ANTAGONISM OF THE EFFECT 0’5 BRADYKlN-m BY NOREPWEPERNE 0N MiCROVASCULRR FLUID FLUX Thesis for the Degree of M. S. MiCHSGAN STATE UNIVERSi'fY JAMES MHN MACiEfiKO 1976 IIIIIIIIIIIIIIIIIIIIIIIIIIII 01591 3951 LIBRARY W 3:03 «W'- MINIMUM"NIWHIHIHNIIHNIIll”!!!MINIMUM 01591 3951 '3 BINDING DY llflAB & SIINS' MON 311105“ INC. LIBRARV mung..- 1 Q. 0‘ I. h .1 . v.,. o... 3217.2 ABSTRACT ANTAGONISM OF THE EFFECT OF BRADYKININ BY NOREPINEPHRINE 0N MICROVASCULAR FLUID FLUX By James John Maciejko Bradykinin (0.8 or 10 meg/min) infused into the brachial artery of the canine forelimb for 60 minutes, causes marked increases in fore- limb weight, lymph flow and lymph total protein concentration. The ‘mechanism is by an increase in the transmural capillary hydrostatic pressure gradient and by a decrease in the transmural colloid osmotic pressure gradient due to an increased microvascular permeability to plasma protein. In contrast, systemically (intravenous) administered bradykinin, even in blood concentrations equal to or exceeding those achieved by the local infusion, failed to increase forelimb lymph flow and lymph total protein concentration. Thus, there exists a route- dependent differential action of bradykinin on transvascular fluid flux. Possible explanations for this differential action between the two modes of infusion are: destruction of bradykinin by the lungs occurring with the intravenous infusion; inactivation by factors in the plasma before reaching the microvessels with the intravenous infusion; an effective antagonism by substances (i.e. catecholamines) released during a sympathoadrenal discharge subsequent to the hypotension pro- duced by the intravenous infusion. .Art' 5 .vol ' use-ea ,...\. I U tum-“7 .an'Ul-J ' 131E I 2‘ "he no on tale : . l‘nyIA .- DU :—-".n no d» 5A.- Bu.) v- 4. James John Maciejko Infusing bradykinin (140 to 280 meg/min) into the vena cava or the left ventricle of the heart slightly increased flow and total pro— tein concentration of the lymph, as compared to the local infusion route. There was little difference in lymph flow and lymph protein concentration between intravenous or left ventricular infusion, al- though left ventricular infusions increased these parameters slightly more than the intravenous infusions. This would seem to indicate that at these dosages of bradykinin, pulmonary inactivation plays a minor role in its destruction. The greater transit time required for bradykinin to reach the microvasculature during a systemic infusion, as compared to a local in- fusion, would allow more time for factors in the blood to inactivate bradykinin. However, this cannot account for the route-dependent dif- ferential actions of bradykinin. This can be explained by the fact that large increases in lymph flow and lymph protein concentration are observed with local infusions of bradykinin into forelimbs perfused at constant inflow. In these experiments, bradykinin must travel through a one to two meter length of polyethylene tubing before reaching the forelimb. Since this distance is greater than the distance bradykinin must travel during systemic infusions and if factors within the blood were destroying bradykinin, then the marked increases in flow and pro- tein concentration of the lymph would not be expected during the local bradykinin infusions at constant inflow. To investigate the possibility of an antagonism by substances released during a hypotensive sympathoadrenal discharge on the micro- vasculature, hypotension was produced for 60 minutes by hemorrhage. 1316 a 23¢" .(I’I mv‘ " ..I C no F11 "UEU. .3 , ’1 Il- ’(J ‘ J u up: us-u‘ l .‘vnn .-.t,.- l 0“. "In: I fcre. “:,} .l. U l I... :5.“ n I L- ”1-! ‘hbs ‘1 .“ 84“, I l ‘Fa‘ nc‘ '11., w.“ j- g (I) i 'm . “CI James John Maciejko This was followed by a 60 minute local infusion of bradykinin (0.8 and 10 meg/min, I.A.) into forelimbs perfused at constant inflow. Flow rate and protein concentration of the lymph increased very slightly to about the same levels as observed with the systemic infusions. To further examine the hypothesis that substances released during a sympathoadrenal discharge antagonize bradykinin at the micro- vascular site, weight, hemodynamic and lymph studies were conducted. Norepinephrine (4 mcg base/min) was infused simultaneously with brady- kinin (0.8 mcg/min) into the forelimb brachial artery. In contrast to bradykinin (0.8 meg/min) infused alone, the concurrent infusion with norepinephrine failed to alter lymph flow and lymph protein concentra- tion. In the weight and hemodynamic experiments at natural inflow, forelimb weight did not change, whereas at constant inflow, forelimb weight increased due to an augmented venous resistance (active venocon- striction by norepinephrine), thereby increasing microvascular pressure and filtration. Hence, it is concluded that norepinephrine prevents the marked increase in extravascular fluid volume that is produced by bradykinin. This antagonism could be due to a direct blockade of the action of bradykinin on the microvascular membrane, a shunting of blood flow from nutritional to non-nutritional channels, or a combination of both. Also, this antagonistic action of norepinephrine may, in part, explain the differential effects of locally and systemically administered bradykinin on lymph flow, protein efflux and microvascular fluid flux. ANTAGONISM OF THE EFFECT OF BRADYKININ BY NOREPINEPHRINE ON MICROVASCULAR FLUID FLUX BY James John Maciejko A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1976 To my parents Without their love, encouragement and support, my education and this thesis would not have been possible. 11 or. 4L» Kasai ZEZCE ACKNOWLEDGMENTS For their many contributions in this endeavor, I express my sincere appreciation to Dr. Jerry B. Scott, Dr. C. C. Chou, Mr. Edward Gersabeck, Mr. Douglas L. Marciniak and Mr. Daniel P. Sak. I also wish to thank Miss Darlene DenHollander for her assis- tance in typing. I am deeply indebted to Dr. George J. Grega, my advisor, for his counsel and valuable assistance throughout this undertaking. iii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . v LIST OF SYMBOLS AND ABBREVIATIONS. . . . . . . . . . . vii INTRODUCTION. . . . . . . . . . . . . . . . . . 1 SURVEY OF THE LITERATURE. . . . . . . . . . . . . . 3 STATEMENT OF THE PROBLEM. . . . . . . . . . . . . . 24 METHODS . . . . . . . . . . . . . . . . . . . 27 RESULTS . . . . . . . . . . . . . . . . . . . 33 DISCUSSION . . . . . . . . . . . . . . . . . . 47 APPENDICES . . . . . . . . . . . . . . . . . . 52 BIBLIOGRAPHY. . . . . . . . . . . . . . . . . . 100 iv H. Table LIST OF TABLES Effects of bradykinin infused intravenously (vena cava) into naturally perfused forelimbs for 60 minutes on lymph flow, protein concentration of lymph and plasma, vascular pressures and hematocrit . . . . . . . . Effects of bradykinin infused into the left ventricle of the heart at constant inflow for 60 minutes on lymph flow, protein concentration of lymph and plasma, vascular pressures and hematocrit . . . . . . . . Effects of locally infused bradykinin (0.8 or 10 meg/min, I.A.) for 60 minutes following 60 minutes of hypotension produced by hemorrhage to lower and maintain aortic pressure near 45 mm Hg. Constant inflow. . . . . . Effects of bradykinin (0.8 mcg/min, I.A.) and bradykinin and norepinephrine (4 meg/min, I.A.) infused locally into naturally perfused forelimbs on weight, blood flows, vascular resistances and vascular pressures. . . . . Effects of bradykinin (0.8 meg/min, I.A.) and bradykinin and norepinephrine (4 mcg/min, I.A.) infused locally into constantly perfused forelimbs on weight, blood flows, vascular resistances and vascular pressures. . . . . Effects of bradykinin alone or infused concurrently with norepinephrine base intra-arterially into the forelimb for 60 minutes on lymph flow, protein concentration of lymph and plasma, vascular pressures and hematocrit . . Effects of bradykinin infused intravenously (vena cava) into naturally perfused forelimbs for 60 minutes on lymph flow, protein concentration of lymph and plasma, vascular pressures and hematocrit . . . . . . . . Effects of bradykinin infused into the left ventricle of the heart at constant inflow for 60 minutes on lymph flow, protein concentration of lymph and plasma, vascular pressures and hematocrit . . . . . . . . . . Page 38 39 4O 41 43 45 53 56 Tame 5-4 . r3. 2. Li “A Table Page A-3. Effects of locally infused bradykinin (0.8 mcg/min, I.A.) at constant inflow for 60 minutes, following 60 minutes of hypotension produced by hemorrhage to lower and main- tain aortic pressure near 45 mm Hg. . . . . . . . 60 A—4. Effects of locally infused bradykinin (10 mcg/min, I.A.) at constant inflow for 60 minutes, following 60 minutes of hypotension produced by hemorrhage to lower and main- tain aortic pressure near 45 mm Hg. . . . . . . . 62 A-S. Effects of bradykinin (0.8 mcg/min, I.A.) infused locally into naturally perfused forelimbs on weight, blood flows, vascular resistances and vascular pressures. . . . . 64 Ar6. Effects of bradykinin (0.8 meg/min, I.A.) and norepinephrine (4 mcg/min, I.A.) infused locally into naturally perfused forelimbs on weight, blood flows, vascular resistances and vascular pressures. . . . . . . . . . . . 69 A-7. Effects of bradykinin (0.8 mcg/min, I.A.) infused locally into constantly perfused forelimbs on weight, blood flows, vascular resistances and vascular pressures. . . . . 74 Ar8. Effects of bradykinin (0.8 mcg/min, I.A.) and norepinephrine (4 mcg/min, I.A.) infused locally into constantly perfused forelimbs on weight, blood flows, vascular resistances and vascular pressures. . . . . . . . . . . . 8O A-9. Effects of bradykinin (0.8 mcg/min, I.A.) infused into the forelimb at natural inflow for 60 minutes on lymph flow, protein concentration of lymph and plasma, vascular pressures and hematocrit . . . . . . . . . . . 86 ArlO. Effects of bradykinin (0.8 meg/min, I.A.) and norepinephrine (4 meg/min, I.A.) infused at natural inflow for 60 minutes on lymph flow, protein concentration of lymph and plasma, vascular pressures and hematocrit . . . . . . . . 89 .A—ll. Effects of bradykinin (0.8 meg/min, I.A.) infused into the forelimb at constant inflow for 60 minutes on lymph flow, protein concentration of lymph and plasma, vascular pres- sures and hematocrit . . . . . . . . . . . . 92 A-lZ. Effects of bradykinin (0.8 mcg/min, I.A.) and norepinephrine (4 meg/min, I.A.) infused at constant inflow for 60 min- utes on lymph flow, protein concentration of lymph and plasma, vascular pressures and hematocrit . . . . . 96 vi CF LH IA IV E gms mcg min mmHs 80.8 BIO 3140 8280 N4 LIST OF SYMBOLS AND ABBREVIATIONS Naturally perfused forelimb. Forelimb perfused at constant inflow. Drug infusion into the left ventricle of the heart. Drug infusion intra-arterially into the forelimb. Drug infusion into the vena cava. Centimeter. Millimeter. Micrometer. Kilogram. Grams. Milligram. Microgram. Milliter. Minute. Angstrom. Millimeters of mercury pressure. 0.8 meg/min of bradykinin. 10 meg/min of bradykinin. 140 mcg/min of bradykinin. 280 mcg/min of bradykinin. 4 mcg/min of norepinephrine base. vii p §_0.0l p §_0.05 p j_0.01 p §_0.05 relative relative relative relative to t0 t0 t0 0 minute control. 0 minute control. 60 minute control. 