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A series of our studies on the dog venous system revealed that cholinergic excitatory innervation was localized in a group of veins: the portal, mesenteric, and hepatic veins and the middle segment of the inferior vena cava. Our studies on pharmacological responsiveness of dog veins also revealed that they could be divided into two groups: the visceral and somatic parts, and the cholinergic excitatory innervation localized to the visceral part. Considering these results and some relevant literature, a hypothesis is proposed on the classification of muscles of the cardiovascular system and some embryological aspects of the parasympathetic cholinergic innervation in the circulatory system are discussed. The embryonic circulatory system of vertebrates can be divided into two parts: somatic and visceral. The body of an embryo is regarded as a double tube and vessels of the visceral part and the heart belong to the inner tube. The muscle of these vessels and the heart are derived from visceral mesoderm, either the coelomic epithelium or mesenchymal cells, in common with muscle of the digestive tube; and thus the parasympathetic cholinergic nerves innervating the muscle of the digestive tube also distribute to these vessels and the heart. The heart and vascular muscles in the visceral part are structures developed early in the course of evolution in invertebrates. Their primary function is to propel the body fluid, and the chief structure containing them is the subintestinal circulatory channel (ventral aorta − heart − subintestinal vein). They exhibit spontaneous, rhythmic activity, showing characteristics of a single unit muscle, and receive parasympathetic cholinergic innervation. On the other hand, the vascular muscles in the somatic part are endothelium-associated muscles developed anew in the vertebrate; do not contract spontaneously, being classified as a multiunit muscle; and lack parasympathetic cholinergic innervation.

Abstract The protective effects of sarpogrelate (SG), a 5-HT2A antagonist, were investigated in perfused guinea-pig Langendorff hearts subjected to ischemia and reperfusion. Changes in cellular levels of high phosphorous energy, NO and Ca2+ in the heart together with simultaneous recordings of left ventricular developed pressure (LVDP) were monitored using an nitric oxide (NO) electrode, fluorometry and 31P-NMR. The recovery of LVDP from ischemia by reperfusion was 30.1% in the control, while the treatment with SG (5×10-7 M) in pre- and post-ischemia hearts produced a gradual increase to 73.1 and 53.6%, respectively. At the final stage of ischemia, the intracellular concentration of Ca2+ ([Ca2+i) and release of NO increased with no twitching and remained at a high steady level. The addition of SG increased the transient NO signal (TNO) level at the end of ischemia compared with the control, but [Ca2+]i during ischemia decreased. Meanwhile, mitochondrial Ca2+ uptake on acidification or Ca2+ content changes of the perfusate was suppressed by pre-treatment with SG or the KATP channel opener diazoxide, but not the KATP channel blocker 5-HD. The myocardial NO elevated with 5-HT in normal Langendorff hearts was suppressed by the treatment with SG. Therefore, the existence of the 5HT2A receptor in a Langendorff heart was anticipated. By in vitro EPR, SG was found to directly quench the hydroxy radical. Thus, these findings suggested that the 5-HT2A receptor induced in ischemia–reperfusion plays an important role in the mitochondrial KATP channel of hearts in close relation with NO and active oxygen radicals.

N-(p-Coumaroyl)serotonin (C) and N-feruroylserotonin (F) with antioxidative activity are present in safflower oil. The protective effects of C and F were investigated in perfused guinea-pig Langendorff hearts subjected to ischemia and reperfusion. Changes in cellular levels of high phosphorous energy, NO and Ca2+in the heart together with simultaneous recordings of left ventricular developed pressure (LVDP) were monitored by an nitric oxide (NO) electrode, fluorometry and 31P-NMR. The rate of recovery of LVDP from ischemia by reperfusion was 30.8% in the control, while in the presence of C or F a gradual increase to 63.2 or 61.0% was observed. Changes of transient NO signals (TNO) released from heart tissue in one contraction (LVDP) were observed to be upside-down with respect to transient fura-2-Ca2+signals (TCa) and transient O2signals detected with a pO2electrode. At the final stage of ischemia, the intracellular concentration of Ca2+([Ca2+]i) and the release of NO increased with no twitching and remained at a high steady level. The addition of C increased the NO level at the end of ischemia compared with the control, but [Ca2+]iduring ischemia decreased. On reperfusion, the increased diastolic level of TCaand TNOreturned rapidly to the control level with the recovery of LVDP. By in vitroEPR, C and F were found to directly quench the activity of active radicals. Therefore, it is concluded that the antioxidant effects of two derivatives isolated from safflower play an important role in ischemia-reperfusion hearts in close relation with NO.

