from postnatal day 0 onward, and SP immunoreactivity was primarily observed in granular cores of the cells suggesting that SP and catecholamine synthesized in the chromaffin cells were released from the granules by adequate stimuli. Very few or a few SP-immunoreactive nerve fibers, acting as a vasomotor effect were found around blood vessels in the superficial cortex from postnatal day 0 onward.

It has been shown that musculoskeletal tissues undergo dynamic tissue remodeling by a process that is quite sensitive to the mechanical environment. However, the detailed molecular mechanism underlying this process remains unclear. We demonstrate here that after denervation-induced mechanical stress deprivation, tendons undergo dynamic tissue remodeling as evidenced by a significant reduction of the collagen fibril diameter. Importantly, the transient up-regulation of osteopontin (OPN) expression was characteristic during the early phase of tendon tissue remodeling. Following this dynamic change of OPN expression, matrix metalloproteinase (MMP)-13 expression was induced, which presumably accounts for the morphological changes of tendon by degrading tendon collagen fibrils. The modulation of MMP-13 expression by OPN was specific, since the expression of MMP-2, which is also known to be involved in tissue remodeling, did not alter in the tendons under the absence or presence of OPN. We also demonstrate that the modulation of MMP-13 expression by OPN is due to the signaling through cell surface receptors for OPN. Thus, we conclude that OPN plays a crucial role in conveying the effect of denervation-induced mechanical stress deprivation to the tendon fibroblasts to degrade the extracellular matrices by regulating MMP-13 expression in tendon fibroblasts.

A transient (lasting for 15 min) bilateral carotid artery occlusion model was created by using male Mongolian gerbils ( n=20, weight 50-60 g). The animals were divided into a group with mild hypothermia (34 degrees C, n=10) and a normothermic group (37 degrees C, n=10). High-energy phosphate metabolism (ATP, PCr, Pi) and intracellular pH were sequentially measured using (31)P-MRS during ischemia and after reperfusion for 1 week. The same animals were also subjected to a histopathological evaluation. During ischemia, there were no statistically significant differences between the two groups in the quantities of the metabolites. However, after reperfusion the rate of metabolic recovery by the mildly hypothermic (MH) group was significantly higher (by 10-20%) than the normothermic (NT) group. The intracellular pH decreased about 0.4 in both groups after ischemia; and after reperfusion the intracellular pH of the MH group returned to baseline levels faster than in the NT group. One week after ischemia, energy metabolism gradually decreased about 10-20% in both groups. In the histopathological evaluation, pyramidal cell damage in the hippocampus was 33% on average in the MH group and 79% in the NT group. The neuronal damage to the cerebral cortex was 26% in the MH group and 61% in the NT group. Astrocyte reactivity in the hippocampus and cerebral cortex was 2.9% and 1.1% in the MH group and 9.7% and 5.2% in the NT group. The results of this experiment indicate that the protective effect of mild hypothermia is due to the high recovery rate of ATP and PCr and the prevention of a secondary decline in high phosphate energy.

Whether cyclical changes in energy-related phosphate metabolites arise during a cardiac cycle in isolated rat hearts and are affected by differences in myosin isozyme composition was determined. Myocardial adenosine triphosphate (ATP), phosphocreatine (PCr), inorganic phosphate (Pi), and intracellular pH in normal, hypothyroid and hyperthyroid rat hearts were measured using the pacing-gated 31P nuclear magnetic resonance technique. Maximal decrease in ATP and PCr, and maximal increase in Pi at the peak-systole in normal rat hearts were observed. In hypothyroid and hyperthyroid rats, similar cyclical changes in phosphate metabolites were observed during the cycle. However, the magnitude of fluctuations was smaller in hypothyroid rats and larger in hyperthyroid rats compared with that observed in normal rats. Cardiac myosin isozyme patterns were also different amongst the experimental groups. The results suggest that cyclical changes and the magnitude of fluctuations in energy-related phosphate metabolites during a cardiac cycle may depend on the cardiac workload and the intrinsic properties in the enzyme kinetics of myosin.

It is often difficult to make a correct diagnosis of ring-like enhanced lesions on Gd-enhanced MR brain images. To differentiate these lesions using proton MR spectroscopy (1H-MRS), we retrospectively evaluated the correlation between the 1H-MR spectra and histopathological findings. We evaluated proton MR spectra obtained from the lesions in 45 patients, including metastasis (n = 19), glioblastoma (n = 10), radiation necrosis (n = 7), brain abscess (n = 5), and cerebral infarction (n = 4). The rate of misdiagnosis was found to be lowest at the threshold level of 2.48 for the (choline containing compounds)/(creatine and phosphocreatine) ratio (Cho/Cr) obtained from the whole lesions, which include the enhanced rim and the non-enhanced inner region. That is, the positively predictive values of a Cho/Cr greater than 2.48 for diagnosing metastasis or glioblastoma was 88.9 and 60.0%, respectively, and the positively predictive value of a Cho/Cr less than 2.48 for diagnosing radiation necrosis or cerebral infarction was 71.4 and 100%, respectively. For further differentiating between metastasis and glioblastoma, information about the presence and absence of an N-acetyl-aspartate (NAA) peak and lipid- or lactate-dominant peak was found to be useful. In 73.7% of metastasis cases a lipid-dominant peak was observed in the whole lesion without an NAA peak in the inner region, whereas the same pattern was observed in only 10% of the glioblastoma cases. Correlation with the histopathological findings showed that a high Cho signal is suggestive of neoplasm. Lipid signal in the non-enhanced central region was correlated to necrosis. Lactate signals were often observed in glioblastoma, abscess and sometimes metastasis, presumably reflecting the anaerobic glycolysis by the living cells in the ring-like enhanced rim. Single-voxel proton MR spectroscopy may serve as a potential tool to provide useful information of differentiation of ring-like enhanced lesions that cannot be diagnosed correctly using enhanced MR images alone.

