A CRYSTALLINE PRESSOR SUBSTANCE (ANGIOTONIN) RESULTING FROM THE REACTION BETWEEN RENIN AND RENIN-ACTIVATOR

1. Renin reacts with renin-activator to form a strong pressor substance which is heat-stable, water- and alcohol-soluble, fluorescent, acid-stable, and alkali-labile. It is a reducing substance and is destroyed by strong oxidizing substances. It forms crystalline salts with oxalic and picric acids. The color reaction for arginine is the only one found to be strongly positive. It is suggested that this substance be called angiotonin. 2. Angiotonin produces a sharp, immediate rise in arterial pressure when injected intravenously. Pithing and dissipation of the anesthetic appear to increase the response. Tachyphylaxis occurs, in contrast to renin, only after many single doses. 3. The responses to adrenaline and angiotonin do not parallel one another. Cocaine, atropine, and stilbestrol do not affect the pressor action of angiotonin. Suprarenalectomy in brief experiments is also without effect. 4. Maximal amounts of angiotonin result when the proportion between renin and activator is roughly 3 to 100. This is not a stoichiometric relationship in the chemical sense. The temperature suitable for good yields is about 38°C., and the time of reaction from 10 to 20 minutes. 5. Renin destroys angiotonin when incubated with it. 6. Angiotonin causes marked contraction of intestinal segments of rabbits without reducing their rhythmic motion. It sensitizes the intestine to further doses of angiotonin and alters the intestine such that renin-activator contracts it. Angiotonin also constricts the vessels of a rabbit''s ear perfused with blood or Ringer''s solution.     

ON THE NATURE OF THE PRESSOR ACTION OF RENIN

1. Tachyphylaxis occurs when renin is repeatedly injected into dogs and cats regardless of whether they are normal, anesthetized, pithed, hepatectomized, suprarenalectomized, nephrectomized, or eviscerated. 2. The pressor response to renin in brief experiments is independent of the height of the arterial pressure or the presence of the suprarenals. Evisceration and large doses of ergotamine reduce the response. It is largely uninfluenced by pithing, intracisternal injection of renin, cocaine, strychnine, caffeine, and infusion of sodium bicarbonate or hydrochloric acid. It may be slightly increased by large blood transfusions or hepatectomy but the result is short lived. 3. There is no parallelism between the pressor responses to carotid sinus stimulation, adrenine, and tyramine on the one hand and renin on the other. 4. Section of the brain may be followed by depressor responses to renin. 5. Intracisternal injection of renin elicits no significant rise in blood pressure or other circulatory manifestations. 6. Continuous infusion of renin produces a prolonged rise of arterial pressure in normal and chronically suprarenalectomized dogs, but the pressure ultimately falls despite continued infusion. 7. Tachyphylaxis develops in the isolated rabbit''s ear perfused with blood and small doses of renin. The same blood perfused through a second ear causes no vasoconstriction when renin is added. Addition of renin-activator restores the ability of renin to cause constriction. 8. Renin alone causes no vasoconstriction when perfused with Ringer''s solution, but renin plus renin-activator restores activity. Tachyphylaxis does not develop when Ringer''s solution is employed instead of recirculating blood. 9. Blood from animals made tachyphylactic by repeated injections of renin is lacking in activator and also fails to cause vasoconstriction in the rabbit''s ear when renin and renin-activator are added. 10. Renin-activator is lost and tachyphylaxis develops more slowly during continuous infusion of renin. Blood pressure may fall after a period of renin infusion despite the pressure in the blood of excess renin. Injection of partially purified activator restores the activator content of the blood as demonstrated in the rabbit''s ear, but no rise in arterial pressure occurs.     

THE CARDIAC FACTOR IN THE "PRESSOR" EFFECTS OF RENIN AND ANGIOTONIN

Upon the isolated hearts of cats perfused with Ringer-Locke solution renin produced no significant effect. Angiotonin, on the other hand, brought about decrease in coronary flow and increase in amplitude of beat without any consistent effect upon heart rate. Both renin and angiotonin augmented the cardiac output and raised the "arterial" pressure in the Starling heart-lung preparation; here too without influence on the heart rate. Electrocardiograms recorded before, during and after the pressor effects of renin and angiotonin in the anesthetized cat showed no abnormalities until the blood pressure had risen above 190 mm. Hg when various types of cardiac arrhythmias appeared. These were prevented, or normal rhythm was restored, by cutting the vagus nerves or injecting atropine. It is concluded that the "pressor" effects of renal pressor substances include direct stimulation of the myocardium and augmentation of ventricular beat. Unless these actions lead to excessive decrease in diastolic volume of the ventricles, the cardiac output will be increased. The significance of this in the production of the pressor effect is discussed.     

