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Cyclic AMP and cyclic GMP phosphodiesterase activities (3' : 5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17) were demonstrated in the isolated intima, media, and adventitia of rabbit aorta. The activity for cyclic AMP hydrolysis in the intima was 2.7-fold higher than that for cyclic GMP hydrolysis. The activity for cyclic AMP hydrolysis in the media was approximately equal to that for cyclic GMP hydrolysis, but in the adventitia, cyclic GMP hydrolytic activity was 2.1-fold higher than cyclic AMP hydrolytic activity. Distribution of the activator of the phosphodiesterase was studied in the three layers. Each layer contained the activator. The activator was predominantly localized in the smooth muscle layer (the media). The effect of the activator and Ca2+ on the media cyclic AMP and cyclic GMP phosphodiesterase was also briefly studied. The activity of the cyclic GMP phosphodiesterase was stimulated by micromolar concentration of Ca2+ in the presence of the activator. However, the activity of the cyclic AMP phosphodiesterase was not significantly stimulated by Ca2+ up to 100 muM in the presence of the activator. Above 90% of cyclic nucleotide phosphodiesterase activity in the whole aorta was found to be derived from the media. A major portion (60-70%) of the media enzyme was found in 105 000 times g supernatant. Cyclic AMP phosphodiesterase in the supernatant was partially purified through Sepharose 6B column chromatography and partially separated from cyclic GMP phosphodiesterase. Using a partially purified preparation from the 105 000 times g supernatant the main kinetic parameters were specified as follows: 1) The pH optimum was found to be about 9.0 using Tris-maleate buffer. The maximum stimulation of the enzyme by Mg2+ was achieved at 4mM of MgC12. 2) High concentration of cyclic GMP (0.1 mM) inhibited noncompetitively the enzyme activity, and the activity was not stimulated at any tested concentration of cyclic GMP. 3) Activity-substrate concentration relationship revealed a high affinity (Km equals 1.0 muM) and low affinity (Km equals 45 muM) for cyclic AMP. The homogenate and 105 000 times g supernatant of the media also showed non-linear kinetics similar to the Sepharose 6B preparation and their apparent Km values for cyclic AMP hydrolysis were 1.2 muM and 36-40 muM and an enzyme extracted by sonication from 105 000 times g precipitate also exhibited non-linear kinetics (Km equals 5.1 muM and 70 muM). 4) Papaverine exhibited much stronger inhibition on the aorta cyclic AMP phosphodiesterase (50% inhibition of the intima enzyme, I5 o at 0.62 muM, I5 o of the media at 0.62 muM and I5 o of the adventitia at 1.0 muM) than on the brain (I5 o at 8.5 muM) and serum (I5 o at 20 muM) cyclic AMP phosphodiesterase, while theophylline inhibited these enzymes similarly. However, cyclic GMP phosphodiesterases in all tissues examined were inhibited similarly, not only by theophylline but also by papaverine.

Agents responsible for the entry of cholesterol-bearing lipoproteins into the arterial wall represent local risk factors in atherogenesis. In an attempt to identify these agents, we inserted a catheter into the ascending aorta of rabbits and 5ml of arterial blood sample was withdrawn. The contraction of the aortic strip of rabbit against Piper and Vane's antagonists with application of this sample was observed. Samples ob-tained after feeding of 1Gm/Kg of cholesterol and after an intravenous administration of thromboxan A2 (TXA2) contracted the aortic strip. Samples from non-treated rabbits or those obtained after intravenous administration of 10μg of epinephrine or norepinephrine did not contract the strip. Previous administration of 20mg/Kg of indomethacin decreased the contraction developed after feeding of 1Gm/Kg of cholesterol. It was suggested that TXA2 might be released into the arterial blood by the ingestion of cholesterol and might be one of the agents responsible for the entry of lipoproteins.