The effects of d -cis- and l -cis-diltiazem on the hydrogen peroxide (H2O2)-induced derangements of mechanical function and energy metabolism, and accumulation of intracellular Na+ were studied in isolated rat hearts. The intracellular concentration of Na+ ([Na+]i) in the myocardium was measured using a nuclear magnetic resonance technique. H2O2 (600 μM) increased the left ventricular end-diastolic pressure, decreased the tissue level of ATP, and increased the release of lactate dehydrogenase from the myocardium. These alterations induced by H2O2 were significantly attenuated by d -cis-diltiazem (15 μM) or l -cis-diltiazem (15 μM). H2O2 (1 mM) produced a marked increase in the myocardial [Na+]i, which was effectively inhibited by tetrodotoxin (3 μM), d -cis-diltiazem (15 μM) or l -cis-diltiazem (15 μM). These results suggest that both d -cis- and l -cis-diltiazem protect the myocardium against the H2O2-induced derangements in the isolated, perfused rat heart. The protective action of d -cis- and l -cis-diltiazem may be due to their ability to inhibit the H2O2-induced increase in [Na+]i, at least in part.

Dibutyryl cAMP (DBcAMP) has a high membrane permeability, and maintenance of the intracellular cAMP concentration may improve the viability of organs. In this study, the effect of DBcAMP pretreatment on warm ischemic injury of rat livers was evaluated. Warm ischemic liver injury was induced in adult Wistar rats weighing 250-280 g by leaving them at room temperature (22-25 degrees C) after cardiac arrest. The hepatic cAMP concentration, %ATP, and trypan blue-positive nuclear ratio were determined after different durations of warm ischemia. In addition, transaminase and endothelin-1 (ET-1) release into the perfusate were examined during 60 min of isolated liver perfusion with Krebs-Henseleit solution. The optimal dose and time of DBcAMP pretreatment were determined to be 15 mg/kg and 60 min prior to warm ischemia, respectively. Data on the trypan blue-positive nuclear ratio and the release of transaminases and ET-1 revealed that warm ischemia first damaged the endothelial cells and then the hepatocytes. DBcAMP pretreatment appeared to protect the liver from warm ischemic injury by increasing the intracellular cAMP concentration and stabilizing the cell membranes of endothelial cells and hepatocytes.

A new endogenous proteinase inhibitor from the cell-free hemolymph of a solitary ascidian, Halocynthia roretzi, was purified by a combination of ammonium sulfate fractionation, hydrophobic interaction chromatography on Ether-Toyopearl and affinity chromatography on Heparin-Sepharose. The purity of the inhibitor was examined by SDS-PAGE, gel-permeation chromatography, reversed-phase chromatography, isoelectric focusing, immunological analysis and amino-terminal amino acid sequence analysis. The inhibitor is a single polypeptide chain whose molecular weight, isoelectric point and the first 10 amino-terminal amino acid sequences are 58 kDa, pI 9.2 and NH2-Thr-Lys-Lys-Asp-Gly-Glu-Glu-Lys-Val-Ala, respectively. The purified protein inhibits plasma enzyme(s) of H. roretzi, and the rate of inhibition to the plasma enzyme(s) activity was accelerated by incubation with dextran sulfate, but the effect was neutralized by further incubation with polycation, such as polybrene or protamine sulfate. The inhibitory activity was not affected appreciably by pH 7-10 but ceased completely below pH 5 or by heating at 50 degrees C for 30 min.

Using31P-NMR spectroscopy, we studied the process of the functional recovery of the regenerating rat liver at 1–5 weeks after a 65% partial hepatectomy from the viewpoint of phosphate metabolism. Portal vein clamping (20 min) or fructose administration (250 mg/kg body weight) was performed to temporarily reduce adenosine triphosphate (ATP) content in the remnant liver. Changes in ATP level were observed for 60 min after declamping the portal vein or after fructose administration. Significant differences in the increased ATP levels after declamping the portal vein or after fructose administration were observed in each hepatectomized group. Until 3 weeks after hepatectomy, ATP levels during 60 min after declamping the portal vein or after fructose administration were significantly lower than those in the nonresected control rats. However, the increase in ATP in the 4-week group was almost identical to the increase in the control group throughout the 60 min. Remnant liver weight already returned to the nonresected control level 1 week after hepatectomy, and remained constant thereafter. On the other hand, the final ATP levels 60 min after declamping the portal vein or after fructose administration returned to the nonresected control level only after 4 weeks following hepatectomy. These results demonstrate that from the viewpoint of ATP synthesis, functional recovery of the regenerating liver to normal can be observed within 4 weeks after partial hepatectomy.

モルモット単一心室筋細胞のNa電流 (INa) に対するbepridilの作用を吸引電極法を用いて検討した.bepridilは-140mVの静止状態から-30mVへの脱分極により活性化されるINaを用量依存性に減少させた (解離定数369μM) .本用量曲線は保持電位を-90mVにすると低濃度側へ平行移動した.2連パルス法によるINaの遮断発現過程実験では, 本剤は4msの条件パルスではINaを14%遮断し, 16ms以上では不活性化状態のINaをより顕著に遮断した.本剤は顕著な使用依存性抑制を示し, INa遮断が定常状態に達するまで迅速であった.定常状態におけるINaの不活性化曲線は, 本剤10μMにより11mV過分極側に偏位したが, 勾配因子は不変であった.不活性化されたINaの回復過程は, -140mVにおいて290msの時定数を示した.以上より, bepridilはNaチャネルの静止状態よりも不活1生化状態に高い親和性を有するが, 活性化チャネルブロックも一部もたらすこと, チャネルからの解離が速いことが明らかとなった.