Inducible nitric oxide (NO) production in macrophages plays an important role in atherosclerosis, the protective effects of vitamin E and its derivatives perhaps being partly mediated by alteration in this parameter. We have investigated the influence of a novel synthesized vitamin E derivative, 1‐0‐hexyl‐2,3,5‐trimethylhydroquinone (HTHQ), on NO production in the RAW 264.7 mouse macrophage cell line. HTHQ dose‐dependently inhibited lipopolysaccharide (LPS)‐induced NO production through reducing LPS‐triggered inducible nitric oxide synthase (iNOS) expression. The phosphorylation and subsequent degradation of IKB caused by LPS in RAW 264.7 cells was markedly blocked. The free radical scavenging activity of HTHQ was only 2‐fold that of vitamin E, whereas its inhibition of NO production was found to be nearly 500‐fold stronger. Our results indicated that HTHQ suppressed NO production in macrophages by blocking IKB degradation and thus inhibiting iNOS expression. The inhibitory activity of HTHQ on NO production did not parallel its free radical scavenging activity, implying a possible involvement of additional functions.

Inducible nitric oxide (NO) production in macrophages plays an important role in atherosclerosis, the protective effects of vitamin E and its derivatives perhaps being partly mediated by alteration in this parameter. We have investigated the influence of a novel synthesized vitamin E derivative, 1-0-hexyl-2,3,5-trimethylhydroquinone (HTHQ), on NO production in the RAW 264.7 mouse macrophage cell line. HTHQ dose-dependently inhibited lipopolysaccharide (LPS)-induced NO production through reducing LPS-triggered inducible nitric oxide synthase (iNOS) expression. The phosphorylation and subsequent degradation of IKB caused by LPS in RAW 264.7 cells was markedly blocked. The free radical scavenging activity of HTHQ was only 2-fold that of vitamin E, whereas its inhibition of NO production was found to be nearly 500-fold stronger. Our results indicated that HTHQ suppressed NO production in macrophages by blocking IKB degradation and thus inhibiting iNOS expression. The inhibitory activity of HTHQ on NO production did not parallel its free radical scavenging activity, implying a possible involvement of additional functions.

Effects of FK409 were investigated in perfused guinea-pig Langendorff hearts subjected to ischemia and reperfusion. Nitric oxide electrode, fluorometry, and 31P nuclear magnetic resonance imaging were used to monitor changes in cellular high-phosphorous energy and nitric oxide and Ca 2content in the heart together with simultaneous recordings of left ventricular developed pressure. After cardioplegic arrest with St. Thomas' Hospital solution, normothermic (37°C) global ischemia was induced for 40 min, and hearts were reperfused for 40 min. FK409 at 10 −8M, which has a minimum inotropic effect on nonischemic hearts, was added to the cardioplegic solution. Treatment with FK409 reduced left ventricular developed pressure during and after ischemia and improved postischemic recovery of left ventricular developed pressure from 55.4 at 40 min of reperfusion in FK409-free hearts up to 80.4 in hearts treated with FK409 (p < 0.01). Flow rate at 1.5 min after treatment with the cardioplegic solution was 27.7 ml/min in hearts treated with FK409 compared with 21.2 ml/min in drug-free hearts (p < 0.01). Treatment with FK409 significantly effected preservation of tissue level of -adenosine triphosphate at the end of ischemia or reperfusion. During ischemia, arrested with the cardioplegic solution, intracellular Ca 2accumulation and nitric oxide release were reduced. At the end of ischemia in FK409-treated hearts, nitric oxide release was 86 greater than in drug-free hearts without reference to the Ca 2concentration. In cardiac surgery, normothermic arrested hearts are subject to damage by oxygen free radicals in reperfusion injury. Therefore, nitric oxide exogenously supplied by FK409 was responsible for the cardioprotective action, presumably by acting directly as an oxygen radical scavenger during reperfusion. A specific nitric oxide donor, like FK409, may have therapeutic use as a nitric oxide–mediated vasorelaxant and additional protective action for reperfusion-injury hearts.