60 minute control. viii INTRODUCTION The pharmacological effects of the venom of Bothrops jararaca (South American serpent) had been under investigation in 1949 by Rocha e Silva and led to the finding that incubation of the venom with the globulin fraction of blood plasma from a dog resulted in a potent vaso- dilator and smoothdmuscle-stimulating substance (47). The material was named bradykinin, owing to its slow muscle contracting action on pig ileum, and the globulin fraction from which the bradykinin was released was named bradykininogen. In 1960, bradykinin was isolated by Elliot (13) from ox serum treated with trypsin and later synthesized by Boissonnas (5). Bradykinin is known to stimulate certain types of smooth muscle, to cause vasodilation, to increase capillary permeability, and to pro- duce pain when brought into contact with pain fibers (34). This study is principally concerned with those actions of bradykinin pertinent to pathological conditions which are manifested by abnormal fluid fluxes causing tissue edema. It is well established that bradykinin, administered locally (0.8 or 10 meg/min, I.A.) increases the efflux rate of water from capillaries and the immediate post-capillary venules leading to massive edema formation with the higher dose. The mechanism of this effect is It .. ‘I‘ A . fl- :1 ('7‘ < (D 5(53559 3331 C01. 3&3! per gfinisu 111‘ jPLALar‘ who: :5 int: dons we fusion 5 trough Nlatur :tansfo half-1i t: the Stunt effect tradyk pressu that : genizc 2 both by a rise in the transmural capillary hydrostatic pressure gradi- ent subsequent to arteriolar vasodilation and by a fall in the trans- mural colloid osmotic pressure gradient owing to an increased microvas- cular permeability to plasma protein. However, when bradykinin is administered systemically (iv), it fails to increase the rate of trans— capillary fluid movement (8). There are obvious differences which may account for the observed route dependent differential actions of brady- kinin on fluid movement. First of all, in the studies pertaining to the intravenous infusions of bradykinin on fluid fluxes, the concentra- tions were too small to equal the concentrations used in the local in— fusion studies. Also, during intravenous infusion, bradykinin passes through the pulmonary circuit before reaching the systemic microvas- culature. It is well established that the lungs metabolize or bio- transform bradykinin (1, 17). Furthermore, bradykinin has a short half-life in plasma, and the transit time from the point of infusion to the microcirculation with systemic infusions could result in de- struction. Finally, locally administered bradykinin has little if any effect on systemic arterial pressure, whereas intravenously infused bradykinin causes marked hypotension. Since the fall in systemic blood pressure is associated with a sympathoadrenal discharge, it is possible that substances liberated subsequent to hypotension effectively anta- gonize the edemogenic action of bradykinin. Consequently, it is the goal of this investigation to construe the mechanisms of the route dependent differential action of bradyki- ninl on transvascular fluid movement. ‘ Due to the excessive cost of commercially available bradykinin ($7.00 to $15.00/mgm), and the large quantities used in the systemic infu- sion studies, these experiments had to be kept to a minimum. Only those systemic experiments of utmost importance were carried out. the ’IEIU file on 0" z A. In SURVEY OF THE LITERATURE Furnishing the tissues of the body with blood in amounts suffi- cient to meet the requirements for oxygen, nutrients and the removal of metabolic byproducts is the foremost aim of the cardiovascular system. The capillaries, which form an interconnecting network of tubes between the arterioles and venules, together with the immediate postcapillary venules, accomplish this function by allowing exchange to occur between the blood and interstitium. Exchange of substances between blood and tissues can occur by filtration, diffusion or pinocytosis. Mbvement of fluid across the capillary wall and into the inter- stitium is based on the balance between hydrostatic and osmotic forces. The degree of filtration or reabsorption is dependent on the sum of these physical forces which can be related in the following equation derived by Starling (52): F - k(Pc - Pi - Np - Ni), where F = the rate of fluid movement; k - filtration coefficient (This coefficient is a measure of the permeability of the microvascular wall to isotonic fluid. It is determined by the product of capillary per- meability and surface area available for diffusion.) (33); Pc = capillary hydrostatic pressure; :ated 3.1 l :igic Tue 1 he: Pi = interstitial hydrostatic pressure; up . plasma colloid osmotic pressure; fii - interstitial colloid osmotic pressure. According to Starling's hypothesis, when the algebraic sum of this equation is positive, filtration occurs; when it is negative, reab- sorption occurs. Capillary hydrostatic pressure (Pc) is directly dependent upon capillary blood volume and compliance. Clough gt El: (7) have indi- cated that capillaries are quite rigid. They report a change of only 0.1 um in radius in capillaries of cat mesentery during systole. This rigidity results from the environment circumjacent to the capillaries. The basement membrane and gel matrix surrounding these microvessels give the capillaries little, if any, compliance (18). Since compliance is relatively constant in capillaries, changes in capillary blood volume are the primary factor in determining Pc. Capillary blood volume is influenced by systemic arterial pressure, venous pressure and the pre and post capillary resistances. The interrelationship of these factors is expressed in the following equation of Pappenheimer and Soto- Rivera (33): Pc - (Pa - Rv)-7R:§h;— + Pv, where Pa = systemic arterial pressure; Pv - venous pressure; Ra = arterial resistance (precapillary); Rv - venous resistance (post capillary). An increase in Pa or Pv will increase Pc. A given increase in Pv, however, has a five to ten fold greater effect on Pc (33). Increasing Rv will raise Pc, whereas increasing Ra will lower Pc. Vessel resis- tances are indirectly related to vessel caliber. This caliber is de- termined mainly by active changes in vascular smooth muscle activity and passively by changes in effective transmural pressure. Effective transmural pressure is the pressure in the interstitial fluid environ- ment of the capillary subtracted from the intraluminal pressure in the capillary. Changes in blood viscosity also affect resistance to blood flow. Blood viscosity is determined by the hematocrit and the dis- solved materials in the plasma. The hydrostatic pressure of the interstitial spaces is deter- mined by tissue compliance and interstitial fluid volume. Classically, it is accepted that this pressure is positive and will therefore, oppose fluid filtration out of the capillaries. However, Guyton (23) using implanted perforated spheres in various tissues, has concluded that this pressure is sub-atmospheric (-7 mmHg). This issue is under much criticism and further investigation is needed to resolve this dilemma. Plasma colloid osmotic pressure (oncotic pressure) is the pres- sure due to the concentration of dissolved proteins in the blood. Under normal conditions, the total osmotic pressure of plasma is about 6,000 mmHg, with the oncotic pressure contributing about 25 mmHg (4). This oncotic pressure is responsible for vascular fluid retention. It is achieved because the plasma proteins are largely confined to the intra- vascular space and therefore, create an active tonicity within the vas- culature. The ions which constitute the bulk of the remaining total Ant,“ 3w 9‘, ti . D H!“ .w'uc W1 .35 P5‘ osmotic pressure pass freely through the capillary membrane, creating no tonicity between the two body fluid spaces (43). Albumin, globulin and fibrinogen constitute the major plasma proteins. Albumin, which is found in the greatest abundance, has an average molecular weight of 69,000 and a concentration of about 4.6 gms Z. Globulin has an average molecular weight of about 140,000 with a concentration of 2.5 gms Z. Fibrinogen is the largest of the plasma proteins, having a molecular weight of about 400,000 but is found in a concentration of only 0.3 gms 2. 0f the total oncotic pres- sure, 19 mmHg are attributable purely to the proteins, and the remain- ing 6 mmHg are due to the cations which bind to the proteins by electro- negative forces. This phenomenon is known as the "Donnan Effect". Since albumin is in the greatest concentration of the three proteins, it constitutes the largest fraction (70%) of the total oncotic pressure. Interstitial fluid colloid osmotic pressure is contingent on the protein concentration of the interstitial fluid. Normal concentra- tion of the interstitial proteins is not uniform; it varies from 0.4 gms Z to 3.3 gms Z, depending on the tissue (33). In skin and skeletal muscle, the average interstitial protein concentration is about 2.0 gms 1, which yields an oncotic pressure of about 5 mmHg. More recent findings suggest that the total protein concentration of interstitial fluid is about 3 gms z and the colloid osmotic pressure about 10 mmHg (58). In the liver, where capillary protein permeability is high, large amounts of the plasma proteins cross the microvascular membrane, producing an interstitial fluid oncotic pressure of about 16 mmHg or more. This corresponds to a minimal protein concentration of about 3.3 gms Z; in fact, this value is often greater. Electron microscopy has revealed that the ultrastructure of the capillaries is not the same in all parts of the circulation (40). Three different types of capil- lary walls have been identified; they are termed continuous, discontin- uous and fenestrated. The continuous capillary wall is the most common type observed in smooth and skeletal muscle, adipose tissue, connective tissue and pulmonary tissue. The capillary wall is a continuous mem- brane of endothelial cells with numerous intercellular channels, 40 to 50 2 wide, connecting the lumen of the capillary with the interstitial space around it. Fenestrated capillaries have intracellular fenestra- tions (openings) in the endothelial wall. The openings are about 0.1 pm in diameter, and may have thin membranes closing them. These types of vessels are found in the intestinal mucosa and the renal glomeruli. In discontinuous capillaries, the endothelial wall is interrupted at intervals by large gaps. These gaps are of such diameter that formed elements of the blood and fluid can freely pass. These capillaries are characteristic of bone marrow, the spleen and the hepatic sinusoids. Discussion of absolute values for interstitial fluid colloid osmotic pressure is under considerable debate. Measurements using im- plantable devices such as perforated capsules that theoretically equil- ibrate with interstitial fluid, may be inaccurate because of the possi- bility of contamination by plasma, or that the fluid sampled may not necessarily contain all the osmotically active substances. The most common method for measuring interstitial oncotic pressure is lymph analysis. This method makes the assumption that lymph is a true re- flection of the interstitial fluid contents. Critics of this view argue [ha {631331 min con: 95) havl size are ten inj Eat the :apillax :23: ex: Kinks. trse re :essel 52th (11' fluid 1 squeeze :ed be $10!