A KINETIC ANALYSIS OF THE RENIN-ANGIOTONIN PRESSOR SYSTEM AND THE STANDARDIZATION OF THE ENZYMES RENIN AND ANGIOTONASE

The physicochemical background of the renal vasporessor system (renin-renin-substrate, angiotinin, angiotonase) is given. The formation and destruction of angiotonin is shown to consist of two consecutive reactions, both of which follow the laws of first order kinetics. Each reaction was studied separately and its reaction constant found to be proportional to the enzyme concentration. Hence these constants should be used to express the activity of the enzymes, renin and angiotonase. The over-all reaction of a mixture of renin and angiotonase such as occurs in kidney extracts with the α-globulin fraction of serum, viz., rapid increase followed by a slow decline in angiotonin concentration, was found experimentally to correspond closely to the theoretical values calculated for such a reaction. The curve obtained also satisfyingly explains the characteristic pressor response to the intravenous injection of renin. An accurate method for the determination of renin in the presence of angiotonase is presented.     

THE RELATION OF THE SPLEEN TO BLOOD DESTRUCTION AND REGENERATION AND TO HEMOLYTIC JAUNDICE : VIII. REGENERATION OF THE BLOOD OF SPLENECTOMIZED DOGS AFTER THE ADMINISTRATION OF HEMOLYTIC AGENTS.

In the splenectornized dog the anemia caused by hemolytic poisons (hemolytic immune serum and sodium oleate) and by bleeding is of a severer grade, runs a longer course, and is accompanied by a less rapid regeneration of the blood than is the case in the normal dog. Also in the splenectomized dog, especially after the use of hemolytic serum, the leucocytosis is greater than in the normal animal. The splenectomized dog almost uniformly exhibits an increased resistance of the red cells to hypotonic salt solution, but after the administration of hemolytic poisons, and especially hemolytic serum, this increased resistance disappears and a decreased resistance persists for long periods of time. The same change occurs in the normal dog, but in the latter the return to the previous degree of resistance is more rapid than in the splenectomized animal.     

THE REACTION OF COW'S MILK TO BLOOD SERUM PRECIPITIN

It seems well established that during normal lactation there passes a slight but well defined quantity of blood protein into the udder. When a specific serum precipitin is applied to diluted milk the reaction occurs at a relatively uniform level of dilution. This line may well be considered the normal level of blood protein elimination. In the main the serum protein level is the same when mastitis streptococci are present as when they are absent, provided they are not too numerous. On the other hand it has been shown that when streptococci are present in large numbers the milk while apparently normal reacts at dilutions considerably higher than the normal. In the non-lactating udder, particularly during the latter end of gestation, serum proteins accumulate. Little and Orcutt have shown that certain antibodies tend to become greatly concentrated within the udder. Howe and others have shown that the colostrum of cows is rich in blood proteins. It is to be noted, however, that colostrum while rich in serum proteins usually reacts with serum precipitin to about one-half the titer of the blood serum itself. It may well be that certain proteins of the blood passing into the acini are reabsorbed, leaving a portion of the antigen within the udder. Thus a gradual accumulation of antigen may occur. Shortly after parturition the udder may be drained with a consequent marked fall in serum content. There, however, apparently exists for the next few days considerable permeability of the capillaries for serum proteins. This is borne out by the reaction of the milk to the serum precipitin, since it may react above the normal level for as long as 10 days or 2 weeks after parturition. During the course of inflammation there may occur a considerable outpouring of blood protein. In the severe cases the exudate may give a titer at as great a dilution as the blood serum. In other milder cases where the inflammation may be confined only to the mucosa of the milk cystern, there is usually little that is abnormal in the appearance of the milk. The precipitin test, however, indicates that serum proteins are present in definitely greater concentration than in normal milk. Of practical interest is the effect of heating the milk on the reaction of serum precipitin. Milk heated to a temperature as high as 66°C. for 20 minutes will react at the same dilutions as the raw milk. Milk pasteurized at 68–69°C. for 20 minutes will no longer react. The precipitin test, then, affords a ready means of detecting milk heated above 68–69°C. It is assumed that the precipitin test may have some further practical application. The evidence presented indicates a well defined serum proportion in the milk which may be increased during inflammation of the udder. The experiments in which mastitis exudate was mixed with varying quantities of market milk indicate that such mixtures can readily be detected with serum precipitin. It seems not improbable that some such procedure might be applied as a presumptive test to the product of small herds or to the mixed milk of a number of cows.     