The effect of a novel Na+-H+exchange inhibitor, SM-20550 [N-(aminoiminomethyl)-1,4-dimethyl-1H-indole-2-carboxamide methanesulfonate] (SM) on the ion transport of myocardial mitochondria was studied using ion fluorometry and superfusion techniques. Isolated mitochondria from the guinea-pig heart were pre-loaded with fluoroprobes of either BCECF AM for H+, SBFI AM for Na+or fura-2 AM for Ca2+. Initially, the treated mitochondria were superfused with a normal medium (MOPS-buffer, pH 7.4, 24°C), subsequently fluorometric experiments on the Na+, H+, Ca2+mobilization across the mitochondrial membrane were performed. The intramitochondrial pH (pHm) was increased by the superfusion of Na+at physiological cytosolic concentrations of 10 mM, indicating the existence of a Na+-H+exchange in mitochondrial membranes. The Na+induced elevation of pHmwas dose-dependently inhibited by SM 1 μM (ΔpHm; 45% as drug-free 100%), and 10 μM (ΔpHm; 70%(, as observed in our experiments with the myocardial sarcolemmal membrane. The selective Na+-H+exchange inhibitor SM reduced such pHmelevations more markedly than that of EIPA [5-(N-ethyl-N-isopropyl) amiloride]. The Na+-H+exchange inhibitors, SM and EIPA suppressed the intramitochondrial Ca2+elevation ([Ca2+]m) brought on by external Ca2+concentration changes: The pretreatment with SM 1 μM, 10 μM and EIPA 10 μM reduced the [Ca2+]minflux by 28.3, 56.5 and 63%, respectively. Additionally, the [Ca2+]melevation induced by acidification of the perfusate was reduced by the prior infusion of SM and EIPA. Pretreatment of mitochondria with SM or EIPA which had beneficial effects on the left ventricular developed pressure (LVDP) in the ischemia-reperfusion injury of Langendorff hearts, reduced the intramitochondrial Na+and pHmlevels, indicating interplay of the inhibitory mechanism of Ca2+-uptake into mitochondria coupled with Na+-H+exchange. These findings suggested that protective effects of Na+-H+exchange inhibitors on reperfused myocardium are due in part to the Ca2+-paradox at the mitochondria level.