Two milliliters of thromboxane A 2 (TXA 2 ) solution was perfused in the coronary arteries by the balloon catheter inserted through right common carotid artery in rabbits. TXA 2 was converted from prostaglandin H 2 (PGH 2 ) with thromboxane A 2 synthetase in platelet microsomes. Serial changes on the ECG and hypotension were observed first hour after the injection in nine out of 11 studied rabbits. Six out of the seven autopsied rabbits showed myocardial ischemia or necrosis histologically. But four control rabbits that were injected with 2 ml inactivated 1.0 μg TXA 2 solution did not show any change. The changes on the ECG were dose-dependent in 1.0, 0.5, and 0.1, μg TXA 2 solution. In addition, the ECG and the blood pressure changes by 1.0 μg TXA 2 solution were suppressed by pretreatment of a non-specific antagonist of TXA 2 , phthalazinol (1 mg/kg). These findings support the concept that TXA 2 is the principal active substance in the filtrate of aggregated platelets that induced acute myocardial ischemia and necrosis in rabbits in a previous experiment.

A platelet-clumping substance was separated from 3,000 ml of rabbit acidified plasma using DEAE-Sephadex column chromatography. Active fraction was eluted accompanying each peak of protein and uronic acid respectively. After rechromatography, white water-soluble powder was obtained. Its yield was 231 mg, thus 77 μ of the clumping substance was obtained from one ml of plasma. It showed the clumping activity against washed or unwashed platelets equally, even in a concentration of 20 μg/ml without calcium ions. The chemical analysis showed 94.5 % protein, 0.6 % uronic acid, 2.4 % hexose and 2.5 % hexosamine. The substance showed metachromatic staining with toluidine blue. The clumping activity was lost after incubation with trypsin and pronase. Saccharose density gradient ultracentrifugation analysis showed that its molecular weight was between 13,000 to 68,000. Using immuno-electrophoresis, a characteristic precipitation line against prealbumin fraction was shown.

The platelet-clumping substance isolated from rabbit plasma by the authors aggregated washed rabbit platelets, while ADP did not. The presence of AMP or adenosine did not inhibit the appearance of platelet aggregation induced by the clumping substance. Pretreatment of phosphoenol-pyruvate and pyruvate kinase on unwashed platelets did not affect the platelet aggregation by the clumping substance. It clumped human platelets and also shrimp thrombocytes. Electron microscopic findings showed that platelets to which the clumping substance was added aggregated in their original form as falt disc shapes with few pseudopods. While fusion of membrane was present, microtubules were observed in its regular form. These results showed the existence of a platelet-clumping substance in blood that is different from ADP, collagen, thrombin, adrenaline etc. and may play an important role in hemostasis and thrombogenesis.

SummaryA new method for assessment of platelet sensitivity to ADP-aggregation was devised. Its reproducibility and the correlations between the values obtained by this method, the optical density (O. D.) method, and the screen filtration pressure (SFP) method were assessed. In summary, this method may be said to have three main points:1. It can be performed without centrifugation, avoiding mechanical stress to platelets, using only 0.8 ml. of blood and inexpensive equipment.2. It may reflect different aspects of platelet function from the O. D. method and the SFP method, despite the positive significant correlations between the values obtained by these three methods.3. It was proved to be highly reproducible and is thought to be useful clinically.By using this method, the effect of sustained isometric exercise by handgripping on platelet aggregability was assessed in coronary sclerotic and cerebral arteriosclerotic patients on placebo and EG-626, a newly synthesized cyclic AMP phosphodiesterase inhibitor. On placebo, an enhancement of platelet sensitivity was observed after isometric exercise in coronary and cerebral arteriosclerotic patients but not in healthy control subjects. The enhancement was prevented by pretreatment of EG-626, administered orally 1.5 hours prior to exercise.

The intracoronary injection of TA2, in a dose of 0.06 to 1.3μg in terms of norepinephrine, induced in rabbits, in a dose dependent manner, mild to severe ischemic myocardial changes, i.e., an angina pectoris-like response, variant angina-like response, myocardial infarction and sudden cardiac death.