A new gating NMR technique triggered by pulses for the cardiac pacing of isolated, perfused rat heart is described. This technique is suitable for obtaining spectra at the same positions over the cardiac cycle with the same RF pulse repetition time and with constant heart rate. Accordingly, each recovered magnetization level at these positions during the cycle can be kept constant. It also allows a qualitative analysis of the myocardial energy level during the cycle under the condition of the same or different cardiac workload (rate pressure product, HR x LVP). This method was used to observe cyclical changes in the concentration of adenosine triphosphate (ATP), phosphocreatine (PCr), P(i), and in pH during the cardiac cycle, and confirmed that the levels of ATP and PCr showed reversible decreases, and P(i) showed an antiphasic increase at the peak systole. On the other hand, no significant changes in pH were noted during the cardiac cycle. This pacing-gated technique is an improvement over other gated NMR techniques triggered by the blood pressure or by ECG traces.

The distribution of substance P (SP) immunoreactivity and the colocalization of SP with other bioactive substances in chromaffin cells and nerve fibers were investigated in the rat adrenal gland at the light microscopic level. In the capsule and cortex, SP immunoreactivity was seen in some nerve fibers around blood vessels and in thick nerve bundles passing through the cortex directly into the medulla. In the medulla, the SP immunoreactivity was observed in a small number of chromaffin cells; these SP-immunoreactive chromaffin cells were either phenylethanolamine N-methyltransferase (PNMT) immunoreactive or immunonegative, indicating that they were either adrenaline cells or noradrenaline (NA) cells. SP-immunoreactive varicose nerve fibers were also found in the medulla and were in contact with a cluster of the NA cells showing catecholamine fluorescence, which suggests that SP from medullary nerve fibers may regulate the secretory activity of the NA cells. Because no SP-immunoreactive ganglion cell was present in the rat adrenal gland, the intra-adrenal nerve fibers were considered to be extrinsic in origin. The double-immunostaining method further revealed that the SP-immunoreactive chromaffin cells also exhibit immunoreactivities for calcitonin gene-related peptide (CGRP), and neuropeptide tyrosine (NPY), suggesting that these peptides can also be released from the chromaffin cells by certain stimuli. The intra-adrenal nerve fibers in the medulla were composed of SP-single immunoreactive, and SP/CGRP-, SP/choline acetyltransferase (ChAT)-, SP/nitric oxide synthase (NOS)-, SP/pituitary adenylate cyclase activating polypeptide (PACAP)-, ChAT/NOS-, and ChAT/PACAP-immunoreactive nerve fibers, which may affect the secretory activity of the NA cells. In the adrenal capsule, the nerve fibers were present around blood vessels and showed immunoreactivities for SP/ CGRP, SP/NPY, SP/NOS, and SP/vasoactive intestinal polypeptide, suggesting that the origin of nerve fibers in the capsule may differ from those in the medulla.

Object. The goal of this study was to investigate the usefulness of proton (1H) magnetic resonance (MR) spectroscopy to evaluate the response of metastatic brain tumors to stereotactic radiosurgery (SRS) in comparison with Gd-enhanced MR imaging and single-photon emission computerized tomography with administration of thallium-201 chloride (201TlCl-SPECT). Methods. Forty patients with a total of 47 metastatic brain tumors were evaluated. The primary lesion was identified in all cases. Stereotactic radiosurgery was effective in 37 lesions. All patients were examined using Gd-enhanced MR imaging before and after SRS. Thalium-201 chloride was administered to 27 patients with 34 tumors and SPECT images were obtained. Proton MR spectroscopy was performed in 36 patients who harbored 43 tumors. On Gd-enhanced MR images, a decrease in the volume of the Gd-enhanced lesion and a change in the enhanced effect in the lesion after treatment were recognized as showing the effectiveness of SRS between 1 and 3 months or more (mean 8.54 ± 3.58 weeks). In 201TlCl-SPECT studies, the ratio of lesion to normal brain decreased from 2 weeks to 2 months (mean 5.03 ± 2.77 weeks) after radiosurgery. On 1H-MR spectroscopy images a high choline (Cho) peak and a lipiddominant (Lip) peak were observed in 25 lesions and a high Cho peak and a lactate-dominant (Lac) peak were observed in 12 lesions before SRS. A decrease in the Cho peak, a disappearance of the Lac peak, and an increase in the Lip peak were observed between 1 week and 1 month (mean 2.76 ± 1.62 weeks) after treatment. Conclusions. Based on histopathological findings obtained at autopsy or at surgery, we assume that a high Cho peak may be observed in viable tumor tissue and a Lip peak in areas of necrosis. The results indicate that 1H-MR spectroscopy is potentially a more sensitive tool in evaluating the response to SRS than 201TlCl-SPECT or Gd-enhanced MR imaging and that it can be used earlier for this purpose than those other imaging methods.