The protective effects of Na+-H+exchange inhibitors SM-20550 (SM) and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) against ischemia-reperfusion injury were investigated in guinea pig Langendorff hearts. The changes in intracellular pH (pHi), high-energy phosphates, and biologic intracellular active ions (Na+iand Ca2+i) were regarded using the 31P-NMR and specific fluorescent signals from the heart tissues together with simultaneous recordings of the left ventricular developed pressure (LVDP). The recovery rate of LVDP from ischemia (40 min) by reperfusion was 36.8% in the control experiments, whereas in the presence of SM 10−7M,a gradual increase to 75.9% (55.5% with 10−8M), in contrast to EIPA (10−7M), 47.5% was observed. SM 10−7Mrestored the ATP level by 70% in 40-min reperfusion, which was already higher than the control in the latter half (20-40 min) of the ischemic period. The recovery rate of phosphocreatine by pretreatment of the heart with SM 10−7Mwas 75% in 40 min reperfusion. The pHiestimated from Pi/phosphocreatine chemical shift became highly acidic in ischemic heart so that SM 10−7Mcaused slight but significant pHireduction from control pHiof 5.89 to 5.75. The level returned to pHiat around 7.38 during 30-40 min reperfusion, and the recovery was significantly greater than the control pHiof 7.24. The fura-2 Ca2+or SBFI-Na+signals during Langendorff ischemia heart increased, and rapidly returned to the control level after the reperfusion. SM suppressed the Na+ior Ca2+ielevation induced in the late stage during ischemia, resulting in LVDP restoration after reperfusion; Diastolic Ca2+in the end period of ischemia, SM 10−7M194% versus drug-free 220.7%, Na+: SM 10−7M121.6% versus drug-free 128.0%. The present results suggest that the selective Na+-H+exchange inhibitor SM is promising as a potent and specific protective agent against ischemia-reperfusion injuries with Ca2+overload induced via Na+-H+, Na+-Ca2+exchange.

4-Hydroxynonenal (HNE), a diffusible product of lipid peroxidation, has been suggested to be a key mediator of oxidative stress-induced cell death. In this study, we partially characterized the mechanism of HNE-mediated cytotoxicity. Incubation of human T lymphoma Jurkat cells with 20-50 μM HNE led to cell death accompanied by DNA fragmentation. Western blot analysis showed that HNE-treatment induced time- and dose-dependent activation of caspase-8, caspase-9 and caspase-3. HNE-induced caspase-3 processing was confirmed by a flow cytometric demonstration of increased catalytic activity on the substrate peptide. HNE treatment also led to remarkable cleavage of poly(ADP-ribose) polymerase (PARP), which was prevented by pretreatment of cells with DEVD-FMK as a caspase-3 inhibitor. The HNE-mediated activation of caspases, cleavage of PARP and DNA fragmentation were blocked by antioxidants cysteine, N-acety-L-cysteine and dithiothreitol, but not by two other HNE-reactive amino acids lysine and histidine, or by cystine, the oxidized form of cysteine. HNE rapidly decreased levels of intracellular reduced glutathione (GSH) and its oxidized form GSSG, and these were also attenuated by the reductants. Coincubation of Jurkat cells with a blocking anti-Fas antibody prevented Fas-induced but not HNE-induced activation of caspase-3. HNE also activated caspase-3 in K562 cells that do not express functional Fas. Our results thereby demonstrate that HNE triggers oxidative stress-linked apoptotic cell death through activation of the caspase cascade. The results also suggest a possible mechanism involving a direct scavenge of intracellular GSH by HNE.

ABSTRACTAcute respiration distress syndrome (ARDS) is a typical complication in toxic shock-like syndrome (TSLS) caused byStreptococcus pyogenes. An isolated perfused rat lung model was used to identify the causative agent of ARDS in TSLS in this study. Some crude preparations separated from the culture supernatants ofS. pyogenesisolates caused rapid increases in the weight of perfused lungs, indicating vascular permeabilization. Six samples from M type 1 and 3 isolates from TSLS and pharyngitis patients showed strong permeabilization activity, whereas preparations from isolates of other M types (although the number of isolates examined was limited) were negative. The active substance was purified to a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using various columns, and the N-terminal amino acid sequence was determined. The resultant sequence of eight amino acids was identical to SpeF (mitogenic factor). Moreover, the vascular permeabilization activity of the purified band was abolished with anti-SpeF antiserum prepared by immunizing with the purified SpeF. This activity was also neutralized by the antiserum prepared by immunizing with a synthetic peptide derived from the published SpeF sequence. These results suggested that streptococcal SpeF is a major cause of permeabilization of lung blood vessels and sufficient for the pathogenesis of ARDS under the conditions of TSLS caused by S. pyogenes.