} 1 20190 Beam argue that changes could occur in lymph composition as it flows from terminal lymphatics upward to larger vessels due to gradients of pro- tein concentration within the interstitial spaces. Renkin and Garlick (45) have shown that dextran molecules of known molecular weight and size are in equal concentration between lymph and interstitial fluid when injected into a tissue. This observation allowed them to conclude that there is no significant protein concentration gradient beyond the capillaries. Garlick and Renkin (20) have performed studies showing that exchange occurs only at lymph nodes and not in the lymphatic trunks. If lymph is sampled before it reaches a node, it should be a true reflection of what is at the terminal lymphatic vessel. Valves exist throughout all lymphatic channels. When a lymph vessel is compressed by pressure, lymph in the channel is squeezed in both directions. Since the valves are only unidirectional to flow, the fluid that will be transported from the terminal lymphatics is that squeezed in the central direction which flows past the valve. Factors that can compress the lymphatics and evoke the movements of lymph are: muscle contraction, arterial pulsations, passive movements of the parts of the body, and compression of the body tissues from the outside (22). During exercise, therefore, lymph flow can increase substantially to about 14 times normal. The most important mechanism by which substances are transpor- ted between the plasma and interstitial fluid is by diffusion. Diffu- sion is a process which is dependent only on the thermal movement of molecules; that is, the greater the concentration difference, the greater the diffusion. This mode of exchange can be described by the Fick Law of Diffusion, which states that the quantity of substance moved per unit time is equal to the free diffusion coefficient of the mole- cule, the concentration gradient, and the area of the capillary mem- brane; These factors are related in the following equation: ds , . dc (it D A dt’ where ds dt - amount of substance moved per unit time; D - free diffusion coefficient for a molecule. (This value is inversely proportional to the square root of the molecular weight.); A - area of capillary membrane; dc dt - concentration gradient. The site of diffusion of a molecule depends on whether the sub- stance is water soluble or lipid soluble. water soluble substances pass through pores in the endothelial cell. For small molecules such as water, ions, and urea, diffusion is free and rapid. However, for lipid-insoluble molecules of increasing size, diffusion becomes pro- gressively more restricted, such that molecules above a molecular weight of 60,000 are almost completely impermeable. Lipid soluble molecules such as C02, 0 and anesthetic gases pass freely through the 2 intact cell. As a result, lipid-soluble molecules pass with great ease and rapidity between the capillary and interstitium. The ease with which a lipid soluble substance passes through the capillary endothelium is dependent on its oil to water partition coefficient. Pinocytosis is a very slow active tranSport process, which is believed not to contribute much to total transcapillary exchange. Elect .- ‘- i A. r I 733W OI ta] as H. gr 10 Electron microscopy has revealed that this mechanism involves vacuoles which traverse across the endothelial cell of the capillary wall. The vacuoles are formed by being pinched off the surface membrane of the cell. They are believed to contain macromolecules which cannot be readily exchanged by either filtration or diffusion. Presently, con- clusive evidence is lacking to support the significance of pinocytosis in transcapillary protein movement. Any physical stimulus or substance which can influence the con- tractile property of vascular smooth muscle or alter the microvascular permeability to plasma protein, can modify fluid movement in the capil- lary bed. Fluid movement is determined by the hydrostatic pressure difference between the blood and interstitial fluid. The contraction or relaxation of vascular smooth muscle will decrease or increase capillary blood volume respectively, thereby decreasing or increasing capillary hydrostatic pressure. Increasing the permeability of the microvascular membrane to protein will result in the escape of plasma protein from the vasculature to the interstitial fluid and raise the oncotic pressure of the interstitium. This augmentation of intersti- tial fluid oncotic pressure will enhance fluid movement out of the capillaries. In the body, vasoactive agents affect fluid movement by exerting a relaxing or contracting effect on the vascular smooth muscle and also by enhancing capillary permeability to protein. Two of the most important of these vasoactive agents are histamine and bradykinin. Over the past five decades, vasoactive substances have been implicated in many pathological conditions, such as inflammation, shock and tissue injury. Of particular interest is the role of vasoactive gfistanc 51'! V85 cf leukc bicod a inc-teas ally co tat; re it is t Bolatc :ation amine :Esetv of the and on Eadie: Cf int tatint thesi: the 1 11min Patio ttnsi >1! ’1 (D I‘: 11 substances in inflammation. The inflammatory response is characterized by: vasodilation, increased vascular permeability, pain and migration of leukocytes (34). Histamine is significantly involved in the mediation of the inflammatory reaction. ‘It is found in the basophilic leukocytes of the blood and the mast cells of tissues. Histamine produces vasodilation, increased vascular permeability, migration of leukocytes and is gener- ally conceded to be the principal mediator of the immediate inflamma- tory response to injury. Although histamine initiates inflammation, it is believed not to sustain the vascular changes because it has been isolated only from tissue exudates in the early stages of acute inflame mation (46). However, Kahlson and Rosengren (30) have shown that his- tamine is also involved in the latter stages of inflammation. They observed that in rats and guinea pigs, the histamine formation capacity of the basophilic leukocytes increases with the onset of inflammation, and once the new histamine is produced, it can be released and continue mediating inflammation. Thus, the histamine present in the mast cells of inflammed tissues is utilized to initiate inflammation, and the his- tamine found in the leukocytes, due to an increase in the rate of syn- thesis, mediates the latter stages of inflammation. Therefore, between the initial and latter stages, no histamine is present, and the plasma kinins are speculated to mediate the inflammatory reaction during this period (60). The observation that urine injected intravenously causes hypo- tension led to the discovery of the plasma kinins. In the late 1920's Frey and associates (58) characterized this hypotensive substance, and 12 also noted that it was found in a number of tissues. The material was first named kallikrein because it was found in abundance in the pan- creas. werle g£_§l, (1937) (57) observed that kallikrein had an in- direct effect by acting as an enzyme that cleaved off a pharmacologi- cally active substance from a precursor present in the plasma. This substance was named kallidin. The discovery of bradykinin came in 1949, when Rocha e Silva (47) observed that trypsin or snake venoms released a peptide from a plasma substrate. The name bradykinin referred to the ability of the peptide to produce slow contraction of guinea pig ileum in vitro. Kallidin and bradykinin together with the plasma kinins are collectively referred to as the kinins. Bradykinin has the following amino acid sequence: HZN - Arg - Pro - Pro - Gly - Phe - Ser - Pro - Phe - Arg - OH. The other kinins exhibit this nonapeptide sequence and differ only in having additional amino acid residues on the N- or C- terminal. Clinical interest in bradykinin has been evoked because of its ability to mimic the main features of the inflammatory response and because it occurs in significant amounts in a wide range of diseases. Bradykinin is cleaved from a precursor termed kininogen, which is found in the plasma az— globulin fraction. The cleavage occurs by a group of enzymes collectively termed kininogenases and includes kallikreins, trypsin, pepsin as well as proteases in snake venoms and bacterial by- products. Of the kininogenases, the most important are the kallikreins, which are widely distributed and divided into tissue kallikreins and plasma kallikrein. Tissue kallikreins are often secreted in an active form from the salivary gland, pancreas, skin (sweat), small and large 13 bowl and the kidney, generally in response to systemic disorders (trauma, heat, infection). Compared with tissue kallikreins, plasma kallikrein differs physiochemically, in that it is formed from an inert precursor (prekallikrein). Activation of plasma kallikrein involves a series of enzymes, which are sequentially converted from pre-enzyme, with the latter successfully activating the next enzyme in the series. Activation is initiated by the Hageman factor (factor XII), which is also involved in blood clotting. The entire sequence of plasma kalli- krein activation is presented in Figure I. Figure I Trauma, Hageman Factor Systemic Infections, Heat, Radiation Plamminogen 4/ Tissue ,7 Kallikrein contact-—+> \/ Pre-kallikrein Plasmin -————e> V pro PF /d11 > V Kininogen Activated Hageman Factor TIF“‘~£>| Plasma Kallikrein.__;> \/ V 1, \1 \/ PF/dil Bradykinin Kallidin Coagulation of Blood Amino >> Peptidase Bradykinin Activated Hageman factor can transform prekallikrein to kalli- krein either by activating plasmin or PF/dil. Plasmin is a proteolytic enzyme involved in the clot-lysing system; it dissolves fibrin and Ill: 1 of 1 v :3 . r ii. 14 subsequently destroys the clot. PF/dil is a plasma factor which can lead to cell lysis and the release of kininogenases. Bradykinin inactivation is very rapid; the half-life in cir- culating blood is less than 15 seconds (11). The kininases are a group of enzymes which inactivate bradykinin. Plasma contains two kininases: carboxypeptidase N, which removes the C- terminal arginine group, and a dipeptide hydrolase that cleaves the proline-phenylalanine bond. Howe ever, inactivation of bradykinin occurs more effectively in the lungs than in the plasma. Friedli g a1. (17) and Alabaster gt; E. (1) have reported up to 952 destruction of bradykinin following a single passage through the pulmonary circuit. Furthermore, the breakdown of bradyki- nin in the lung appears to be related to age. Friedli gt El: (17) have shown that a high degree of inactivation occurs in mature ewes (932), whereas newborn lambs show significantly less inactivation (68%). Fetal lambs at birth show 46% inactivation, and pre-term fe- tuses (110-128 days gestation) demonstrate no bradykinin inactivation in the pulmonary vascular bed. Bradykinin is conceded to be a prominent mediator of inflamma- tion, because of its ability to cause vasodilation, increased vascular permeability, pain and local accumulation of leukocytes, when injected intradermally (48). Furthermore, the probable activation of Hageman factor and other prekininogenases by contact with injured tissues, would explain the formation of kinins in inflammation. However, there is difficulty in demonstrating bradykinin in inflammatory exudates because of its rapid inactivation by kininases (34). Bradykinin has been claimed to also play a possible role in many other disease states. ‘ .eve by a 11:11 tcxi (Le the 15 In carcinoid tumors, which have metastasized to the liver, bradykinin concentrations range from 9-25 meg/100 ml of hepatic venous blood, as compared to a normal range of 0.1-7.9 meg/100 ml of hepatic venous blood (59). Bradykinin is believed to cause vasodilation and increased blood to the area of the tumor, supporting the tumor's enhanced meta— bolic requirements. In endotoxin induced shock, bradykinin is believed to decrease peripheral vascular resistance, which causes decreased blood pressure (59). Endotoxin is believed to activate the Hageman factor, which causes enhanced bradykinin levels. In studies using the unanesthetized Rhesus monkey, bradykinin levels in arterial blood samples increased during endotoxin shock (41). Bradykinin increased from a control value of 0.0 mcg/ml to 0.011 mcg/ml, after a 30 to 40 minute infusion of 10 mcg/kg Escherichia coli endotoxin. Bradykinin levels were shown to increase in rabbits subjected to endotoxin shock by also activating the Hageman factor (15). In these studies, assayed kininogen levels decreased 40% from control, after injection of endo- toxin, due to the release of bradykinin. In traumatic shock states (i.e. traffic accidents) the kininogen level is lowered in man from the normal value of 15 to 20 meg/ml plasma to 14.5 to 9.5 meg/ml plasma, indicating the release of bradykinin (55). Therefore, on a molar basis, up to 32,500 mcg of bradykinin could theoretically be released in the average man (blood volume - 5,000 ml). Bradykinin has two important effects on the cardiovascular system which have previously been eluded to. The first is a pronounced hypotension with systemic administration, and the second is an increase in the capillary membrane's permeability to protein, such that water efflux occurs, with local administration. ”is 51-M- _-111. _---..111 ---11.