Two distinct oligosaccharide ligands that function in both the allogeneic contact/cytotoxic reaction (ACR) by hemocytes and gamete fertilization were identified by means of the monoclonal antibodies (Mabs), CRB1 and CRB2, which inhibit both the ACR and fertilization in Halocynthia roretzi. The CRB1 epitope, determined to be the “core” α1–6 fucosylated oligosaccharide epitope (CFOE) in hybrid-type N-glycans, was detected on the cell surfaces of all types of hemocytes as well as on those of follicle cells and the vitelline coat of eggs. The CRB2 epitope, which was indicated as an outer-side sulfated and phosphorylated ojigosaccharide epitope (OSPOE) in complex-type N-glycans, was demonstrated on the cell surfaces of some types of hemocytes and egg follicle cells. Both epitopes, OSPOES and CFOE, may function as ligands in the initial attachment and binding, respectively, in the sperm-egg interaction. These epitopes may also function in the same way in the self-recognition of the ACR. We propose that the fucose residue in CFOE is among the key molecules, and perhaps the most important, involved in self-recognition in the ACR and fertilization.

The viriform cell (VC) in the tunic of Halocynthia roretzi is rod shaped (about 15x5 µm) and is characterized by intracellular eosinophilic and electron-dense granules (about 1–2 µm in diameter). The VC is confirmed to be a native tunic cell by the DNA fingerprint method. Light and electron microscopic observations and immunohistochemical findings demonstrated that VCs appeared in the tunic and hemocoel of about 3-month-old specimens but were not seen in younger juvenile specimens (2 weeks after metamorphosis). There were more VCs in the tunic matrix of 1-year-old specimens than in 3-month-old specimens. It seems that the cells migrated from the hemocoel through the epidermis. In mature specimens, the density of VCs was very high not only in the inner tunic matrix but also in the tunic of growing attachment villi. When the villi became brown, broken VCs were observed at the area just under the cuticle, where the subcuticle forms later. VCs also crowded at the repairing surface of the tunic after artificial injury. These findings suggest that VCs may play roles in subcuticle formation and wound healing.

The effects of in situ freezing and the combination of in situ freezing and stress-shielding on the microstructure and ultrastructure of the patellar tendon were studied with use of 20 mature rabbits. The patellar tendon was frozen in situ with liquid nitrogen to kill fibroblasts and then was completely released from stress by chronically pulling a stainless-steel wire installed between the patella and the tibial tubercle. Microstructurally, the freezing treatment induced separation of collagen fiber bundles and fibroblast necrosis at 3 weeks, although the separation disappeared at 6 weeks. Ultrastructurally, small collagen fibrils with a diameter of less than 90 nm were predominant; at 6 weeks, the area occupied by collagen fibrils had decreased. In the frozen-shielded tendon, numerous large spaces were observed in the matrix at 3 weeks. This treatment increased the number of fibrils with a diameter greater than 360 nm and decreased the number of collagen fibrils per unit of area and the area occupied by collagen fibrils at 3 weeks. This study demonstrated that in situ freezing and the combination of in situ freezing and stress-shielding leads to a smaller volume of collagen fibrils per unit of cross section of the patellar tendon by mechanisms that remain to be defined.

A new endogenous serine proteinase from the cell-free hemolymph of a solitary ascidian, Halocythia roretzi, was purified by a combination of ammonium sulfate fractionation, hydrophobic interaction chromatography on TSKgel Toyopearl HW 65 F, ion exchange chromatography on TSKgel DEAE-Toyopearl 650 M, affinity chromatography on Arginine-Sepharose 4B, gel filtration on TSKgel Toyopearl HW 65F and hydroxyapatite chromatography on Bio-Gel HT. The serine proteinase is a single polypeptide chain whose molecular weight and isoelectric point are 39 kDa and about 7.6 pI, respectively. The most susceptible substrate was Boc-Leu-Gly-Arg-4-methyl-coumaryl-7-amide (MCA), and activity was optimal at pH 8. The enzyme was relatively stable at high temperatures; about 50% activity was retained even at 60 degrees C for 30 min in 50 mM Tris-HCl, pH 8.0, containing 0.5 M NaCl, and 0.05% Brij-35. The enzyme was characterized by the inhibitory effects of synthetic or natural inhibitors, substrate specificity toward 26 peptidyl-MCAs, proteinase activity toward natural proteins and complex formation with a serine proteinase inhibitor (58 kDa) previously found in H. roretzi hemolymph, indicating that the enzyme was a member of serine proteinases and strongly inhibited by the 58 kDa serine proteinase inhibitor as well as human antithrombin III. We also demonstrated the clotting enzyme activity of the purified serine proteinase toward bovine fibrinogen and Limulus coagulogen, a fibrinogen-like clottable protein of horseshoe crabs.

Uveitis was induced in rats by endotoxin administration. The temporal correlation between ocular inflammatory reaction and lens metabolic change was examined. The levels of lens phosphate compounds, adenosine triphosphate (ATP), choline phosphate (CP), and inorganic phosphate (Pi), were measured using 31P-nuclear magnetic resonance (NMR) spectroscopy. Anterior chamber inflammation reaction appeared 12 h after administration, peaked at 24 h, and disappeared by 72 h. Concurrent with the active inflammation, lens Pi increased significantly 22 +/- 17% (p < 0.05) and 23 +/- 15% (p < 0.05), 12 and 24 h after administration, respectively, and returned to normal while the inflammation abated. No significant changes were observed in CP and ATP levels in the lens during the course of inflammation. The time course of the inflammation appeared closely related to the change in Pi level, which may be part of the protective mechanism of the lens against inflammation.