Arterial endothelial cells, which are capable of phagocytizing carbon particles of the same size as beta- and pre-beta-lipoprotein, were found only in endothelial cells of arterial segments susceptible to atheromatous changes in susceptible animal species, and the distribution closely corresponded to the susceptibility. The distribution of such endothelial cells is dense in large arteries, in the openings to their branches, especially in downstream portions, of rabbits, hens, and cocks; however, the distribution is relatively scanty in arteries of rhesus monkeys and is very scanty in dogs. Carbon particles were also rare in the suckling rabbit and tended to increase with age. They were not found in Wistar rats but were found in spontaneously hypertensive rats, which showed a characteristically diffuse distribution, even in relatively small arteries. The carbon particles, phagocytized, were released to the subendothelial space but were difficult to pass through the internal elastic lamina and tended to stagnate there for more than one month. The authors therefore call these cells hyperreactive endothelial cells. Various vasoactive substances, such as angiotensin II, histamine, and serotonin, significantly enhanced the phagocytic activities of arotic endothelial cells in rabbits; epinephrine and norepinephrine also slightly enhanced these activities. Various smooth muscle relaxants, such as ATP, pyridinol carbamate (ATP synthesis-enhancing substance), cycli-AMP, dibutyryl cycli-AMP, phthalazinol (cyclic-AMP phosphodiesterase inhibitor), iproveratril (calcium entry-inhibiting substance), colchicine, and vinblastine, with their different modes of action, commonly inhibited phagocytic activities, a finding that suggests a significant role for contractile protein in the permeability problem of atherogenesis. The atheromatous lesions of cholesterol-fed rabbits exhibited a striking increase in hyperreactive endothelial cells, accompanied by a marked rise in the activity of low-Km cyclic-AMP phosphodiesterase activity in atheromatous lesions and adjacent muscular layers, especially in rabbits with rapidly progressing atheroma.

The effects of sustained isometric handgrip exercise on platelet aggregability and the left ventricular function were assessed in 29 patients with ischemic heart disease (Table 1) on placebo and EG626, a newly synthesized cyclic AMP phosphodiesterase inhibitor. The platelet aggregability was assessed by using a new device (Sano, 1974) which avoids centrifugation and needs only a small amount of citrated blood (Fig. 1). The left ventricular performance was estimated by a non-invasive technique, systolic time intervals. Parameters of systolic time intervals (LVET, PEP, ICT and PEP/ET) were expressed as indices following Motomiya's multiple regression equations (Table 2).RESULTS (1) Platelet Aggregability: On placebo, an enhancement of platelet aggregability was observed after isometric handgrip exercise in patients with ischemic heart disease but not in healthy control subjects. The enhancement of platelet aggregability was prevented by pretreatment of EG626, administered orally 90 minutes prior to the exercise (Fig. 1).(2) Left Ventricular Function (Fig. 3): a) At Rest: Systolic blood pressure was significantly lower on EG626 pretreatment than on placebo (p

Polyglucose newly synthesized by phosphorylase in endothelial cells of rabbit aorta was studied electron microscopically by Takeuchi and Sasaki's method to confirm the presence of phosphorylase activity in endothelial cells of aortic wall. The polyglucose stained irregularly with lead, forming irregular particles 200–500 A in diameter. It was found in both endothelial cells and smooth muscle of the aorta. Native glycogen was not recognized in endothelial cells, but was present in the smooth muscle of the media. On the contrary, both polyglucose and native glycogen were observed in the intermyofibrillar sarcoplasm of heart muscle, which was simultaneously studied as a control.

Atherosclerosis was produced in rabbits by feeding 1% cholesterol pellets for 15 weeks to 35 male rabbits divided into two equal groups – the placebo control and phthalazinol group – with a comparable serum cholesterol level. The phthalazinol group was given 20 mg/kg of the compound daily. 13 rabbits of both groups were on 15 weeks of the treatment and the remaining 22 rabbits were treated for 30 weeks. The treated group exhibited a statistically significant increased removal of cholesterol from atherosclerotic aortas and improvement in pathological changes. This enhancing effect was more marked in the animals treated for 30 weeks. Such evidence indicates that there are substances capable of enhancing the removal of cholesterol from atheromatous lesions. In light of the pharmacological properties of phthalazinol, the possible role of thromboxane A2 released from platelets adhered and aggregated on atheromatous plaques, in the progression of atherosclerosis is worthy of continued investigation.