-1_-l1.1_1|._.le 511 it: Yet 16 The intravenous (systemic) infusion of bradykinin results in a fall in systemic arterial pressure. See Figure II. Figure II Dosage of Bradykinin Reference Species Blood Pressure Mode of Infusion 10 min infusion 40 Dog 2 mcg/kg + of 412 from control into femoral vein 40 Cat 10 mcg/kg + of 552 from control " 40 Chimp 1.5 mcg/kg + of 282 from control " Bolus injection control inf, into jugular, 16 Dog 1.2 meg/kg 120 mmHg 55 mmHg femoral or brachial vein control inf. 16 Dog 0.5 mcg/kg 120 mmHg 75 mmHg " control inf. 1.5 min infusion 8 Dog 12.4 meg/min 113 mmHg 79 mmHg intravenously In the experiments where bradykinin was infused over a 10 minute period, blood pressure slowly waned to control levels in 3 to 6 minutes. Where bradykinin was administered in bolus injections, blood pressure returned to normal almost immediately. Since cardiac output is increased (12, 16, 42), the hypotension results from a decreased peripheral vascular resistance. Total peripheral resistance decreases only because of an increase in blood vessel reg: pres stro inc: dial ind dir met 1111 the til enh 17 radius, since hematocrit does not change or slightly increases (50), and vessel length is suspected not to change significantly. The de- creased resistance occurs mainly at the arteriolar level, owing to the relaxation of vascular smooth muscle. Several investigators, however, have noted that the hypotension manifested during the intravenous in- fusion of bradykinin is not maintained (14, 42, 50). This is due to a waning of the effect of bradykinin on peripheral vascular resistance. This suggests bradykinin could evoke compensatory responses, such as enhanced sympathoadrenal activity, enhanced kininase activity, or auto— regulation, which would antagonize the steady decline in arterial blood pressure (24). The increase in cardiac output results from the elevation of stroke volume and perhaps stroke frequency (24). Stroke vOlume is increased mainly because of the decrease to ejection. Harrison gt El' (26) have observed in the open-chest dog, that stroke volume may also 'increase by reason of small gains in left ventricular contractile force. Bradykinin in physiological concentrations has little effect on stroke frequency in the isolated heart (24). The increase in car- diac frequency seen with intravenous infusions results probably from indirect mechanisms, since no evidence exists showing that bradykinin directly enhances the frequency rate of the heart. One possible mechanism is activation of the baroreceptors as a result of the reduced hypotension. This activated mechanism will enhance sympathetic dis- charge (norepinephrine) and thus increase the heart rate and contrac- tile force. Epinephrine secretion by the adrenal medulla is also enhanced through the baroreceptor mechanism and will cause an increase 11:5 wt 6 4 ’3‘: we“ 10 18 in stroke frequency. Lewis (34) has shown that bradykinin acts direct- ly on the adrenal glands to cause the release of catecholamines. While this phenomenon seems likely to increase cardiac frequency during sys- temic bradykinin infusion, the question as yet has not been critically examined. Surprisingly, there are little data on the effects of systemr ically administered bradykinin on fluid flux and extravascular fluid volume. MOst investigators simply assumed that local and systemically infused bradykinin would exert qualitatively the same effect on fluid filtration. Data in the literature suggest that this may not be so. Daugherty gngl, (8) noted that intravenous infusions of bradykinin (20.6, 41.2 and 82.4 meg/min) did not change weight and failed to increase small vein pressures in collateral-free, innervated canine forelimbs. The infusion period for each of the bradykinin concentra- tions employed averaged about 1.5 minutes. These studies indicate that intravenously administered bradykinin is unsuccessful in promoting edema formation in both skin and skeletal muscle, because of a failure to increase transmural capillary hydrostatic pressure and/or decrease transmural oncotic pressure. However, it is possible that results would be different if bradykinin were infused systemically over a longer period of time and at a higher dosage range. Absolute plasma volume during the systemic infusion of brady- kinin has not been measured. However, because of the similarity in the effects observed between systemic bradykinin and systemic histamine infusions (marked hypotension), it would not be surprising if bradyki- nin also failed to affect plasma volume. Deyrup (9) injected histamine C8: are 19 (3 to 12 mcg/kg) subcutaneously into the thigh of the canine hindlimb. Plasma volume changes were assessed as an indication of histamine's effect on transcapillary fluid flux. The results show that plasma volume was unchanged or moderately increased, and in a few cases it was slightly reduced. She also observed no evidence for increased capillary permeability, since the escape of albumin-bound dye T—1824 from the vasculature did not increase. Local infusions of bradykinin clearly cause rapid efflux of fluid from many systemic vascular beds. The increased net transvascu- lar fluid efflux has been inferred from the development of edema, increases in organ weight, and increases in flow rate and protein con— centration of lymph in forelimbs infused with bradykinin (2, 8, 10, 32). The increased net fluid filtration is attributable to both a rise in the transmural capillary hydrostatic pressure gradient and a fall in the transmural colloid osmotic pressure gradient. Small vein pressures and blood flow in skin and skeletal muscle are markedly increased by bradykinin in the canine forelimb (8, 32), suggesting that capillary hydrostatic pressure is greatly increased. The increased microvascular pressure is attributable to an augmented capillary inflow subsequent to arteriolar vasodilation. However, direct measurements of capillary hydrostatic pressure have never been made. The fall in the transmural colloid osmotic pressure gradient is attributable to an increased microvascular permeability to plasma protein. Flow rate and total protein concentration of lymph draining a vascular bed increases markedly from control during the infusion of 11er : ace flue t t 1111 {GEEK 131m evid- pre: the sen she bra Que tha 20 bradykinin (0.8 or 10 meg/min) (32). With the higher dosage of brady- kinin, the lymph protein concentration approaches plasma protein values. The increased protein efflux is usually attributed to an increased pore size by a direct action of bradykinin on the immediate post-capillary ‘microvascular membrane (19). Microscopic studies demonstrate the exis- tence of gaps appearing between adjacent endothelial cells, perhaps due to "rounding up" of the cells, thereby creating an increased intra- cellular cleft (35). The theory that bradykinin causes an increased pore size has recently been challenged by Renkin and his co-workers (6, 29, 44). These investigators believe that augmented pinocytosis is the major route of protein efflux from the microvasculature. However, definitive evidence for this hypothesis is still lacking. There is some controversy in the literature surrounding the possibility that increased capillary hydrostatic pressure increases microvascular permeability and therefore that the increased protein efflux is due to an indirect action of bradykinin. Various studies have presented evidence consistent with the concept that the microvas- cular surfaces become more permeable to macromolecules as microvascular pressure is increased. The increase in permeability occurs mainly at the level of the venous capillary and venule. Rowley (51) has pre- sented findings consistent with the "stretched pore phenomenon" (52), showing that the increased capillary hydrostatic pressure observed with bradykinin, forces the opening of microvascular pores and the subse- quent loss of plasma protein. He believes that this is the major way that bradykinin acts on the microvascular membrane to decrease the 364' "4 ‘91 Ital-pl 11: YRS! 3&1 pro for sti 21 transmural oncotic pressure gradient, since he observed no increase in macromolecular efflux when capillary hydrostatic pressure remained con- stant. Recent studies by Kline 35 El: (31) have presented data sugges- ting that increased microvascular pressure is not associated with in-. creases in microvascular permeability to plasma proteins as is seen with bradykinin. In fact, bradykinin increased protein efflux greatly in forelimbs perfused at constant flow. Under this condition, micro- vascular pressure either failed to increase relative to control or decreased slightly; yet marked protein efflux occurred (32). Further- more, there was no evidence of venous constriction in any of the experi- ments reported by these investigators (32). Thus, local infusions of bradykinin cause edema by two contributing mechanisms. From experi- ments comparing weight gain, lymph flow and protein concentration in forelimbs infused with bradykinin (0.8 or 10 mcg/min, I.A.) at natural or constant inflow, it was estimated that a larger proportion of the edema was due to a decreased transmural oncotic pressure gradient or a direct action of bradykinin on the microvascular membrane promoting protein efflux (32). The increased capillary hydrostatic pressure, forcing fluid (water) out of the microvasculature and into the inter— stitium, exerts the lesser effect in edema formation. It is also thought that capillary surface area increases with locally infused bradykinin. The increased surface area would enhance the volume of fluid filtered per unit time, thereby adding to the edema (24) . Daugherty 22 El: (8) noted that in comparing intra-arterial and intravenous infusions of bradykinin, that large increases in hindlimb 22 weight occurred during intra-arterial administration, whereas during intravenous infusion, weight did not change. Since locally administered bradykinin increases microvascular permeability and subsequently causes net fluid filtration even when capillary hydrostatic pressure remains constant at a normal level, it is bewildering why net fluid filtration does not occur during systemic administration of bradykinin. There are several possible explanations for this route-dependent differential action. First of all, by infusing bradykinin intravenously or down— stream to the lung, it must pass through the pulmonary circuit where up to 95% destruction can occur, thereby allowing very little or no drug to enter the capillary beds. Secondly, if bradykinin was given intravenously in large enough quantities so that a significant amount escaped pulmonary inactivation and reached the arterial vasculature causing acute hypotension, catecholamines would be released.