We compared the abilities of three solutions (bicarbonate-free glucose-phosphate Ringer's solution (EP II), lactated Ringer's solution, and physiologic saline solution) to preserve pig cornea by measuring phosphate metabolites with 31 P NMR spectroscopy. EP II preserves corneal viability better than others, and preserving extracted cornea also is better than preserving the whole eye

This study was designed to investigate whether vagal innervation and mucosal prostaglandins (PGs) participate in gastric adaptive cytoprotection. Rats were divided into three groups; sham operation (control), truncal vagotomy or splanchnicotomy. In the first experiment, 100% ethanol (EtOH) was orally administered 15 min after pretreatment with 20% EtOH to all 3 groups. One hour later, the gastric mucosa was examined macroscopically. In a second experiment, the mucosal PG contents 15 min after administration of either 20% EtOH or saline were measured by high performance liquid chromatography. In truncal vagotomized rats, the adaptive cytoprotection caused by exposure to 20% EtOH in control and splanchnicotomized rats was not observed and an increase in hemorrhagic lesion severity was seen. In the control and splanchnicotomized rats, PGE2 contents were elevated following 20% EtHO treatment, as compared to those in the saline-treated rats. However, PGE2 contents in vagotomized rats were not altered by EtOH exposure, and were significantly lower than in the control and splanchnicotomized groups, whereas PGF2 alpha and PGD2 contents were significantly higher after EtOH administration as compared to those in saline-treated rats. These results suggest that vagal innervation is essential for adaptive cytoprotection and that the vagotomy-induced decrease in PGE2 and increases in PGF2 alpha and PGD2 following 20% EtOH administration, may be caused by a disturbance in adaptive cytoprotection.

We have investigated the compositional modulation of AlGaAs layers grown in tetrahedral-shaped recesses (TSRs) on n-type GaAs (111)B substrates using scanning electron microscopy, photoluminescence and cathodoluminescence. Growth hardly occurred between the TSRs, and complicated facets were seen in the TSR. Gallium-rich regions were formed around the edges between sidewalls rather than at the center of sidewalls and at the bottom region rather than the top region. To observe the facetting process during growth, GaAs/AlGaAs multilayers were also grown in TSRs. Aluminum compositional modulation is dependent not only on the difference in migration distances of Ga and Al atoms but also on the differences in the incorporating process among different facets.

The KrF laser ablation of Al, Cu, Si, and SrTiO3 targets has been studied using an electrostatic deflection energy analyzer and a quadrupole mass filter. Beam like ions appear to accompany ablation. The average kinetic energies of Si+ and metal ions are about 45 eV and 60 eV, respectively. The energies of ions are ranked in the order of Si3. The mean ion energy of Cu+ is not affected by the laser fluence range of 2-7.5 J/cm2.

Effects of fertilization and foliar spray treatment on the ascorbic acid content of lettuce and garland chrysanthemums were investigated.1. The growth of garland chrysanthemums was not affected by the treatment of different compositions in nutrient solution, but that of lettuce was greatly influenced and dry weight decreased in 1/2N, 1/2P, 1/2K, 1/2Ca, and 1/2Mg regimes. The ascorbic acid content of both vegetables were inclined to decrease in 3/2N, 3/2K, and 1/2Mg regimes and to increase in 3/2P, 1/2Ca, and 3/2Mg regimes of nutrient solutions. Urea foliar spray treatment largely increased the ascorbic acid content.2. The fresh weight of lettuce grown in 1/4 fold of standard nutrient solution was decreased 27-46% as compared with that of 1/2 fold or more concentrations, but the ascorbic acid content was singularly increased at that time. There are positive correlations between the ascorbic acid and sugar content, and these relationships were discussed. A total of four or five foliar spray treatments of 0.5% of urea solution every 5 days was expected to increase the ascorbic acid content.3. The treatment of very low concentration of s-triazine herbicide did not increase the growth of lettuce. But foliar spray treatment of 2.5% simazine solution increased the ascorbic acid and sugar content, and decreased the NO3-N content.

Bradykinin was injected retrogradely into a branch of the splenic artery of lightly anesthetized dogs, and the reflex hypertensive response was used as an indicator of the nociception. The reflex hypertensive response to low doses of bradykinin (less than 1 nmol), but not to high doses (3-5 nmol), was markedly suppressed by infusion of indomethacin (0.54 mumol/min) into the splenic artery. During indomethacin infusion, the reflex hypertensive response to low doses of bradykinin was potentiated dose dependently by the following arachidonic acid metabolites (ED50): prostaglandin (PG)I2 (2.9 nmol) greater than or equal to PGH2 (4.4) greater than PGE2 (14) = thromboxane (TX)A2(15) greater than PGD2 (greater than 100). Leukotrienes B4, C4 and D4 showed practically no activity. This potentiating effect lasted up to 20 min after injection, particularly with PGE2. These results may not exclude the role of PGI2 as a major candidate for involvement in bradykinin-induced nociception, although a selective TX synthetase inhibitor (OKY-046) showed a weak analgesic effect (only 1/30 that of indomethacin).

7-Ethoxycarbonyl-6,8-dimethyl-4-hydroxymethyl-1(2H)-phthalazinone (EG-626) was reported as an antagonist of thromboxane (Tx) A2 in the contraction of rabbit aorta. It was, however, observed that EG-626 did inhibit the contraction of superfused rabbit aorta, but also did inhibit that of rabbit coeliac artery, rat stomach strip and rat colon induced by TxA2, PG endoperoxides, angiotensin II and PGF2α in non-specific manner. EG-626 had no effect on the biosynthesis of PG endoperoxides as well as TxA2. These results indicate that EG-626 is not a TxA2 antagonist, but has a general inhibitory effect on the smooth muscles. This inhibitory effect of EG-626 may be explained by the inhibition of phosphodiesterase.