We noted that eight outbreaks of Shigella sonneifrom an unknown source occurred sequentially in Aichi Prefecture, Japan, between October 1992‐June 1993. For comparative purposes we analyzed 53 outbreak‐related isolates of Shigella sonneiusing different subtyping methods and studied the epidemiology of the outbreaks. It appeared from our study that DNA‐based techniques such as plasmid typing and pulsed‐field gel electrophoresis (PFGE) were more useful tools for subtyping Shigella sonneithan colicin typing and the antimicrobial susceptibility test. Moreover, according to PFGE analysis, four genetically related isolates of Shigella sonneiwere responsible for the eight sequential outbreaks. To further investigate the epidemiology of outbreaks, 58 sporadic isolates of Shigella sonneifrom overseas travelers with shigellosis during the same period were also examined. We found that some sporadic isolates from travelers in Asia were genetically related to those of the outbreak‐related isolates, indicating that genetically related isolates prevailed in Asia during this period, probably because of the extensive movement of people or food.

ABSTRACTThe complete nucleotide sequence of a novel enteric virus, Aichi virus, associated with nonbacterial acute gastroenteritis in humans was determined. The Aichi virus genome proved to be a single-stranded positive-sense RNA molecule with 8,251 bases excluding a poly(A) tail; it contains a large open reading frame with 7,302 nucleotides that encodes a potential polyprotein precursor of 2,433 amino acids. The genome contains a 5' nontranslated region (NTR) with 712 bases and a 3' NTR with 240 bases followed by a poly(A) tail. The structure of the genome, VPg–5' NTR–leader protein–structural proteins–nonstructural proteins–3' NTR–poly(A), was found to be typical of a picornavirus. The VP0-VP3 and VP3-VP1 cleavage sites were determined to be Q-H and Q-T, respectively, by N-terminal amino acid sequence analyses using purified virion proteins. Possible cleavage sites, Q-G, Q-A, and Q-S, which cleave P2 and P3 polyproteins were found to be similar to those of picornaviruses. A dendrogram based on 3Dpolproteins indicated that Aichi virus is genetically distinct from the known six genera of picornaviruses including entero-, rhino-, cardio-, aphtho-, and hepatovirus and echovirus 22. Considering this together with other properties of the virus (T. Yamashita, S. Kobayashi, K. Sakae, S. Nakata, S. Chiba, Y. Ishihara, and S. Isomura, J. Infect. Dis. 164:954–957, 1991), we propose that Aichi virus be regarded as a new genus of the family Picornaviridae.

Abstract: The present study was undertaken to evaluate the effects of vasodilators on the development of neurogenic pulmonary edema. Pulmonary edema was induced by injecting fibrinogen and thrombin into the cisterna magna of vagotomized rats (fibrin-induced pulmonary edema). Before the intrathecal injections, rats were pretreated with intravenous injection of one of the following vasodilators: phentolamine, isoproterenol, nifedipine, diltiazem, isosorbide dinitrate, or substance P. Each vasodilator reduced the incidence of fibrin-induced pulmonary edema and lung-water ratio dose-dependently except nifedipine and diltiazem. There was a uniform relationship between the lung-water ratio and the prefibrin mean arterial pressure obtained under administration of different doses of the each drug. A similar relationship was obtained even if the drug used was different. Treatment with nifedipine or diltiazem, however, diminished the blood pressure but provided less protection against the development of pulmonary edema. The blood volume in edema-positive lungs was minimally different from that in edema-negative lungs. These results suggest that the neurogenic pulmonary edema may be effectively prevented by most vasodilators except Ca++-blockers.