- The mechanism for this release would be by increased sympathoadrenal dis- charge resulting from the activation of the baroreceptor phenomenon and by a direct action of bradykinin on the adrenals to cause epinephrine secretion. The release of the catecholamines could effectively antago- nize the microvascular effects of bradykinin. Finally, there are many other substances released in response to hypotension such as renin, an- giotensin II, vasopressin (ADH), aldosterone, etc., which may also an— tagonize the effects of bradykinin on the microvascular membrane. The catecholamines released during hypotension (49) are also secreted in situations of hypoxia, asphyxia and emotional stress. They are vasoconstrictors, and there is considerable disagreement in the literature as to their effect on transvascular fluid flux. In general, veil ghri 1018 teat that seat hOU1 .eg int and pet of 23 the catecholamines constrict the capacitance vessels (veins) and the resistance vessels (arteries and arterioles). One exception is epine- phrine, which in low concentrations dilates precapillary vessels in skeletal muscle (27). When norepinephrine is infused into naturally perfused forelimbs and ileum segments, small vein pressures have been observed to increase, decrease or remain unchanged. The weight of the organ (forelimb or ileum) altered directly with the change in small vein pressure. This association between small vein pressure and organ weight can be explained in terms of the varied effects of the norepine- phrine on pre and post-capillary resistances. If the venules constrict more than the arterioles, capillary outflow would be impeded. This would increase capillary hydrostatic pressure and cause increased fil- tration of fluid and thus an increase in organ weight. If the arteri- oles constrict more than the venules, capillary inflow would be impeded and hydrostatic pressure would decrease, thereby favoring net fluid reabsorption. Studies performed by Mellander and Nordenfelt (37) have shown that capillary surface area available for diffusion and capillary per- meability to proteins were unaffected by norepinephrine. Jarhult (28), however, has observed that in denervated skeletal muscle of the lower leg of the cat hindlimb perfused at constant inflow, norepinephrine increases capillary surface area for diffusion. In contrast, Appelgren and Lewis (3) have reported a decrease in capillary surface area and permeability in naturally perfused human skeletal muscle when solutions of 0.4 meg/ml of norepinephrine were infused locally. intr1 csmo ares ital 2W8 feat iStI act kin- the' eit aCt STATEMENT OF THE PROBLEM It is well established that bradykinin administered locally, intra-arterially, increases the rate of transcapillary fluid movement, as evidenced by edema formation, by decreasing the transmural colloid osmotic pressure gradient and increasing the transmural hydrostatic pressure gradient. However, when bradykinin is administered system- ically (iv), it fails to increase the rate of transcapillary fluid movement. This suggests that the edemogenic action of bradykinin is route dependent. Studies by Friedli (17) and Alabaster (1) have shown that bradykinin is inactivated up to 95% through the passage of a single pulmonary circuit. This is due to the high concentration of kininases in pulmonary tissue. These investigators also suggest this may be why intravenous bradykinin fails to promote edema. However, if bradykinin was administered in large amount, so that a significant quantity would reach the arterial side of the vasculature, or if bradykinin was admin- istered upstream to the lungs (left ventricle) to bypass pulmonary in- activation, transcapillary fluid movement may be modified. If brady- kinin fails to promote edema via this mode of systemic administration, then it is quite possible that an autoregulatory mechanism is activated either indirectly, owing to the hypotension and/or directly, due to the action of bradykinin on a tissue (adrenals), antagonizing its effect. 24 :tze ' tions the 11 51 (D A. fete: flui< POte Upon also 25 Marciniak g£_§l, (36) have shown that histamine fails to pro- mote edema in the dog forelimb when administered systemically into the left ventricle in concentrations which would exceed the brachial artery blood concentrations achieved by local infusion. These studies indi- cate that histamine not only fails to promote edema, but rather causes net extravascular fluid reabsorption. This is evidenced by a decrease in forelimb weight, which is only partially attributable to a reduction in intravascular blood volume. Marciniak g£_§l, (35) have also shown that when histamine and norepinephrine are infused simultaneously by local infusion, forelimb weight, lymph flow and lymph total protein fails to increase. This antagonism could be due to a shunting of blood from nutri- tional to non-nutritional channels, a direct blockade of histamine on the microvascular membrane by norepinephrine, or a combination of both. The antagonistic effect of norepinephrine could also explain the dif— ferential effects of locally and systemically administered histamine on fluid flux. Locally administered histamine (4 or 64 meg/min, I.A.) fails to alter systemic arterial pressure or will minimally decrease it after edema develops, whereas systemically administered histamine (400 to 800 mcg/min) causes a fall in blood pressure. The hypOtension would act as a stimulus for sympathoadrenal discharge with the resul- ting catecholamine release. Since bradykinin is similar to histamine by causing marked hy- potension upon systemic administration and promotes edema formation upon local administration, it is possible that the catecholamines may also antagonize the edemogenic effects of bradykinin. This study 26 attempt to determine the mechanism of the route dependent differential action of bradykinin on fluid filtration. The possible role of destruc- tion of bradykinin in the blood and antagonism of the microvascular actions of bradykinin by catecholamines will be investigated. This ‘will be accomplished by infusing bradykinin upstream and downstream to the pulmonary circulation, by infusing bradykinin locally during sys— temic hypotension, and by simultaneously infusing bradykinin and cate- cholamines locally into canine forelimbs while monitoring lymph flow, lymph protein concentration and/or forelimb weight. Since very little data is found in the literature, relevant to the effect of systemically administered bradykinin on fluid and protein efflux and edema formation, the effects of systemically administered bradykinin on these parameters will also be thoroughly investigated. METHODS Mongrel dogs of either sex, weighing approximately 20 kilograms were anesthetized with sodium pentobarbitol (30 mgm/kg) and respirated by positive pressure ventilation (Harvard Respiration Pump, Harvard Apparatus Co., Inc., Millie, Maryland). After surgery, ten thousand U.S.P. units of sodium heparin were administered intravenously to pre- vent blood coagulation. The collateral-free, innervated forelimb, perfused at natural or at constant inflow, was used as the test organ for studying the effects of bradykinin and norepinephrine on extravascular fluid volume and hemodynamic parameters (21). Bradykinin was infused alone (0.8 mcg/min) or infused at this dose simultaneously with norepinephrine (4 meg/min) into the brachial artery. The surgical procedure consisted of sectioning the skin cir- cumferentially about 5 cm above the elbow of the right forelimb with electrocautery. The brachial artery, the brachial and cephalic veins, and the forelimb nerves (median, musculocutaneous, radial and ulnar) were isolated and coated with an inert silicone spray to prevent drying. The muscles and remaining connective tissue were then sec- tioned with electrocautery. The humerus was cut, and the ends of the marrow cavity were packed with bone wax. Therefore, blood entered the 27 dom: fit: bed: the All SUE tin 10 28 limb only through the brachial artery and exited only through the bra- chial and cephalic veins. The brachial and cephalic veins were partially transected and cannulated at the level of the elbow with short sections of polyethy-. lene tubing (PE-320). The sections of tubing were 20 cm in length with a 900 angle about 3 cm from the end. The terminal 3 cm angle of the tubing was inserted into the veins. The cannulas were secured at the same level as the veins, and the outflows were directed into a reser— voir. The reservoir was maintained at a constant volume, via a varia- ble speed Holter pump (Model RE-161, Extracorporeal Medical Special- ties, King of Prussia, Penn.), which continually returned blood to the animal, via a cannulated jugular vein. In these experiments, the median cubital vein, which is the major anastomosis between the brachi- al and cephalic veins, was ligated. Thus, brachial venous outflow was predominantly from muscle, whereas cephalic venous outflow was pre- dominantly from skin. Although this procedure does not completely isolate the skin and skeletal muscle, the amount of separation is suf- ficient to permit comparison of resistance changes in the two parallel beds (39). Blood flow (ml/min) was measured by timed collections from the brachial and cephalic venous outflows into graduated cylinders. All blood flows were converted from ml/min to ml/min/lOO grams of tis- sue, based on the total weight of the forelimb after the experiment. Brachial and cephalic vein pressures were monitored by inser- ting PE-60 polyethylene tUbing into side branching vessels, located 3 to 5 cm distal to the elbow. Systemic arterial pressure was measured by inserting PE-24O polyethylene tubing into the common carotid artery. <' s 3'. 5 ‘ 'I :38 EVE: art. bra- die He bra 29 This cannula was inserted in an upstream direction into the arch of the aorta. Pressure in a skin small vein was measured by cannulating up- stream one of the small surface veins on the dorsal side of the paw with PE-60 tubing. The femoral vein was cannulated in each animal for the administration of heparin, sodium pentobarbitol and saline, when- ever they were required. All pressures were monitored with Statham pressure transducers (Model P23Gb, Statham Instruments, Inc., Oknard, California), connected to a recording Sanborn oscillograph (7700 series, Hewlett-Packard Co., Palo Alto, California). In the experiments utilizing naturally perfused forelimbs, a small side branch of the brachial artery above the level of the elbow was isolated and cannulated upstream with PE-SO polyethylene tubing for local (intra-arterial) infusions of bradykinin. When bradykinin and norepinephrine were infused simultaneously, two small side branches of' the brachial artery were cannulated. These cannulas were inserted in an upstream direction, so that the tip was located at the bifurcation of the side branch off the brachial artery. In experiments using forelimbs perfused at constant flow, the brachial artery was isolated, tied off and transected about 5 cm above the elbow. Blood was obtained from a cannula inserted into the femoral artery and pumped at a constant controlled flow into the transected brachial artery. A Sigmamotor pump (MDdel T68H, Sigmamotor Inc., Mid- dleport, New York) was used to keep the inflow constant. Perfusion pressure was measured by a cannula inserted into a side branch of the brachial artery distal to the site of inflow and was set during the £02 at: f) H. '1 Sit 11! 