Anti-aggregating activity of 7-ethoxycarbonyl-6,8-dimethyl-4-hydroxymethyl-1(2H)-phthalazinone (EG 626) was tested using rabbit platelets in vitro. EG-626 alone, when added before, prevented platelet aggregation induced by ADP, as did PGI2, papaverine and dipyridamole. Spontaneous disaggregation was also accelerated when EG-626 was added after the maximal aggregation induced by ADP. EG-626 alone also inhibited platelet aggregation induced by collagen and arachidonic acid. ID50s of these agents in ADP-induced aggregation were 7-9 nM for PGI2, 223 microM for EG-626, 266 microM for papaverine and 957 microM for dipyridamole. When EG-626 was used in combination with PGI2, a threshold dose (50 microM) of EG-626 potentiated the antiaggregation effect of subthreshold dose (3 nM) of PGI2 upto 100% inhibition in collagen-induced platelet aggregation. The marked potentiating effect of EG-626 was accompanied by an accumulation of cyclic AMP in the platelets. These effects might be due to inhibition of phosphodiesterase. Papaverine and dipyridamole, other phosphodiesterase inhibitors, also potentiated the anti-aggregating activity of PGI2. The activity of papaverine, however, was one eighth of EG-626 and that of dipyridamole was much less. The most effective combination of PGI2 and EG-626 to induce 50% inhibition was obtained with 20% of ID50 of each agent, whereas that of PGI2 and papaverine or dipyridamole was 39 or 41%, respectively.

The activity of prostaglandins (PG) in producing vascular permeability was quantitated by dye extraction method in skin of anaesthetized rabbits. PGE1 and PGE2 (0.01–10 μg) produced increase in vascular permeability. Activity was approximately equal to that of histamine (Hist) and 1 20 of that of bradykinin (BK) on a weight basis. The activity of PGF1α and PGF2α was only 1 20 of that of PGE1 or PGE2. In spite of the relatively low potency of PGE1 and PGE2 in the rabbit, near threshold doses (0.1 or 1 μg) of PGE2 could potentiate permeability responses to bradykinin (0.1 μg) by 10 or 100-fold, respectively. Equivalent doses (0.1 or 1 μg) of histamine could not potentiate the bradykinin responses. Arachidonic acid (AA) at 1 μg, produced a 10-fold potentiation in the permeability response to bradykinin (0.1 μg). Pretreatment of the rabbits with indomethacin (20 mg/kg, i.p.) reduced the responses of BK (0.1 μg) + AA (1 μg) down to a similar magnitude of those seen with bradykinin alone. However, indomethacin did not block responses to either, BK alone, BK + PGE2, or BK + Hist. Various doses (1, 10, 100 and 300 μg) of arachidonic acid alone also produced increase in cutaneous vascular permeability, although its potency was only 1 3 – 1 8 of that of PGE2. This activity of arachidonic acid was attributed in part to its bioconversion to PGE2, since its activity was significantly reduced by the prostaglandin antagonist, diphloretin phosphate (DPP) (60 mg/kg, i.v.) and by indomethacin (20 mg/kg, i.p.), which blocks conversion of arachidonic acid to prostaglandins. Arachidonic acid may owe some of its permeability increaseing effects to histamine release, since its effects were also reduced by the antihistamine, pyrilamine (2.5 mg/kg, i.v.).

Rat carrageenin-induced pleurisy was used as an acute exudative inflammatory model. The crude ethanol extract of the pleural fluid at 5 hr after carrageenin injection caused the very slow contraction of guinea-pig ileum, which was antagonized by FPL 55712 (1 microgram/ml). The ethanol extract was cleaned by LH-20 and was rendered for separation of LTC4 and LTD4 by reversed-phase high-performance liquid chromatography (HPLC). Two peaks which showed the same retention time on HPLC as those of LTC4 and LTD4 had the contractile activity of guinea-pig ileum and the ratios of the contractile activity to the height on HPLC agreed with those of synthetic LTC4 and LTD4. Two peaks of delta 6-trans-LTB4, 5S, 12R-(E, E, E, Z)-diHETE and 5S, 12S-(E, E, E, Z)-diHETE, were detected, but the appreciable amount of LTB4 was smaller than that of each delta 6-trans-LTB4 in the pleural fluid at 5 hr.

Isolated rat aortae were incubated at 22 degrees C in tris-buffered saline (pH 7.4). The incubation medium was changed every 10 min, and the amounts of prostacyclin (PGI2) in the medium were immediately bioassayed as an inhibitory activity against rabbit platelet aggregation induced by ADP. The addition of arachidonic acid to the medium increased the generation of PGI2 but this was followed by a gradual decrease even in the presence of the same amount of arachidonic acid. The decrease of PGI2 generation from exogenous arachidonic acid was prevented by tryptophan, which is required by PG hydroperoxidase with heme compound as cofactors. MK-447 and its analogues, which are phenolic compounds and exerted tryptophan-like action on the PG endoperoxide biosynthesis by bovine seminal vesicle microsomes, also prevented the decrease of PGI2 generation in isolated rat aortae. The phenolic compounds enhanced PGI2 generation from endogenous arachidonic acid. These results indicate that the phenolic compounds enhanced PGI2 generation in vascular tissue, acting as a tryptophan-like cofactor of PG hydroperoxidase.