The levels of cyclic adenosine 3′,5′-monophosphate (cyclic AMP) in the aortic intima and media of man and other species were measured microbiochemically by Lowry's quantitative histochemical technique and Gilman's competitive protein binding assay. Rather high levels of cAMP were found in the intima of all species examined as compared with that in the media. The role of cAMP in intima is discussed in relation to atherogenesis.

Thepresence of IgG and beta-lipoprotein in thoracic and abdominal aortas of 33 rhesus monkeys under various conditions was observed by immunofluorescence and interference filter technique. In untreated and placebo control monkeys, a definite fluorescence due to IgG was seen in highly limited areas of the subendothelial space and the innermost layers and a weak but positive fluorescence due to beta-lipoprotein was sometimes show in highly limited spots of the subendothelial space, but not the medial layers. In challenged animals with cholesterol (1 g/kg p.o.) or epinephrine (1 mug/kg i.v.) or norepinephrine (10 mug/kg i.v.) or angiotensin II (1 mug/kg i.v.) or cholesterol (1 g/kg p.o.) plus epinephrine (1 mug/kg i.v.), definite fluorescence showing the entry of beta-lipoprotein and IgG from the vessel lumen into the subendothelial space and then into the medial layers of the arterial wall was clearly shown by serial sacrificing of animals after each challenge. The passage of beta-lipoprotein through the internal elastic lamina into the medial layer, was characteristically delayed as compared with that of IgG. In pretreated animals with pyridinolcarbamate (PDC) (10 mg/kg p.o.), EG467 (1 mg/kg p.o.) and Premarin (5 mg/kg i.v.), the acute entry of those plasma proteins was significantly inhibited.

Forty hospitalized patients with angina pectoris, 25 men and 15 women, 37 to 75 years of age, were subjected to Master's two-step test. Three hours before the test, 1.0 Gm. of pyridinolcarbamate or placebo was given orally using a double-blind technique. Before, immediately after, and 3, 5, 7, 10, and 30 minutes after exercise, electrocardiographic changes and clinical symptoms were observed. In 20 of the above subjects prothrombin activity, calcium clotting time, and the platelet and adhesive platelet count was measured. Thirty-three of the 40 patients pretreated with placebo developed ischemic ECG findings immediately to 10 minutes after the exercise test. Seventeen of the same 40 subjects pretreated with pyridinolcarbamate developed ischemic ECG findings. The difference between placebo and pyridinolcarbamate was statistically significant at p For the second exercise test the 17 patients who had received pyridinolcarbamate for the first test were pretreated with placebo, and 13 showed positive ischemic ECG changes. The 23 patients who had received placebo for the first exercise test were pretreated for the second test with pyridinolcarbamate, and 10 developed positive ECG changes. There was no significant difference noted between the two exercise tests in regard to the appearance of ischemic ECG changes. The appearance of anginal pain after exercise was prevented by pretreatment with pyridinolcarbamate (p In comparison to other 39 healthy volunteers, the patients with angina pectoris showed enhanced blood coagulability and lower platelet count. In healthy persons, enhanced blood coagulability was observed one minute after the exercise test (p p

The result of a new type of antithrombotic treatment with nialamide in a group of 42 patients suffering from acute myocardial infarction was reported. In the control group of 21 patients 8 cases died, while in the nialamide group of 21 patients 1 case died. The treatment is promising through the assessment of the result by Pathologic Index Rating (Schnur1)). The theoretical basis for the use of new type of antithrombotic treatment with MAO inhibitor was also detailed.