30 control period, 5 to 10 mmHg below aortic pressure. Local (intra- arterial) administration of drugs was by direct infusion into the pump circuit behind the Sigmamotor pump. When surgery was completed, the forelimb was suspended on a wiremesh platform attached to a strain guage balance. The output from the balance was amplified and recorded on the Sanborn oscillograph, which thus monitored changes in forelimb weight throughout the experi- ment. The system.was calibrated by adding known weights to the plat- form. The addition of a 2 gram weight caused a pen deflection of 10 to 20 mm on the chart paper. Vascular resistances were calculated as follows: total skin resistance - Pa - Plsv/Fs/IOO gms of forelimb, total muscle resistance - Pa - lev/Fm/IOO gms of forelimb, skin large vein resistance - Pssv - Plsv/Fs/lOO gms of forelimb, where Pa - systemic arterial pressure, Plsv - cephalic vein pressure, Fs/lOO gms - cephalic flow per 100 grams of forelimb, lev - brachial vein pressure, Fm/lOO gms - brachial flow per 100 grams of forelimb, and Pssv - skin small vein pressure. In the lymph studies, intact canine forelimbs perfused either naturally or at constant flow were used to collect lymph and measure lymph protein concentration. In the right forelimb, small incisions using electrocautery were made superficial to the brachial artery, the cephalic vein (above the elbow) and the second superficial dorsal metacarpal vein. A small incision was also made over the femoral tri- angle. A lymph vessel in the area of the cephalic vein was isolated and cannulated with PE-lO polyethylene tubing about 10 cm in length and In 3nd pl 8': mm CE 01 w- 31 beveled at the cannulating end. All other lymph vessels in this area, which drain primarily the forelimb skin and paw, were tied off (40). Lymph was collected at 10 minute intervals in miniature 0.3 m1 gradua- ted cylinders, constructed from plastic pipettes. Lymph total protein concentration was measured by the spectrophotometric method of Weddell (56) on a Beckman DB Spectrophotometer (Model 24, Beckman Instruments, Inc., Fullerton, California). Local infusion of the drugs was accomr plished by the same routes of administration used in the previous studies. Bradykinin (0.8 meg/min or 10 meg/min) was infused alone or infused (0.8 meg/min) simultaneously with norepinephrine (4 meg/min) intra-arterially. The drugs used in these experiments were bradykinin (Shwartz/ Mann, Division of Becton, Dickinson and Company) and levarterenol bi- tartrate (norepinephrine; Winthrop Laboratories, Special Chemical Dept.) in solutions of isotonic saline. They were administered intra- arterially or intravenously at a volume delivery rate of 0.2 ml/min with a Harvard Apparatus infusion/withdrawal pump. In two experimental series, bradykinin (0.8 mcg/min or 10 mcg/ min, I.A.) was infused locally for 60 minutes into forelimbs perfused at constant inflow, following hemorrhagic induced hypotension. Hemorr- hagic hypotension was produced by removing the necessary amount of blood (via a PE-360 polyethylene catheter inserted into the femoral artery), to lower and maintain blood pressure at approximately 45 mmHg for 60 minutes. Lymph flow’was also monitored in these experiments. In one series of experiments, systemic bradykinin administra- tion (140 meg/min for 30 minutes, followed by 280 mcg/min for 30 32 minutes) was accomplished by infusing intravenously via a catheter in- serted into the femoral vein up to the inferior vena cava. In another experimental series, PE-240 polyethylene tubing was inserted down the right common carotid artery into the left ventricle of the heart. Initially, the catheter was connected to a pressure transducer and successful placement was confirmed by a typical left ventricular pressure tracing. Bradykinin (140 meg/min for 30 minutes, followed by 280 mcg/min for 30 minutes) was then administered by infu- sion into this catheter. Arterial blood samples (5 ml) were withdrawn from the cannula monitoring systemic arterial blood pressure. Samples were taken during a control period, followed by collections at 30 minute intervals throughout the experiment. Total plasma protein concentrations in grams per cent and hematocrits were determined from these samples. All data were statistically analyzed by Analysis of Variance (Randomized Complete Block Design), and the means were compared to con- trol by the Least Significant Difference Test (53). RESULTS Table 1 In naturally perfused forelimbs, intravenously infused brady- kinin (140 to 280 meg/min) produced a minimal increase in both lymph flow rate and lymph total protein concentration. Plasma protein con- centration was not changed, while the hematocrits were increased signi- ficantly. Skin small vein pressure did not change, and systemic arte- rial pressure decreased moderately, although transiently returning near control levels by the end of the infusion period. Table 2 In forelimbs perfused at constant inflow, systemic infusions of bradykinin into the left ventricle resulted in moderate reductions in perfusion pressure and systemic arterial pressure. Lymph flow rate and lymph total protein concentration were moderately increased. Plasma protein concentration was unchanged, and the hematocrit ratios were elevated. Skin small vein pressure minimally decreased. Table 3 Hypotension induced by hemorrhage markedly decreased systemic aortic pressures. Hemorrhagic hypotension produced no effect upon 33 34 skin small vein pressure, lymph flow rate, lymph total protein concen- tration and hematocrit ratio. Perfusion pressure was markedly elevated, while plasma protein concentration decreased minimally. The local in- fusion of bradykinin (0.8 or 10 meg/min, I.A.) initiated at minute 60 failed to produce any significant alterations of skin small vein pres- sure. Aortic pressure increased minimally, while perfusion pressure decreased substantially, relative to minute 60. Lymph flow rate slightly increased; however, the increase was not significant until the last 20 minutes of the infusion of the higher dose of bradykinin (10 meg/min). Lymph total protein concentration was elevated minimally with the higher dosage of bradykinin and did not change with the lower dosage. Hematocrit was essentially not affected further by the local infusion of bradykinin. Table 4 Table 4 shows the effects of bradykinin infused alone (0.8 mcg/ min, I.A.) and in combination with norepinephrine (4 mcg base/min, I.A.) in naturally perfused forelimbs on weight, vascular pressures, resis- tances and blood flows. Bradykinin infused into the brachial artery slightly decreased systemic aortic blood pressure only during the lat- ter 20 minutes of the infusion period, whereas blood pressure slightly increased with the concurrent infusion of bradykinin and norepinephrine. Forelimb weight moderately increased with bradykinin, yet decreased and slowly waned to control with the concurrent infusion. Skin small vein, cephalic vein and brachial vein pressures and the cephalic and brachial venous outflows increased during the first 5 to 10 minutes of 35 the bradykinin infusion period, and then slowly declined back to the control levels throughout the remainder of the experiment. With the simultaneous infusion, the vein pressures increased moderately and were maintained during the infusion period except for the cephalic vein pressure which did not change; both outflow rates decreased. Total skin and muscle resistances decreased within the first 5 minutes of the bradykinin infusion and then slowly attenuated back to control. No change was observed in skin large vein resistance with bradykinin in- fused alone. All resistances were markedly increased with the con- current infusion of bradykinin and norepinephrine. Table 5 Table 5 shows the effects of bradykinin infused alone (0.8 mch min, I.A.) and in combination with norepinephrine (4 mcg base/min, I.A.) in forelimbs perfused at constant inflow on forelimb weight, vas- cular pressures, resistances and blood flows. Systemic aortic pressure failed to change while perfusion pressure markedly decreased and then slowly increased to a level above control with the infusion of bradyki- nin into the brachial artery. The simultaneous infusion of bradykinin and norepinephrine increased systemic aortic pressure and markedly in- creased perfusion pressure. Forelimb weight increased with both the single and concurrent infusions; however, the concurrent infusion pro- duced a considerably greater augmentation of this weight. Skin small vein, cephalic vein and brachial vein pressures together with the cephalic and venous outflows did not change with bradykinin infused alone. The vein pressures markedly increased with the concurrent 36 infusion, while the cephalic venous outflow decreased and the brachial venous outflow increased. Total skin and muscle resistances decreased within the first 5 minutes and then slowly waned back to control levels with bradykinin infused alone; no change was observed in skin large vein resistance. With the simultaneous infusion, total skin and skin large vein resistances increased, whereas total muscle resistance did not change. Table 6 In naturally perfused forelimbs, local infusion of bradykinin (0.8 meg/min, I.A.) produced no effect upon aortic pressure. When in- fused in combination with norepinephrine (4 mcg base/min, I.A.), aortic pressure was moderately increased. Skin small vein pressures were markedly increased with both infusions, although the augmentation was stronger with the concurrent infusion. Bradykinin alone caused marked increases in lymph flow and lymph protein concentration, but with the simultaneous infusion of norepinephrine, no change was observed in either of these parameters. During the simultaneous infusion of brady- kinin and norepinephrine, a moderate increase in arterial hematocrit was observed and no change occurred in plasma protein concentration. In forelimbs perfused at constant inflow, bradykinin (0.8 mcg/ min, I.A.) did not change systemic aortic pressure or skin small vein pressure. Perfusion pressure decreased significantly over the first 20 minutes of the infusion but then increased, reaching levels signi- ficantly above control, during the latter 10 minutes of infusion. Bradykinin infused simultaneously with norepinephrine (4 mcg base/min, 37 I.A.) produced marked increases in all vascular pressures. Brady- kinin infused alone increased lymph flow rate and lymph total protein concentration; however, only a very slight increase in lymph flow and no change in lymph total protein concentration was observed during the combination infusion. Plasma protein concentration did not change with either mode of infusion, while arterial hematocrits increased slightly only in the combined infusion of the drugs. 38 .oafiu onus on opwumaou mo.o.w a I + .osuu ouou ou o>wuoaou Ho.o.w a u « «ea «me an uauooumsmm AN mamuwv m.m ~.m m.m caoooum mammam Au msmuwv em.m «q.m «H.m eo.m +m.~ e.~ m.~ n.~ asmuoum Sauce eases Aces oH\Hsv «mo. «e0. +mo. No. Ho. #0. go. #0. Boom soap sash: Amm say musmmoum Ha fig Hg OH Ha HH fig HH sao> Hausa swam hum sav ouomooum +qo~ amofi «flea «Nod «Hod «ma NHH NHH oooam Howuouum auscummm cm on as on ON 0H 0 can Amouooaav mafia owum coda oowuom sowmomsH Houusoo .Aolav uwuooumams mam monommoua umHsomm> .mamoan use sasha mo oowumuuoooooo aaououm .Boam nasha so mounsaa em you unflaaouom monomuon hHHmuouos ousa Am>oo mao>v mamooao>muuaa venous“ awsaxmvmuo mo muoommmun.~ manna 39 .oaau ouou ou opauoamu mo.o.w a I + .oawu ouou ou o>fiumaou ~o.o.w a u « «we «So on uwuuoumamm AN maoumv m.q o.e m.q saououm mammam AN mamuwv «o.m «n.~ Rw.~ «e.~ o.~ m.~ w.H m.H cfiououm Hmuoe nosha Aces o~\HaV «Na. «Ha. +mo. +mo. oo. «o. No. no. mood scam enema Amm sav ousmmoum Rafi «ofi «oH «OH «Hg Na ma ma sfio> Hamam oaxm moi Roi Ram «mm «mm «as Baa Sue A»: aav «Anemone aoamauuam Awm aav ouommoum «me see «as «we «Hm «ow mma mum eooam Hefiuouu< oaamumsm om om oo cm om 0H 0 oH1 Amousoasv mafia owwm oeam oouuom sowosmoH Houuoou .Aolav ufiuooumaon mam mmusmmmua uoaoumm> .mamman one amaha mo soaumuuaoosoo awmuoum .soam nasha so moussaa_oo pom soamaa uooumooo um ammo: may mo maowuuso> puma man can“ venoms“ afiaaxhomun mo uncommMI1.N manma 40 .BOussga co ou o>wuogou no.0.“ a I c .oaHu ouou Ou o>gumaou mo.o.w a n + .mmusaga om ou m>gumHmu go.o.w a I 3 .oagu ouon ou o>guogou go.o.w a I a «no «me me O: oe Ogm +ne +ne «we we mm w.om ugHOOuoBom «q.s «0.4 «m.q +m.e ~.m can “N mamumv +o.m +n.m m.m o.¢ m.e m.om agououm maomgm 3am.n +w.~ ~.~ m.~ ¢.~ m.~ q.~ q.~ o.~ m.~ m.~ q.~ ~.~ m.~ egg on mamuwv g.~ a.g o.~ g.~ g.~ g.~ m.N m.~ ~.N g.~ ~.N g.