The actions of leukotriene (LT) C 4 and D 4 on the systemic arterial pressure and the insufflation pressure in guinea pigs and rabbits were examined. In guinea pigs, 0.3 – 3 nmole/kg of LTC 4 and 0.1 – 1.0 nmole/kg of LTD 4 administrated from left jugular vein caused dose-dependent increase of the airway resistance measured by the Konzett-Rossler method and a triphasic blood pressure response; an initial hypotension, a secondary hypertension and a third long-lasting hypotension. All of the hypertensive phase and 100 – 150% of the increase of the airway resistance by LTC 4 and LTD 4 were inhibited by a selective thromboxane synthetase inhibitor, OKY-1581 (10 mg/kg, i.v.) and only the hypertension was observed. Indomethacin (10 mg/kg, i.p.) also inhibited not only the airway resistance increase, but also the prolonged hypotension by LTC 4 and shortened the duration of the hypotension by LTD 4 . It is suggested that thromboxane might be involved in bronchoconstriction and hypertensive effects by LTC 4 and LTD 4 and that hypotensive prostaglandin might be involved in the hypotensive phase after LTC 4 and LTD 4 . In rabbits, the increse of the airway resistance by LTC 4 and LTD 4 (upto 100 nmole/kg, i.v.) was negligible and only the hypotension was observed.

A single platelet thrombus was formed in an arteriole of the hamster cheek pouch by electrical stimulation followed by topical application of ADP. The sizes of the thrombi were continuously recorded with a photocell placed on a TV monitor screen and quantified by areas on the record. Repeated application of small doses of ADP (5–15 nmol/10 μl) resulted in very reproducible formation of the thrombi, and the size of the thrombi was reduced dose-dependently by topical application of PGI2. Three drugs were tested in this model. Cycloxygenase inhibitor (indomethacin 10 mg/kg, i.p.) increased the formatiion of thrombi, while a smaller dose (3 mg/kg) did not have any significant effect. This could be explained by inhibition of the generation of endogemous PGI2, since aggregation of hamster platelets by ADP was not inhibited by indomethacin in vitro . EG-626 (phthalazinol, a phosphodiesterase inhibitor) (300 mg/kg, i.p.) decreased the size of thrombus. AI-122 (1.0 mg/kg, i.p.) which has been proven to enhance PGI2 biosynthesis from isolated rat aortae, also decreased the formation. Thus, drugs such as EG-626 or AI-122 are quite promising as anti-thrombic drugs.

A lithium and carbamazepine combination was reported in five patients with bipolar disorder (DSM-III), who had not received benefits from conventional treatments. Patients 1, 2 and 4 showed favorable antimanic responses to carbamazepine used in combination with lithium carbonate. Patient 3 did not respond well to either lithium or carbamazepine alone, but improved dramatically on the simultaneous administration of the two drugs. The prophylactic benefit was observed in two patients. These clinical results suggest that a lithium and carbamazepine combination may be more efficacious in the treatment of acute manic episode and the prevention of affective recurrences than each drug alone.

Twenty-four drugs, consisting of antipsychotics, tricyclic antidepressants, other centrally acting drugs, and related compounds, were studied for their effects on aggregation of rabbit platelets. Phenothiazine neuroleptics, haloperidol, sultopride, tricyclic antidepressants, sulpiride, atropine, and propranolol showed a selective inhibitory effect on the collagen-induced aggregation, but not on aggregations induced by arachidonic acid (AA) or adenosine diphosphate (ADP). These drugs are thought to inhibit the liberation of AA from phospholipids in platelet membranes, suggesting that they might inhibit phospholipases. Mepyramine, promethazine, phentolamine, and clozapine inhibited the aggregations evoked by collagen and AA, but failed to inhibit the ADP-induced aggregation. These drugs might inhibit the generation of prostaglandin endoperoxides or thromboxanes. Phenobarbital, phenytoin, procaine, lidocaine, flurazepam, trihexyphenydil, and lithium carbonate did not inhibit any kind of aggregation at the concentrations used. The clinical and pharmacological significance of these findings is discussed; it seems that the inhibitory effects of antipsychotics and antidepressants on platelet aggregation are closely related to the specific clinical and psychotropic effects of these drugs, but not to other actions.

Injection of γ-carrageenin into t he pleural cavity of rats caused the accumulation of the pleural exudate. When levels of prostaglandins (PGs) and thromboxane (TX) B 2 were quantified by gas chromatography-mass spectrometry as their methyl ester (ME)-dimethyllisopropylsilyl (DMiPS) ether or ME-methoxine-DMiPS ether derivatives, 6-keto-PGF 1α reached the maximum at 1 hr after carrageenin, then PGE 2 and TXB 2 showed peaks at 3 hr and waned off before 9 hr. he PGF 2α level was kept low, but PGD 2 , PGE 1 and PGF 1α were not detected. Aspirin (100 mg/kg, i.p.) significantly decreased the PG and TXB 2 levels and suppressed the rate of plasma exudation until 5 hr, but did not at 7 hr, when it was measured by the amount of exuded pontamine sky blue injected intravenously. OKY-025 (300 mg/kg, i.p.), a selective TXA synthetase inhibitor, and tranylcypromine (20 mg/kg, i.p.), a PGI synthetase inhibitor, could not extensively inhibit the accumulation of the exudate. These results suggest that the cyclooxygenase products of arachidonic acid, particularly PGE 2 , definitely play an important role in the exudation during the first 5 hr.