In our previous study, three types of intra-cellular potentials were found in a small area in the region adjacent to the postero-inferior margin of the membranous portion of the interventricular septum of the isolated dog heart. From the electrophysiologieal behavior of these cells, it was presumed that the cells of the third type were the point of block for retrograde conduction in cases of unidirectional A-V conduction, and that those of the first type lay upstream from the point of retrograde conduction block, whereas those of the second type lay- downstream beyond the blocking point. In this study, histological examination was used to find the origins of these three types of potentials. When a potential of one or another type was found the mieroelectrode tip was gently cut off and later localized histologically. It was found that all three types of potential originated from the ventricular portion of the specialized museilar tissue, that the origin of the third type was just above or at the bifurcation of the bundle of His, and that the origin of the first type was distal while that of the second type was proximal to the origin of the third type. Thus, the deduction from the electrophysiological behavior was proved by the histological examination. This gave further evidence for our belief that there is a sensitive point for retrograde conduction block in unidirectional A-V conduction just above the bifurcation of the bundle of His.

The effect of peroral application of pyrldinolcarbamate (30 mg/kg/day) was studied in rats which were kept either on a standard laboratory diet, or on a diet containing a large amount of fat (40%) and cholesterol (5%). PDC significantly increased the lactate dehydrogenase activity in the aortae of rats kept on a standard diet, and the malate dehydrogenase activity in the aortae of rats kept on high-fat diet. In experiments in vitro, PDC increased the activity of the aortic lactate and malate dehydrogenase. This effect was most pronounced at higher concentrations of PDC in the incubation medium (3000μg/ml). In the rat myocardium, activity of the enzymes studies was not noticeable influenced by PDC administration. Under the given experimental conditions, pyrldinolcarbamate increased the activity of enzymes participating in the citrate cycle and glycolysis. In view of the present knowledge of the role of the vascular wall metabolism in atherogenesls, this effect could contribute to greater resistance of the vessel wall against the development of atherosclerotic changes.

In rabbits the repetitious administration of a small amount of a high molecular substance, i.e., bacterial polysaccharide prepared from a strain of Shigella flexneri 2b , which is capable of eliciting intravascular blood coagulation, precipitating fibrinogen, clumping the platelets and leukocytes, inducing swelling of endothelial cells and the adherence on them of electrodense plasma substance and platelet-leukocyte thrombi, induced a subendothelial fibroblastic proliferation, deposition of a metachromatic substance, and fragmentation of the internal elastic membrane in the coronary artery and in some of the other arteries (e.g., of the lung), and grossly visible infarct-like lesions in the myocardium of the left ventricle and also microscopically recognizable scar and fibrotic and degenerative foci in the myocardium beneath the endocardium of the left ventricle. Repetitious administration of the bacterial polysaccharide in combination with subsequent administration of a small amount of adrenaline, which by itself induces no recognizable change of the blood vessels and the heart, except for a few and microscopically small degenerative foci in a limited area of the myocardium, induced visibly large infarct-like lesions in the myocardium of the left ventricle and strong damage to the artery, especially of the medium-sized and small arteries and arterioles located inside the myocardium of the left ventricle, as in the case of animals treated with polysaccharide, but far stronger. The aorta showed also a similar change in the intimal layer and slight irregularity in the medial myofibers. Otherwise, no sclerosis of the Monckeberg type was found. The other organs, including the kidney, showed no recognizable change, except in one animal which showed gastric ulcer perforating into the abdominal cavity. The damage to the heart and the blood vessels, caused by the bacterial polysaccharide, was considered as a common phenomenon induced by some of the high molecular substances, and not as a species-specific phenomenon. Such damage was reduced effectively by the concurrent oral administration of magnesium chloride.