~ o.N w.g w.om afiououm gouoa naskg aaug. sswo. same. no. so. we. No. we. go. «0. go. go. go. go. cgm Ange og\Hav +50. +50. co. co. co. no. no. No. no. no. No. No. No. No. w.om Comm scam sashg +3m~ cow cg gg gg cg m m a a 0g 0g Ng Ng Ogm Amm say ousmmoum +3ag mg mg mg mg gg Ng og Cg m m m gg Ng w.om cgm> ggoam ogxm +3wmg +3oMg 30mg 3N~g augg BNOg «wag «Nag «Neg «gog aqog smog mog meg cgm Awm say «smog +35Mg 3mmg smug 30mg smug «ng «mwg «wag {cog song smog ggg ogg m.om ouommoum sogmomuom ems «so «as «we «we ceea emm «we «we Rae «es «we wgg egg ogm Awe say «no «no «we «we «on «mm «mm «me «On «me «me «we oNg mgg w.om ousmmoum ooogm ngumuus ogsmumhm omg cgg OOg om om cm CO on oq om om 0g 0 0g: Ammuscgav saga sogmsmaH awagxhomum mwonuuosmm gouucoo .soguag unnumaoo .mm as me Home ousmmoua owuuom samuouma mam uoBOH ou owonuuoaon an couscoum dogwoouonh: mo mousaga ow mafisogHOM mousaaa om now A.¢.H .cfia\woa og no m.ov agsgx%omup vomswca hggmoog mo muoommm11.m manna 41 AN N oog x as x was x mm 38v +NNN «BNN +Nom egos +ogm «Nmn +NNN Hg gg «z N.om 1 NH Ng NN Ng m +N +N gg Ng N.om museumgmom aNem Neuoa eg «a «N «m +e In «N N N «z N.om gmaeum o0g\aNa\gav e m e N cg INN «mg N N N.om sogmuao msoam> Negeumum «N ea «g «N 1N «N In Cg og «z N.om geese» oog\aNa\Nav NN Ng SN SN INN ANN «NN Ng NH N.om sogguso maoeu> oggaaaoo INH INN +mg +eg +mg INN +mg m m «z N.om gum see ON ON ON ON +Ng INN «SN N a N.om assesses aNo> Negaumum a e m e o e m m a «z N.om Ame aav m a m m n +N +N m m m.om muammmum agm> oggmgamo «mN «aN «SN eNN INN «mm «gm og ON «2 N.om gum aav mg mg mg mg +NN «NN INN Ng Ng N.om «Dammuua aNo> Ngeam aNxm «mgg meg eegg «egg Nag +Ngg Ngg meg mog «z N.om gum see ousmmuum +NNN +NNN NNN NNN amg NNN ng NNN NNN N.om coogm gmguouu< ugBoumNm +N +g +N1 +m- «on «N1 Rog- o o «z N.om Amaeumv ANN «Ng «4g INN «gg «cg «N o o N.om usage: cg «mango so we on mg cg m N o m1 AmmuaaNae cage moauom aoqmomaH Houuaou 1" L .Aolav mousmmoua umgoomm> use mooowumgmou umgoomm> .mBOgu voogn .uswgoa so moaggouom oomomuoa hggousuma ouag hagwooa vomsmog A.¢.H .swa\moa «v mogunmoognouoo use sgsaxhooun use A.<.H .sga\wos m.oV ogsgxhvouo mo museummnl.q ognma 42 .oagu ouou ou mpgumgou mo.o.w a I + .oagu ouon ou m>gumgou go.o.w a I « N N ooN SN +Nm mo +NoN +NoN +ooN Ne N N «z + N.oN x -Na x aNa wam aav N N N N N N N N N N.om museumNmmN :Ne> aNxm owNmN NN N ooN «NNN +moN +noN «NNN NNNN 0N mm NN NN «z + N.om x -Na x eNa x we see NN SN NN NN NN INN «oN ON SN N.om momeumNmoe «Nunez Neuoa on me on mN oN m N o n- NaousaNav maNN vowuom soamowaH Houuaou .eeeaNuaoonu.e «News 43 «NN «NN «NN «NN «NN «NN «NN N N Nz N.oN NNaNNN ooNNENENNav NN NN NN NN NN NN NN NN NN N.oN SONNueo Naoaus NNNNNNNN «N «N «N «N «N «N «N NN oN Nz N.oN NNaNNN ooNNNNBNNaN NN NN NN NN NN NN NN NN NN N.oN SONNN=o mecca» NNNNNNNN «NN «NN «NN «NN «NN «NN «NN N N Nz N.oN NNN aav N N N N N N N N N N.oN «Nammuum NNN> NNNNNNNN «NN «NN «NN «NN «NN «NN «NN N N Nz N.oN NNN aav N N N N N N N N N NNN 3333 5Q 03230 «NN «NN NNN «NN «NN «NN «NN NN NN Nz N.oN NNN sac NN NN NN NN NN NN NN NN NN N.oN NNNNNNNN NNN> NNeaN cNNN «NNN «NNN «NNN «NNN «NNN «NNN «NNN NNN NNN Nz N.oN NNN aav «NNN NNN NNN NNN +NN «NN «NN NNN NNN N.oN NNNNNNNN aonaNNmN «NNN «NNN «NNN «NNN «NNN «NNN «NNN NNN NNN Nz N.NN NNN aav NNNNNNNN NNN NNN NNN NNN NNN NNN NNN NNN NNN N.oN NooNN NNNNNNHN NNaauNNN «oN «NN NNN NN oN N N- o o Nz N.oN NNaNNNV «NN «NN +N N N N N o o N.oN NNNNos EN eNaNNo NN NN oN NN oN N N o N- NNoNaaNaV «ANN UOHuom doamfide HOHudoo .AoIsv monomooum umasomo> use mooomumgmou umHSONN> .mBOHm voogn .uswaos so mosggouom vomomuoa hgusmumooo ouaN maamooa monomaa A.<.H .aNa\woa NV oswunaocNaouos use sgsaxmvoun use A.<.H .sNa\moa m.ov sfiowxhooup mo enougmmnu.n oases 44 .08.: open ou 02330“ 3.0 w. n I + .063 ohms ou mime-non 8.0 1v. a I e N N ooN «N «N «N «N «N «N «N N N Nz + N.NN « 1Na « eNa «NNN see N N N N N N N N N N.oN euaNNmNNNN aNm> «NNNN NNNN NN N ooN NN NN NN NN NN NN NN NN NN st+ N.oN « 1N3 x aNa x NN aav NN NN +oN +oN «N «N «N NN NN N.oN NumNNNNNmN «Nome: NNNoN NN N ooN «NN «NN «NN «NN «NN «NN «NN NN NN Nz + N.oN x 1N5 x aNa x N: aae NN oN N N +N «N «N oN oN N.oN wuaNNNNNNN NNNN NNNoN NN NN oN NN oN N N o N1 NNuusaNav oaNN UOHHQQ d—OfimndeHH HOHUQOU .NusaNuaouun.N NNNNN 45 «no. «no. «mo. «no. «mo. No. go. go. «2 + o.om mo «cg. «mg. «mg. «Mg. +mo. No. go. go. o.om NaNa oN\Nav go. go. go. go. go. «No. go. go. Nz + w.om m2 Comm 30am nosxg «NN. «NN. «we. «NN. «NN. mg. «0. No. w.om +og +og «mg «mg «mg «mg gg «g «z + m.om no N ON ON N oN oN NN NN N.oN NNN aao anommon «mm *mm «om NNN «om «mm m o N2 + w.om m2 cgo> gamam swxm «mg {mg *mg NNN «mg «mg og og m.om «NNN «NNN «NNN «NNN «NNN «NNN NNN NNN Nz + N.oN No NNN aav +o~g NNN mgg Nog «mm «mm Ngg mgg m.om ousmmoum sogmomuom «mmg «mmg «Nag «omg song «wag ngg mgg «z + w.om mu mug +ng mwg mug NNN mmg NNN oNg w.om Aw: sav unannoum vooam «NNN «NNN «NNN «NNN «NNN «NNN NoN NoN N2 + N.oN N2 NNNNNNNN NNSNNNNN gog Nog Nog Nog cog mog mog Nog o.om oo on ON on om og o ogl Amouoawsv Cage voguom sogmsmog gouuooo .Aolav uguooumso: use mousmmouo umasoomp «osmogo one nmshg mo soguouusooooo swououo .BOHm noshg co moussgs oo now ASNHOHOM Una ousg Nggmguouuo umuuag omen odaunooagoouoa pugs Ngusouuoocoo oomowsg No oooao sasaxmoouo mo ouoommm11.o Ogooa 46 .osgu onus on opguwaou mo.o.w o I + .oafiu ouou ou O>Numgou go.o.w o I N «NN +NN on Nz + o.om mo oN om on o.om uguooumsmm NNN «NN mm «2 + o.om mz o.om w.N m.m m.e N2 + o.om mo m.m ~.N g.N o.om SN 25qu N.N m.N N.N Nz + o.om mz swououm msomHm o.m o.om +N.~ m.~ ~.N ~.N g.~ o.~ m.g m.g «z + w.om mo «~.m «~.m «m.m «m.~ N.~ m.~ o.~ o.g o.om AN msmuwv N.~ ~.~ ~.~ m.~ N.~ m.~ g.~ o.N N2 + m.om mz ogououm gmuoe nosmg «o.e «N.N «g.e «o.N ~.N «N.m N.~ N.N o.om oo on oN on oN og o og1 Ammuocwav wage ooguom sogmomsH Houuaoo .NuaaNNaoo11.N NNNNN DISCUSSION These data suggest that the edemogenic action of bradykinin in the canine forelimb is route dependent. When bradykinin is adminis- tered systemically (140 to 280 meg/min, i.v.) into naturally perfused forelimbs, flow rate and protein concentration of the lymph increased slightly, as compared to local bradykinin (0.8 meg/min) and failed to produce visible or tactile signs of edema (Table 1). One could easily explain these results by the fact that bradykinin is destroyed in the pulmonary circulation. However, although it is well documented in the literature that bradykinin is destroyed in the lungs by kininases (l, 17), it is possible to introduce a large enough quantity of bradykinin intravenously to exceed the saturation point of the kininases in the lungs, or infuse bradykinin downstream to the lung, to bypass pulmonary inactivation. Therefore, bradykinin was infused (140 to 280 meg/min) into the left ventricle of the heart in constantly perfused forelimbs (Table 2). Minimal increases in lymph flow and lymph total protein concentration were also observed, as compared to local bradykinin (0.8 meg/min); however, left ventricular infusions increased these para- meters slightly more than the intravenous infusions. This might sug- gest that at these systemic dosages of bradykinin, pulmonary inactiva— tion plays a very minor role in its destruction. 47 there blooc bed. bradj tota tons a on fore brad tors be r pro1 at 1 str' nin not of 31'1 br- in 48 With the possibility of pulmonary inactivation eliminated, there still existed the feasibility that factors within the arterial blood may destroy bradykinin before it reached the studied vascular bed. Since in forelimbs perfused at constant inflow, locally infused bradykinin (0.8 mcg/min) exhibited increases in lymph flow and lymph total protein concentration, this possibility was expelled. During constant perfusion, locally administered bradykinin must traverse over a one to two meter length of polyethylene tubing before reaching the forelimb. This distance is greater or equal to any distance that bradykinin would travel during a left ventricular infusion. If fac- tors inherent in the blood were inactivating bradykinin, it would not be expected to observe the marked increases in flow rate and total protein concentration of the lymph as is noted with the local infusions at constant inflow. This does not imply that bradykinin is not de- stroyed in the plasma. As noted previously, the half-life of bradyki- nin in plasma is about 15 seconds; however, this degradation rate is not sufficient to account for the route dependent differential action of bradykinin. Since the differential actions of local and systemic (left ven- tricle only) bradykinin on lymph flow, protein efflux and fluid fluxes cannot be explained by involvement of the lung or inactivation in the plasma, it seems likely that they result from different actions on mi- crovascular pressure, permeability to plasma proteins and/or surface area. An obvious difference between local and systemic infusions of bradykinin is the marked hypotension observed only with the systemic infusion (140 to 280 meg/min). The decreased systemic arterial blood Bi ki LI in do 511 ch. in th is al is ti We 11101 101 ME 49 pressure would induce a sympathoadrenal discharge, releasing catechola- mines that may effectively antagonize the effects of bradykinin. Therefore, it was decided to determine if another type of hypotension known to elicit catecholamine release, could antagonize changes in lymph flow and protein concentration produced with bradykinin in fore- limbs perfused at constant inflow. Hypotension was induced for 60 minutes with hemorrhage prior to initiating a local infusion of brady- kinin (0.8 or 10 meg/min, I.A.) for an additional 60 minutes (Table 3). Lymph flow was slightly increased with the lower dosage and minimally increased with the higher dosage. The increase observed with the lower dosage is most likely due to a rise in microvascular pressure, since skin small vein pressure rose, and lymph protein concentration did not change. However, with the higher dosage, lymph protein concentration increased slightly during the latter ten minutes of the infusion, sug- gesting that the increased lymph flow seen with the higher dosage of bradykinin resulted from both an increase in the microvascular pressure (inferred from increased skin small vein pressure) and a decrease in the transmural colloid osmotic pressure. Interestingly, this increase is not nearly as great as is observed with locally infused bradykinin alone. The antagonism is not peculiar to bradykinin hypotension, but is apparently related to the sympathoadrenal discharge. To test the hypothesis that the catecholamines are antagonis- tic to the action of bradykinin in the microvasculature, experiments were performed to compare differences in forelimb weight changes, he- mOdynamics, lymph flow and lymph total protein concentration between local bradykinin infusions alone, and simultaneously with norepine- phrine. The dosage of bradykinin used was 0.8 meg/min, while the do ni ph V8 V6 bl PE Ur ft fr 50 dosage for norepinephrine was 4 mcg base/min. In forelimbs perfused at natural flow, bradykinin (0.8 mcg/min) infused locally into the brachial artery for 60 minutes markedly aug- mented lymph flow, lymph protein concentration and forelimb weight (Tables 4 & 6). The weight gain represents increased extravascular fluid, since all segmental vascular resistances were constant or in- creasing from the two minute point onward. A constant or increasing vascular resistance suggests that vessel caliber was either constant or decreasing. Therefore, vascular volume changes cannot account for the increases in forelimb weight. Lymph total protein increased by an increase in microvascular permeability to protein; however, the mecha- nism for this direct effect remains speculative (32). When norepine- phrine was infused simultaneously, the changes in lymph flow, lymph total protein concentration and forelimb weight were completely pre- vented. All segmental resistances increased, indicating a decrease in vessel caliber and reduced blood flow to the forelimb. To determine the possible contributions of reduced forelimb blood flow in the naturally perfused forelimbs during the simultaneous infusions of bradykinin and norepinephrine, the experiments were re- peated, using forelimbs perfused