The activity of synthetic LTC4 was tested in guinea-pig ileum and was 200 times more potent than histamine in contraction of the ileum (3 × 10−11 M- 3 × 10−9 M). The activities of LTC4 and LTD4 in increased vascular permeability in guinea pigs, rats and rabbits were compared with those histamine, bradykinin and prostaglandin (PG) E2. LTC4 was approximately equipotent to bradykinin on a molar basis in guinea pigs and rats and 5–100 times more potent than histamin. LTD4 was about 10 times more potent than LTC4 in guinea pigs and as equipotent to LTC4 in rats. On the contrary, in rabbits, neither LTC4 (upto 30 nmole/site) nor LTD4 (1 nmole/site) induced the dye exduation. These results show that species difference is present in activity of LTC4 and LTD4 in vascular permeability. Furthermore, in guinea pigs, the vascular permeability increased by LTC4 was not affected after pretreatment with pyrilamine (2.5 mg/kg, i.v.), and LTC4 and LTD4 did not potenciate the activity of bradykinin in vascular permeability.

To clarify the vasoconstrictory action of leukotriene D4 (LTD4) on the injured coronary arteries, 8 mongrel dogs were denuded by basket balloon catheter (denuded group) and LTD4 (0.33μg) was directly administered into left anterior descending coronary artery. Hemodynamic parameters as well as epicardial ECG were recorded after LTD4 administration. Another 8 non-denuded dogs were served as control.Furthermore, the influence of denudation to LTD4-induced coronary spasm was determined by coronary arteriography (CAG) . In results, a significant reduction of coronary blood flow (from 1.03±0.13 to 0.49±0.05 ml/kg/min. P

Rat pleurisy was induced by intrapleural injection of lambda-carrageenin. The pleural exudate began to be accumulated 1-3 h after carrageenin administration and showed a peak at 19 h. The levels of prekallikrein and high-molecular-weight (HMW), but not low-molecular-weight (LMW), kininogen in the pleural fluid were markedly decreased when compared with those in plasma. Prekallikrein in rat plasma was activated by incubation with carrageenin in vitro. Captopril increased the plasma exudation significantly at 1-5 h. Depletion of prekallikrein and HMW kininogen in rat plasma by preadministration of bromelain caused marked inhibition of the plasma exudation at 1-24 h. The rest of the plasma exudation after bromelain was further decreased by simultaneous pretreatment of rats with both bromelain and aspirin. These results clearly indicate that plasma prekallikrein was activated in the pleural cavity and bradykinin released was responsible for plasma exudation during the entire course of this pleurisy.

Purified kerosene was fractionated for 5°C rise in boiling point. Vapor-liquid equilibria of each fraction containing a minute amount of diethyl sulfide of w-propyl mercaptan were measured at 1 atmosphere. The equilibrium ratios of the minor components in each fraction are expressed as functions of the boiling point of the fraction.

Prekallikrein activity in plasma was assayed using a synthetic peptidyl fluorogenic substrate (carbobenzoxy-L-phenylalanyl-L-arginine 4-methylcoumarinyl-7-amide), after activation of prekallikrein by acetone and kaolin. For total kininogen assay, the pretreatment of plasma at pH 2.0 was the best to eliminate bradykinin potentiators and kininase activity, before addition of trypsin to convert kininogen to bradykinin. Assay method of high molecular weight (HMW) kininogen was established by conversion of HMW-kininogen to bradykinin through activation of Hageman factor by glass powder and that of low molecular weight (LMW) kininogen was also by treatment of HMW-kininogen-depleted plasma in the same way as that for total kininogen.

The effects of chlorpromazine (CPZ) and imipramine (IMP) on platelet aggregation in vitro were investigated. Both CPZ and IMP inhibited epinephrine-induced secondary aggregation in a dose-dependent fashion and changed adenosine diphosphate (ADP)-induced irreversible aggregation into a reversible response in human platelets. With rabbit platelets, CPZ (0.6-20 microM) and IMP (6-60 microM) showed a selective inhibitory effect on the aggregation by collagen, whereas, even at 200 microM, this effect was found neither in arachidonic acid (AA)-induced aggregation nor in ADP-induced one. CPZ and IMP also inhibited human platelet aggregations induced by collagen, but not by AA. The antiaggregating action of CPZ and IMP was thought to be due to their inhibitory effects on AA liberation from platelet membranes, suggesting that they might inhibit participating enzymes (phospholipase A2, C or lipase) or the process of the activation of the enzymes.

Intrapleural injection of 2% λ-carrageenin caused the accumulation of exudate up to 19 hr. The rate of plasma exudation, measured by the exuded dye amounts for 20 min in the pleural cavity after intravenous injection of pontamine sky blue, showed a peak at 5 hr. Aspirin (100 mg/kg, i. p.) suppressed the dye exudation up to 5 hr, but did not at 7 hr. This inhibition coincided with the decrease of the PG and TXB2 levels, which were measured by gas chromatography-mass spectrometry, in the pleural exudate. In vitro experiments, MK-447, a phenolic compound, stimulates PG endoperoxide biosynthesis at lower doses and inhibits it at higher doses, acting as a tryptophan-like cofactor required by PG endoperoxide synthetase. This drug (0.3, 1.0 and 3.0 mg/kg, i. p.) suppressed the dye exudation dose-dependently up to 5 hr, but did not at 7 hr even at a higher dose, in combination with the dose-dependent decrease of the pleural level of PGE2, which was reported to be a major PG among PGs and TXB2 in the exudate in inducing the plasma exudation (Harada et al ; Prostaglandins, 23 : 881, 1982). Thus, the anti-inflammatory action of MK-447 can be explained by inhibition of PGE2 generation, giving no consideration to the role of oxygen-derived free radicals as a prime mediator in inflammation.