The present study was undertaken to evaluate the effects of a newly synthesized nitro compound (E-4701) on the systemic circulatory system with special reference to venous return and vascular compliance. Dogs were anesthetized with sodium pentobarbital. After opening the chest, cannulae were inserted into the superior and inferior vena cavae and into the right atrial appendage. The venous flow from the caval veins was redirected to a blood reservoir with an outlet at a constant height. The blood was pumped into the right atrium at a constant flow rate. E-4701 had a hypotensive effect, and also caused a decrease in reservoir blood volume; i.e., a decrease in venous return. Venous return via the superior vena cava was increased, whereas return via the inferior vena cava was decreased. Similar effects on the systemic circulatory system were observed with nitroprusside. The lowest dose of nitroprusside that caused a significant reduction in blood pressure was the same dose as that which caused a decrease in reservoir blood volume. However, a low dose of E-4701 caused a significant reduction in reservoir blood volume without affecting the systemic blood pressure. Arterial and venous compliances were increased by both E-4701 and nitroprusside. Nitroglycerin and isosorbide dinitrate increased venous compliance. but did not affect arterial compliance. The results suggest that E-4701 caused an almost equipotent reduction in blood pressure and venous return by dilating the arterial and venous vascular beds. The capacitance vessels may be more sensitive to E-4701 than the resistance vessels.

The contribution of cytosolic ion and energy milieu changes to ischemia/reperfusion injury was investigated in isolated guinea-pig hearts and mitochondria, with fluorometry and 31P nuclear magnetic resonance (NMR). The fura-2 Ca2+signal during ischemia in the guinea-pig Langendorff heart changed triphasically (phases I, II, and III) and rapidly returned to the control level after the reperfusion. These triphasic changes during ischemia were affected by various agents that affect the cytosolic ion milieu: the combination of asebotoxin-III and dihydroouabain (which increase intracellular Na+) caused an increase in Ca2+levels in the final stage (phase III) with a manifestation of contracture after the reperfusion of the heart. Inhibitors of the H+-Na+exchange such as 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) produced a significant restorative effect on the contractility of the reperfused heart with increased proton and decreased Na+and Ca2+in the cytosol. The mitochondrial matrix Ca2+([Ca2+]m) preloaded with abnormally high Ca2+levels was markedly increased by perfusion with either a physiologic concentration of Ca2+or an acidified perfusate. These [Ca2+]mincreases were reduced by the H+-Na+and H+-K+exchange inhibitor (EIPA; omeprazole), respectively. These findings will help to explain the Ca paradox at the mitochondria level (i.e., mitochondria for Ca2+pumping play an essential role in the cellular homeostasis of Ca2+for the maintenance of cell functions of the heart, acting like a Ca2+scavenger in the cytosol). Factors that induce Ca2+overload on mitochondria via sarcolemmal Ca2+influx and any exchange mechanisms with Na+, K+, Ca2+, and H+will lead to a loss of contractility, associated with the extremely reduced level of free energy change predicted from the reduced ATP · PCr/Pi ratio by 31P NMR.

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The plasma leakage in neurogenic pulmonary edema (NPE) is generally thought to be a result of an elevation in the intravascular pressure induced by a shift of blood volume from systemic to pulmonary circulation. Besides such a mechanism, a possibility that the central nervous system directly affects the pulmonary vascular bed to increase the vascular permeability was demonstrated in this study. NPE was induced with brain ischemia in anesthetized dogs. Supplementary blood was collected from another dog and mixed with 10 % solution of dextran 40. Right and left atria (RA and LA) and pulmonary arterial trunk were cannulated. At first, we made a right heart bypass preparation. The venous return to the reservoir from the systemic circulation via RA was suddenly changed to that from the pulmonary circulation via LA. By this procedure, a complete artificial pulmonary perfusion preparation was made, while the brain circulation ceased. Perfusion pressure was 20-30 cmH2O, and left atrial pressure was in the range of 7-11 cmH2O. Initial peak inspiratory pressure was 8-10 cmH2O. Even under these conditions which were considered not to induce a hydrostatic pulmonary edema, edema still occurred, indicating an increased vascular permeability. Elimination of lung innervation inhibited the decrease in the reservoir blood volume, suggesting a physiological role of lung innervation on the pulmonary vascular permeability.