Electron microscopical analysis of endothelial linings of the aortic lumen and of the vasa vasorum was made in man, rhesus monkey, dog, rabbit, rat, chicken and pigeon with reference to permeability of the arterial wall, because cholesterol and other substances accumulating in atheromatous lesions have been known to be transported mainly by infiltration into the lesions from the blood stream across the endothelial lining. Porous capillaries were found in vasa vasorum of man, rabbit and pigeon and a well developed vacuole system in the endothelial cells of man and chicken. For the present experiment of atherosclerosis the rabbit has been chosen. In the succeeding experiments we found substances capable of inhibiting the edematous arterial reaction such as pyridinolcarbamate and its derivatives as well as estrogens and glucocorticoids. We have tested these substances for the prevention of atherosclerosis of cholesterol-fed rabbits and also for the treatment of established atherosclerosis of the animals. The first important finding was the correlation between their inhibiting potency against the edematous arterial reaction and their property of preventing atherosclerosis. The second Important finding was appearance of atrophy, necrosis and calcification of arterial wall along with the atheroma-preventive effect of estrogen and prednisolone and the absence of such effects in pyridinolcarbamate, while the latter substance enhances the regeneration of smooth muscles in the lesions. Dosages of 10 to 30 mg per kg of pyridinolcarbamate given by mouth daily have shown the atheromatous mass with foam cells, necrosis and degenerative foci being replaced by regenerated smooth-muscle, collagen and elastic fibers, and the cholesterol content of arterial wall was definitely reduced in the course of 6 to 30 weeks of the treatment, without any untoward effect. Histoenzymatic analysis revealed the increased enzymatic activity of glycolytic enzymes Including glucose-6-phosphate dehydrogenase in the arterial wall of animals treated with pyridinolcarbamate and the decreased enzymatic activity of glycolytic enzymes in that of animals treated by estrogen. The results reported in this paper show that the author's edematous arterial reaction has opened a way toward finding antiatherosclerotic agents which may prove to be useful in the prevention and treatment of human atherosclerosis. ACTA PATH. JAP. 19: 15˜43, 1969.

An antibradykinin and antilysylbradykinin agent, pyridinolcarbamate (2,6-bis-hydroxymethylpyridine- bis N- methylcarbamate), has a potent preventive effect on the edematous arterial reaction, and has been shown to possess an antiatherogenic property on the basis of the results obtained in the present series of experiments with 118 rabbits. The feeding of a high-cholesterol diet to the animals over a prolonged period of 13 to 15 weeks was employed to induce the experimental atherosclerosis, and the daily administration of pyridinolcarbamate in doses of 5 and 10 mg. per kilogram was shown to exert a powerful effect in preventing the apperance of atheromatous changes (p The antiatherogenic property of this compound is not related to an influence on the level of cholesterol in the blood; in fact, hypercholesterolemia is not depressed by the drug. The antiatherogenic property has been presumed to correlate possibly with the kinin-antagonistic effect of this compound.

Incubation at 37°C for 30 min or the addition of CaCl2 (5 mM) generated platelet-clumping activity in citrated plasma and EDTA plasma previously acidified to pH 5.5 and in which heparin was added to give a concentration of 0.5 mg/ml. Similar treatment could not produce platelet-clumping activity in saline or denatured plasma that had been heated to 56°C. Heparin, chondroitin sulfate B, or heparan sulfate produced platelet-clumping activity in BaSO4-adsorbed serum over a given pH range. The effective pH range for each compound was; heparin 0.5 mg/ml, pH 5.16 ± 0.09 to 6.31 ± 0.14; chondroitin sulfate B 0.5 mg/ml, pH 5.62 ± 0.09 to 6.88 ± 0.06; 2 mg/ml, pH 5.17 ± 0.16 to 7.18 ± 0.16. The effect of heparan sulfate in generating platelet-clumping activity was estimated only qualitatively at pH 6.0 and proved to be positive. Chondroitin sulfate A, chondroitin sulfate C, and hyaluronic acid failed to yield platelet-clumping activity in BaSO4-adsorbed plasma. The minimal concentrations of heparin and chondroitin sulfate B necessary to induce platelet-clumping activity in the BaSO4-adsorbed serum were 0.5 mg/ml and 0.1 mg/ml, respectively.