MECHANISM OF THE LYSIS OF PNEUMOCOCCI BY FREEZING AND THAWING,BILE, AND OTHER AGENTS

Pneumococci, living or dead, are soluble in bile when: (a) the autolytic enzymes are still present in a potentially active form; (b) conditions are favorable for enzymatic action. Bile solubility of pneumococci involves as a necessary step one, or a few, of the many stages of the autolytic complex. These observations hold true for the disruption of pneumococci by freezing and thawing, by previous desiccation with cold acetone, and by dilute solutions of antiseptics. A possible mechanism is discussed to account for these forms of lysis.     

STUDIES ON THE BLOOD VESSELS IN THE MEMBRANES OF CHICK EMBRYOS : PART III. ANATOMY AND PHYSIOLOGY OF THE BLOOD VESSELS AT DIFFERENT AGES

On the basis of the anatomical studies presented the following inferences or conclusions are drawn. 1. In the course of development there appear in the vascular membranes of chick embryos arterial vessels of all calibers, namely, capillaries, small arteries with 2 or 3 cell layers, and large arteries formed of endothelium, longitudinal and circular layers of muscle and adventitia. 2. In none of the stages are elastic fibers developed. Only in the most central portion of the umbilical artery, in that portion namely which is to be regarded as belonging to the embryo, are elastic fibers discoverable. 3. The structure of capillaries is histologically the same at all stages. The small arteries of embryos 10 days old resemble histologically those of 18. At no stage of development are appearances of degeneration nor of fat to be found in arteries. When the physiological results of our investigations are compared with the anatomical ones the following comments may be made. In respect to Paragraph 1 of the anatomical results we may remark that when we study the different forms of the wall of arterial vessels the most delicate vessels consisting of single cells exhibit the greatest irritability. Those which are built of 3 to 4 muscle layers are less irritable. Stouter vessels appearing for the first time at 10 days of incubation require stronger stimuli to bring about the same reaction. In respect to Paragraph 2 of the anatomical results we may make this comment. The absence of elastic fibers in all arteries of the embryonic membranes throughout the period of their development is important in defining a physiological property of the larger vessels. The medium and larger vessels, beginning with the 4th day of incubation, contract differently from normal adult human arteries. In the contracted state they appear in cross section not as small replicas of larger circular structures, but take on a new form. During the course of contraction they become flat and appear band-like as would a garden hose when it is compressed by a weight. In examining a vessel so contracted one sees on rotating the vessel either a broad side or a narrow one. It is for this reason that such arteries appear alternately narrow as a line or broad as a band. It is not until the narrow artery is elevated with a hook that its uniform band-like nature becomes evident. The absence of elastic tissue, the presence of which in all probability is mainly responsible for the usual shape of arteries on cross section, permits one to see how the phenomenon which has been described may come about. Concerning Paragraph 3 of the anatomical conclusions we have this to say. According to the histological investigation a stage of degeneration is wanting in the blood vessels of the embryonic membrane in a sense in which one is accustomed to see such changes in other blood vessel systems during the course of life. On the day of hatching the constituent cells and fibers of the arteries of all calibers are anatomically the same as in their early development. These vessels do not die as the result of aging: The nutrient fluid ceases to flow because of contraction of the umbilical vessels. The blood vessels die in complete possession of their physiological irritability and anatomical integrity. The unaltered irritability of blood vessels of the same caliber at all ages is consonant with their unaltered anatomical structure.     

THE RELATION OF THE SPLEEN TO BLOOD DESTRUCTION AND REGENERATION AND TO HEMOLYTIC JAUNDICE : XV. THE RESISTANCE TO HEMOLYTIC AGENTS OF DOGS IN WHICH THE SPLENIC BLOOD HAS BEEN DIVERTED FROM THE LIVER.

1. Dogs whose splenic veins or portal vein (Eck fistula) have been transplanted into the inferior vena cava, or whose splenic veins have been ligated, show a lessened tendency to jaundice similar to that exhibited by splenectomized animals. 2. Although the previously existing anemia and the concomitant increased resistance of the red cells of these animals are undoubtedly factors in the greater resistance to hemolytic agents, the lessened tendency to jaundice is, in part at least, due to a mechanical factor dependent on the course of the blood supply to the liver. 3. The additional anemia caused in the test animals by hemolytic agents is usually less than in the controls, although the total fall from the original normal may be greater. This applies to the splenectomized as well as the other test animals and is a modification of our former statements in regard to the severity of the anemia in splenectomized dogs. 4. Although the destruction of blood in these animals is less than in the normal controls, the repair of the same takes considerably longer than in the controls. This confirms similar results previously obtained in splenectomized animals. 5. The white cells exhibit much the same changes as they do following the administration of hemolytic agents to splenectomized or normal animals. As these changes are not unlike those following uncomplicated splenectomy or the operations here discussed, they cannot be considered as characteristic of any of the above procedures, but perhaps as an accompaniment of any temporary increased blood destruction. 6. The reaction of test and control animals is substantially the same, whether the jaundice is caused by toluylenediamin or hemolytic immune serum.     

STUDIES ON THE BLOOD VESSELS IN THE MEMBRANES OF CHICK EMBRYOS : PART IV. MODIFICATION OF IRRITABILITY OF THE BLOOD VESSELS

When faradic stimulation was undertaken of vessels irrigated with Ringer''s solution, which alternately contained and was free from carbon dioxide, it was observed that the reaction was far less when the solution contained carbon dioxide. A reversal of the effect could be obtained many times. It appears, therefore, that when Ringer''s solution contained carbon dioxide in the concentration described, the irritability of the vessels to electrical stimuli decreased, although carbon dioxide by itself and in the absence of the application of the stimuli, appeared to be void of effect upon the vessels. The rare, divergent results were traced to technical errors. We attempted to discover whether the observed decrease in irritability of the vessels might not be due to the absence of oxygen. For this purpose we irrigated the vessels with Ringer''s solution alternately containing nitrogen and oxygen. When nitrogen caused any change this was due to an influence on the rate of the heart and not on the irritability or reactivity of the arteries. In whatever way we tried we were unable to bring about a change in reactivity of the arteries by creating a condition of oxygen lack independently of a change in the rate of the heart beat. We attempted to study also the effect of other acids beside carbon dioxide on the changed reactivity of the arteries. Irrigation with various concentrations of lactic acid was without result. We also employed solutions buffered with potassium and sodium phosphate. When the irrigation was undertaken with these solutions having a pH range varying from 7.7 to 5.9 we observed neither a direct action nor one which modified the preparation in such a way as to change its susceptibility to faradic stimulation. Important investigations have been published recently by Atzler and Lehmann (2) on the direct influence of the hydrogen ion concentration on the behavior of blood vessels. Hammett and Zoll believed that, as the result of their experiments in which they attempted to bring about stimulation with solutions of concentrated carbon dioxide, they were able to exclude the possibility of action due to acid alone and therefore ascribed to carbon dioxide a specific effect. In our own experiments the method of irrigation does not permit an inference whether, or how far, an acid effect plays a rôle in the carbon dioxide experiments. For beside the question of hydrogen ion concentration and of buffering, the question of the penetration of substances from the surface to the contractile elements of the wall of the vessels requires to be considered. Carbon dioxide has an ability, beyond that of all other substances, to penetrate through tissues (3). It may be owing to this property that we could influence the reactivity of the blood vessels with it and it alone. This possibility must be further investigated. In these experiments, however, it was our object to show only that it was possible to influence the irritability of blood vessels experimentally. The conclusion is justified by our experiments that carbon dioxide in small concentrations reduces the threshold of irritability for electrical stimuli of the blood vessels of the embryonic membrane.     

THE PRESENCE OF RENIN ACTIVITY IN BLOOD VESSEL WALLS

The preparation, of an extract of hog blood vessels and organs containing renin activity has been described. The extract hydrolyzes natural or synthetic renin substrate to form a vasopressor material. This reaction could not be distinguished from the hydrolysis of renin substrates by kidney renin. The activation energy, effect of substrate concentration on velocity, inhibition by antirenin, and pH optimum of the extract and of kidney renin are the same. Renin activity is found in the adventitia and media of the aorta, the liver and, to a lesser extent, in other vascular organs.     

ON THE RELATION OF THE CATALYTIC ACTIVITY OF THE BLOOD TO THE NUMBER OF RED BLOOD CELLS IN HEALTH,AND TO THE NUMBER OF WHITE BLOOD CELLS AND THE BODY TEMPERATURE IN PERITONITIS

The catalytic activity of the blood of normal rabbits varies almost directly with the volume and number of red blood cells. This explains to a certain extent at least why animals of the same general degree of nutrition, and of the same litter, should have about the same activity since they are likely to have the same number of red blood cells, and why healthy large animals should read high while small poorly nourished ones should read low. Accompanying the hyperpyrexia resulting from puncture of the corpus striatum of a rabbit''s brain, there is no change in either the catalytic activity of the blood or the white blood count. In experimentally produced peritonitis, the catalytic activity of the blood always rises, and is, therefore, absolutely independent of body temperature and white blood cells since one or both of these may rise, fall or remain stationary while the catalytic action increases.