One-shot treatment of atherogenic substances, such as cholesterol, animal fat, adrenalin or cigarette smoking to animals induces “acute vascular injury” characterized histologically by swollen endothelial cells and edematous changes in subendothelial layer and upper part of media of aorta (Shimamoto et al., 1960), the cause of which is still unknown. Most of these atherogenic substances or procedures are known to increase the level of free fatty acid (FFA) in serum and the possibility of FFA as a direct attacker to induce this edematous change in arterial wall was studied. Oral administration of oleic acid (2 g/kg), intravenous injection of angiotensin II (10 γ/kg or 1 γ/kg) or heparin (200 unit/kg), intramuscular injection of AGTH (10 unit) or concomitant administration of cholesterol (2 g/kg) and epinephrine (1 γ/kg) or oleic acid (2 g/kg) and epinephrine (1 γ/kg) were performed in 21 rabbits and histological features of aortic wall of each treated rabbit were compared with those of 6 placebo control rabbits, all of which were sacrificed 2 hours after treatment. All treated rabbits exhibited a statistically significant increase of serum free fatty acid, compared with that before treatment. Serum cholesterol level did not change after these treatments. Histological examination, however, revealed a typical edematous change in aortic wall of rabbits treated with angiotensin II or cholesterol and epinephrine. Those treated with ACTH, heparin or oleic acid, did not exhibit such pathological changes in their aortic wall. These different histological findings suggest that the elevation of plasma FFA was not a direct offender to acute vascular injury in aortic wall.

Many investigators failed to demonstrate vasa vasorum in the inner third of media in aortic wall with routine histological techniques and it had been believed that the inner third of media may be an avascular area and the nourishment of this area may mainly be provided by the blood running through the aortic lumen. Recently, the introduction of microangiographic technique enabled the study of microcirculation in the vessel wall instead of routine histological technique. J. A. CLARKE, using an X-ray microscopic technique, demonstrated vasa vasorum of human thoracic aorta, revealing the patterns of distribution to the inner third of media just beneath the internal elastic lamina. The purpose of this paper is to describe a finding on the patterns and distributions of vasa vasorum in the aortic wall of dog. Materials and Methods Twelve healthy male dogs weighing 8.5 to 1 3.5kg anaesthetized with sodium pentobarbiturate (5 w/v% 10ml. i. v.) were used. Before the sampling of aortic specimen sodium heparin (500unit/kg) was injected intravenously. The abdomen was opened, the jejunum and ileum were retreated out of the way, and the vena cava inferior and the abdominal aorta were carefully isolated from the surrounding tissues. The both vessels were ligated at the level L2 to L4, and each was proximally cannulated with polyethylene tube. All blood in the area under the study was removed by a through flushing with Ringer's solution containing heparin (200 units/ml) at 37'C. Fifty per cent Micropaque solution was injected under manual pressure into the aortic tube in a dose of 300ml at first and then into the venous tube in a dose of 200 ml. Five minutes later, the chest was opened and the aorta was extirpated quickly with the heart. The aorta was isolated and aortic lumen was washed with running water for 3 minutes. A 10 per cent neutral buffered formalin solution was used for the fixation of the material for 48 hours or more. After flxation, the aorta was made into cross or longitudinal section of 0.5 mm to Imm thick, and examined by X-ray microscope. A model of SOFTEX was used as an X-ray source. Exposure time varied 20 to 30 minutes depending on the thickness of specimen, at 7kV at 3mA.

SummaryADP-induced platelet aggregation in citrated platelet rich plasma (CPRP) was examined using a photoelectric system. To exclude a variation in intensities of platelet aggregation in repeated measurements and to compare intensities of different samples, the maximum deflection of the optical density of CPRP induced by adding ADP solution was divided by a deflection of the optical density of the platelet free plasma and its value, shown as a percentage, was defined as an intensity of ADP-induced platelet aggregation. In this method, the linearity was found in the dose response curve of the platelet aggregation induced by 10−6, 3 × 10−6 and 10−5 molar of ADP with statistical significance. These variation coefficients were less than 5% in the responses induced by the higher doses of ADP. Changes in the ADP-induced platelet aggregation after a Master’s two step test were examined in 13 patients with angina pectoris 3 h after oral administration of placebo or 1 g of pyridinolcarbamate. Under placebo pretreatment, an enhancement of platelet aggregation was observed 1 min after the exercise test with statistical significance (P < 0.01 ∼ 0.05). In the cases of the same subjects pretreated with pyridinolcarbamate, such change was not observed at any time. Using a parallel line assay, an inhibitory effect of pyridinolcarbamate against enhancement of ADP-induced platelet aggregation after the exercise was also recognized with statistical significance (P < 0.01). In the 10 healthy volunteers, there was no statistically significant enhancement of ADP-induced platelet aggregation using any concentration of ADP 1 to 10 min after the exercise test.

In the theory of electrocardiography the direction of myocardial fibers has been almost ignored. In order to evaluate whether this is proper or not, directional difference of conduction velocity in the ventricular syncytium was examined by capillary microelectrodes. Conduction velocity in the direction of myocardial fibers was found to be usually several times larger than that vertical to them. This difference is significantly large and cannot be ignored even though rapidity of conduction through Purkinje fibers is considered. This statement became more applicable in various abnormal conditions. Thus it is concluded that the direction of myocardial fibers should be given more attention in discussion of propagation of excitation waves in the cardiac ventricle.

Pyridinolcarbamate; Anginin (B23) is a venous bradykinin antagonist, which antagonizes competitively and reversibly the venoconstrictive effect of bradykinin and its active homologues. This compound has also been shown to antagonize the congestion, exudation and hemorrhage induced by brady-kinin and its active homologues and to remove the edematous change from experimental atheroma and to promote the normal fibrous healing of atheroma.This compound has also exhibited clinically a beneficial effect in the atherosclerotic diseases of the brain and the heart, however, the evaluation of the effectiveness of this kind of drug in these morbid conditions had to depend upon rather the indirect evidences. In order to see directly the effect of pyridinolcarbamate on the arterial lesion the atherosclerosis of extremities seems much more suitable. The authors have first attempted this compound in the treatment of atherosclerotic occlusion in lower extremities of 2 hos-pitalized patients; 33 years old and 57 years old men.The first patient aged 33 years suffered from a marked cyanosis of his right foot with a history of 6 months and the pulsations of right popliteal, posterior tibial and dorsal pedal arteries were completely absent and arterio-graphy revealed the complete obstruction of popliteal artery immediately above the knee joint. Pyridinolcarbamate was given 1Gm. daily and on the 3rd day of the medication the cyanosis disappeared dramatically and the intermittent claudication exhibited a slight improvement. On the 14th day of the medication the right dorsal pedal pulsation was recognized and on the 15th day the pulsation became visible through the skin and at the same time the pulsation of right popliteal artery began to be recognized. The volume of pulsation increased gradually but relatively rapidly and concomi-tantly with the remarkable improvement of intermittent claudication and on the 25th day of the treatment there was no differnce of skin temperature be-tween right and left foot and the pulsation exhibited almost normal volume in dorsal pedal artery. On the 26th day the pulsation of his right posterior tibial artery also reappeared. Under the medication of this compound no relapse and no untoward effects of the drug have been noted for 3 months period of the observation.The second case aged 57 years suffered from intermittent claudication and the absence of the pulsation of bilateral popliteal, dorsal pedal and pos-terior tibial arteries and the arteriography revealed the occlusion of the middle part of the right femoral artery.The treatment with pyridinolcarbamate for 2 weeks in a daily dose of one Gm. induced a slight improvement of intermittent claudication and on the end of third week a marked improvement was noted and at the same time the pulsation of his right dorsal pedal artery reappeared. His inter-mittent claudication exhibited a marked improvement concomitantly with the appearance of his dorsal pedal artery. A definite shortening in crest time in plethysmogram was noted in the bilateral second toes on the 130th day of the treatment. Thereafter his condition has been continuously good and he had no relapse and no untoward effects of the drug during the whole observation period of 5 months.The effect of pyridinolcarbamate seems to originate from the improve-ment of atheromatous changes by the compound in these patients.