at constant inflow (Tables 5~& 6). Under this condition, the local intra-arterial infusion of bradykinin for 60 minutes increased lymph flow, lymph protein concentration and forelimb weight. Since vascular resistances were constant or rising (minute 5 onward), increased weight is due to increased extravascular fluid. The simultaneous infusion of norepinephrine into the brachial artery essentially prevented these changes in lymph flow and total pro— tein concentration. The forelimb weight increased more with the 51 simultaneous infusion of norepinephrine and because no change was ob- served in the lymph total protein concentration, it must be ascribed to the rise in microvascular pressure (inferred from the increase in skin small vein pressure), attributable to the intense norepinephrine vaso- constriction (total skin and skin large vein resistances increased). Since lymph flow rate increased only slightly as compared to the fore- limb weight increase (60 gms), the weight gain might be attributable to an augmentation of intracellular fluid volume. This effect is observed in exercise where increases in organ weight are due primarily to rises in intracellular water content (25). Another possibility for this dis- crepancy is that lymphatic drainage may have been obstructed, thereby accumulating extracellular fluid. However, this possibility seems quite remote, since there is no reason to suspect lymphatic blockage. Thus norepinephrine infused locally into the brachial artery prevents the marked protein efflux by bradykinin independent of reduced fore- limb blood flow. For the doses of bradykinin and norepinephrine used, a shift in blood flow occurs in the constantly perfused forelimbs. Blood flow increases in the brachial vein, which drains largely muscle tissue and is reduced in the cephalic vein, which drains largely the skin. This suggests that norepinephrine causes a shunting of blood flow from skin to muscle. The antagonism of the bradykinin induced protein efflux by nor- epinephrine could be due to a blocking of the actions of bradykinin on the microvascular membrane, a shunting of blood flow from nutritional to non-nutritional channels, or a combination of both. Additional ex- perimentation is needed to resolve these questions. APPENDICES APPENDIX This appendix lists, in the form of tables, all the individual observations for the experiments performed in this study. Also listed are the means, standard error of the mean, and statistical significance. The data in the appendix tables corresponds to the mean values in Tables 1-6 as follows: Table Number Appendix Table NUmber 1 A1 2 A2 3 A3, A4 4 A5, A6 5 A7, A8 6 A9, A10, A11, A12 52 53 N... N+ N+ N+ N+ N+ N+ NH 3H8 32:53 gg gg gg cg gg gg gg gg momma M m m N o n M o M M o M M o M M og o gg m og gg gg Mg Ng Mg Mg Mg Mg Mg Mg Ng NN NN NN NN NN NN NN NN N: as NN NN NN NN NN NN NN NN «Nag-NNN 50> NNmaN 5on NH NH NH NH NH NH NH NH mouse 3353 +¢og «NOg «gog «Nog «gOg «mo Ngg Ngg momma No no No no no no oog oog no no OCg no oog 00g Mgg Ngg so no mm ms nu ow No no ugg hog MOg oMg MNg o~g MMg MMg oMg mug MMg mug ONg MOg o~g o~g Aww 38v ousmmoum Neg Neg 00g 00g no no egg egg oooam gmguouu< afiaoumhm ow om ow 0M om og o ogl Ammuasfiav mafia oMNm oegm UOHHON fiOHM—JHGH HOHUQOU .AoIaV uguooumsms one monsmoouo Nogsomm> «managa one noahg go sogumuuaoosoo samuouo «sogm goshg so mouosga om NOH moawgouom oomsmuoo Nggou=uos ouog Am>oo oso>v Ngmoooo>ouuog vomsmag agagxhomug mo muoommuul.g< ogooa 54 N.N N.“ N.“ N.N N.“ N.“ N.“ N.N.- .Nohm BNNEBN «N.N «N.N «N.N «N.N NN.N N.N N.N N.N Names N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N NNNaNNNN N.N N.N N.N N.N N.N N.N N.N N.N NNNNNNN NNNoN NNaNN No.“ No.“ No.“ No.“ OH NH OH NH not... 335: «No. «No. NNN. No. No. No. No. No. enema No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. NaNa NNNNaN No. No. No. No. No. No. No. No. NNNN SNNN NNaNN ON ON ON oN NN oN o NN- gmuusaNav oaNN NNNN oNNN UOHHQM flOfingHn—H HOHugHOU .NoaaNNaou-1.N< NNNNN 55 .osgu ouou ou o>guogou mo.o.w a I + .oseu ouou 0o o>Nuogou go.o.w o I « NM+ «%fl g+ uouuo mumoamum No MN MM momma oM MM MM we no MM we go oM MN MS MM Mm mo Mo oq No gq uHuuoumamm N .H M .H N .H uouuo Boone: m.m ~.M M.M memos N.m g.m g.M ~.m M.m ~.M M.m g.m N.m o.m o.M g.o «.0 M.o o.o AN msmuwv N.N N.N N. N 538m 3%: on on 0e OM ON 0g 0 Ogr Amousawav mafia comm oegm ooguom sogmsmdg gouusou ; .vosdwua0011.g< magma 56 m+ m+ 0+ N“. n“. N+ 1H §+ uouuo unaccoum NNN NNN «NN «NN «NN «NN NNN NNN Neaoa NNN NNN NNN NNN oN NN NNN NNN NNN NN NN NNN NN NN NNN NNN NNN NNN NNN NNN NNN oNN NNN oNN NNN NNN NNN ooN NN NN NNN NNN NNN NN ON ON ON NN NNN ooN NNN aav NNN NNN NN NN NNN oNN NNN NNN NNNNNNNN NONNNNNNN NH NM NM NM NH NM NM NH Note 3353 «NN «NN «NN «NN «NN «NN NNN NNN Names NN NN NN NNN NN NoN NNN NNN NN ON ON NN NN NN NNN NNN ON ON oN NN oN NN NNN NNN NN oN oN NN oN oN NNN NoN NN oN NN oN NN NN NNN NNN NNN aav NNNNNNNN NN NN NN NN NN NN NNN NNN NooNN NNNNNDNN ONaoNNNN NN oN oN ON ON ON 0 NN- NNNDNNNaN oaNN NNNN NNNN ooguom sogmomsg gouuaoo F ”d |“ .AeIav uguooumsmn use monommouo umgsomm> «mammgo mam noskg mo sowumuuaooaoo agououo «Dng goshg so mousafia oo pom BOngg acouosoo um unmon man go ogoguuao> uwog onu ousg vomomsg sgagxmvmuo mo muoomumru.m< ogooa 57 Nomw No .H 8% No.“ No.“ go.H o... o... uouuo vuogoamum «NN. «NN. NNN. NNN. No. No. No. No. Names No. No. No. No. No. No. No. No. NN. NN. oN. NN. No. No. No. No. No. No. No. No. No. oN. No. No. NN. NN. oN. No. No. No. No. No. NN. NN. NN. NN. NN. No. No. No. NaNa oN\NaN No. No. No. No. No. No. No. No. ouNN SONN :NaNN NM NM NM NM NM 0H NM NM not... 3383 egg «og «og «cg egg Ng Mg Mg memos N N NN oN oN NN NN NN NN NN NN NN NN NN NN NN NN NN oN NN NN NN NN NN NN NN NN NN NN NN NN NN oN oN oN N oN NN NN NN NNN aav N N N N N oN oN oN «CNNNNNN aNo> NNNaN aNNN oN oN oN oN oN ON 0 NN- NNNNNNNBN oaNN NNNN oNNN UOMHOM flOHmSHGH HOHUGOU .NesaNNaouuu.N< NNNNN Table A2.-Continued. 1 Infusion Period Control 8280 8140 50 60 40 20 30 10 -10 Time (minutes) 2.9 3.5 2.3 2.6 4.1 3.0 2.9 2.7 2.9 2.5 2.6 3.1 2.3 2.1 2.0 1.9 1.6 2.3 1.9 1.3 2.0 1.9 1.5 1.6 1.7 Lymph Total Protein 1.9 2.2 (grams Z) 2.2 2.2 2.9 2.3 2.5 2.5 1.6 2.0 2.1 1.6 2.1 2.1 1.7 3.3 2.5 1.8 2.0 1.8 2.0 2.8 2.7 58 2.7* 2 2.8* 1 2.4* 1 2.0 O‘I—l O O N+1 mv-I O O N+1 1.8 11.1 means standard error OQNNMO\ Q'fidx'ffifi' QGNOON O O O I O O ~¢~¢¢fi~¢~31n WQv-IO‘UDO oooooo «stews-teen Plasma Protein (grams Z) Ira—4 ~¢ +| \ocu <-+I mm <-+l means standard error 59 .osgu ouou ou m>gumgou Mo.o.w o I + .oaNu ouou ou o>gumgou go.o.w o I « g+ g+ g+ Nouuo MHoMaoum «MN «NN MM Booms MN MN MM Ne Me MM no me go MN he MN MN MN MM oc MM MM ufiuooumsmm oM on ow oM om og o ogr Amouasgav wage oMNM oNgM magnum sONmouoH gouuooo I d- 1.! . 32:281.. NNN NNNNN. 60 N+ NH N+ N+ N+ N+ N+ N+ N+ NH NH N+ N+ N+ .858 335: ENN NN NN NN NN NN NN oN S N N N NN NN memos Og N M M M n M .M m N m M Og Og M M Og Og Og Og Og M M M n M Og Og M M M M M M M M m N M M Og Og M M n M M n n M n M n M Og Ng Am: EEO NM MM OM mm OM OM MN Mg Mg Mg Mg gg Mg Mg mummmmum NN NN NN NN NN NN NN NN NN NN NN oN NN NN NNN-.5 NNmsN 5st NH NNH NNN... NNH NM NM NH NM NM SH NNH 2H NM NM Note 335: «3MMg +3sMg 3MMg 3MNg 3OMg 3M~g «MMg «MMg «Mug «MMg «MMg «MMg ggg OgM momma Mgg Mgg ONg Mgg NNg ONg MMg MMg OMg OMg OMg OMg OOg OOg Mgg Mgg ONg ONg Mgg ogg Ohg ONg OMg MMg OMg OMg MM MM NOg OOg OOg Ogg Mug ONg Mug OMg OMg Mug OMg Mug MOg MOg MMg OMg OMg OMg OMg MNg Mug oMg OMg OMg OOg OMg MOg MM OMN MNM OON hug OMg OMg MgN OgN MMg OON OON OON MNg ONg AMM 66v Mug Mgg Ogg MM ONg MMg OON MON OON MMg MMg MMg OMg OMg ousmmoum scamswuom 3H NH NH NH NH NH NH NH NH NH NH NH NH NH sou—.8 3353 «MM «NM «MM «MM «MM {MM «MM «NM «OM «NM «MM «MM ONg Mgg momma NM MM NM NM Ms NM OM NM NM OM NM NM ogg hog OM MM NM NM MN On on MM OM NM NM OM NOg NOg MM MM OM OM MM OM OM OM MM MM NM MM Mgg Mgg MM MM NM NM mm MM NM OM MM NM NM MM MOg mOg AMM aav MM NM BM On On NM MM OM NM OM NM NM MMg OMg ouommmum MooHM Ogg Ogg MOg NM MM 5M NM MM NM NM OM MM OMg OMg gmguoun< ogaoumhm ONg Ogg OOg OM OM OM OM OM OM OM ON Og O Ogl Amouaswav wage Moguom sogmsmog mummuuoaom gouuaoo .MM as MM uous ousomouo oguuoo sgousgos Moo uosog ou owonuuoso: MM Mooooouo dogwoouoohs mo mauoaga OM MoNBOggou «mouosga OM now aogwsg usoumooo um A.<.H .oNa\Moa M.Ov sgsgxhomug Monomsg MggBOOg mo mucoummrr.M< ongH 6]. 63:38 OM ca 02330... MO.O w: a I c 632:! OM 3 02330..— g0.0 H a I a 68.3 cum» 3 2,333 M0.0 Iv. M I + .063 0.3» cu ozuwgwu gO.O .vu M I « an «M NH M+ g+ uouuo uuuMaauu +nM +MM «NM NM mm manna NM on NM NM an Mn an Mn oM an oM NM MM NM an en Mn Nn NM an NM oM 0M NM MN NN mm cm NM NM “NuuouMeMm M.H M.M N.N N.N M.M uouuo 33cm...» +M.n +N.n N.N o.M M.M Mange N.N N.N M.n N.N M.M o.n M.n m.n ~.N N.N o.M o.M M.n M.M _.n N.M M.M N.M m.M M.M M.M N.N M.M N.N M.M AN mauumv N.N M.N M.M N.M M.M uMououm nauMNm M.M N.“ N.“ N.“ N.“ N.“ N.“ N.N n..+. N.“ M.“ N.N M.M n.“ ~85 3233.. M.N N.N o.N N.N M.N ~.N N.N N.N N.N g.N N.N M.N o.N N.N «some o.n _.N M.N M.M M.M o.N a." M.M M._ N." N." M.M M._ m." _.N N.M N.M N.N M.M M.M N.M N.~ o.N o.N N.N M.M n.— N.” N.N N.N N.N o.m M.M o.n N.N ~.n N.N N.N o.M N.N N.N M.m N.N N.N c.N N.N M.N ~.N N.N N.N N.N o.N H.N N.N M.N o.N O.“ N." n.g M.M M.M N.N N.N n.N M.N M.M N.“ ~.N M.N M.M AN oauumv N.“ a.” o.N N." N.N M.M M.M N.“ M.M N._ M.M M.M M.M M.M aMououm Nauoa can»; Mo.+ Mo.+ 8.“ Mo.H No.“n 8.+ 8.+ 8.+ 8.+ 8.“ SH 8.“ SH 8.“ 8:8 83:3» +No. +No. co. co. Mo. no. me. No. no. No. No. No. No. No. canoe Mo. Mo. do. Mo. Mo. No. No. Mo. Mo. Mo. Mo. Ho. No. go. Mo. Mo. Mo. No. do. Mo. do. Mo. Mo. Mo. Mo. do. Mo. Mo. do. Mo. no. Ho. Mo. _o. No. No. no. we. N0. M0. no. no. No. NH. mo. od. co. no. no. No. No. No. No. No. Mo. do. MN. NN. MN. NN. NH. no. mo. Me. No. no. we. Mo. Mo. Mo. gaMa oMMNaO Ho. No. Me. Ho. Ne. Mo. No. Ne. No. No. No. Mo. No. No. Mama saga sag»; oNM oMg cod oa om oN oo on oM ON ON ON o o“- Amouaaaav oaNs venom coi‘sfi—H 0523.350: gouuaoo .voaaauaoouu.n< uNpMa 62 M.gg+ M.M+ N.M+ M.N+ O.N+ M.g+ M. N.O+ N.g+ M.g+ M.g+ M.g+ O.g+ g.g+ uouuo Mumvcmum 3+MN OON Mg gg gg Og M M M M Og Og Ng Ng mamas M M M M M Og N M M M M M gg Ng gN MN NN Og gg gg M Og Ng Ng Mg Mg Mg Ng Og Og M N N N N N M N N N Ng Ng M N N M N M N N M M M M M M NMM EEO OM OM OM MN ON Ng Ng gg Mg Mg Mg Ng Mg Ng unannoum Mg Ng Ng gg Og .Og M M M M M M gg gg awo> Hgmam aaxm M.MN+ M.Mg+ g.Mg+ M.Mg+ M.Mg+ N.gmfl M.gg+ N.M+ M.gg+ M.gg+ M.Ng+ M.Ng+ M.M+ M.M+ uouum MuaM:Mum +3MMg +3MMg 3MNg 3NNg aNgg 3NOg «MMg «NMg «NMg «gMg «MMg «MMg MOg MOg mamma OMg OMg OMg MNg MNg Mgg MMg MMg MMg MMg MMg ONg NM MM MM MM MM OM MM OM OMg OMg ONg ONg OMg MMg MOg MOg OOg MOg NM NM NM MN MMg OMg OMg MNg MNg MMg ONg Ngg MM MM OM MM OM MN MMg MMg OMg OMg MMg MMg Ogg Ogg OMN OgN MMg MNg OMg OMg MMN MgN OON MMg OMg ONg Ogg Ogg AMM EEO ONg OMg OMg MMg OMg MNg MMg MMg MMg OON ONN OMg Ngg Mgg muammwum aoamswuom M.M+ g.M+ g.N+ M.M+ N.M+ M.N+ g.M+ M.N+ M.N+ N.g+ M.N+ M.N+ g.M+ N.M+ uouum MHMMGMum «MM «MM «MM «MM «MM O«MN «MM «MM «MM «NM «MM «MM Mgg Ngg mauve OM MM OM ON MM OM OM MM OM OM OM NM NOg MOg MN MN MN MN ON OM MM OM OM OM MM MM Ogg Ogg NM MM OM OM OM NM MM MM OM NM NM NM MNg NNg MM NM NM NM MM MM MM OM MM MM OM MM ONg ONg AMM EEO MN MN MM MM OM OM ON NM MM NM MM OM ONg ONg musmooum MOOHM NN NN OM OM MN MN MM MM MM NM NM NM MNg MNg Hmaumuu< uaamuwNm ONg Ogg OOg OM OM .DWI OM OM OM OM ON Og _ O Ogl Ammuaawav mafia coupon abumamaH uuuauuoamm Houuaoo .MM 33 MM aqua ouawuuum owuuoa nauuawna was Hosod cu «Munuuoafis Na vuoavoun downaquMNn Mo muuaawa OM MawsoaaoM .oousawa OM qu soamaw unaumcoo um N.<.H .aNa\Mua Ogv awnaxhvoun Mmmauau NHHaooa mo muuuwmmll.M< manna 623 63:33 OM 3 053g.» M0.0 .w a I a 66.3 33 3 3333 M0.0 Iv. a I + w Joan—Nae OM on 0351?.» 86 H a I 3 .053 0.5» cu obuunaou g0.0 M I « M.N“ M.NH N.NH N.NH N.M+ ~83 3.33. «NM MnM NM oM OM Mauve NM oM mm NN Nn NM NM MM MM MM MN mm Nn mm mm on on Mn MM mM on NM NM on an NM NM NM on Nn uMuoouMaum M.M M.M. M.M M.N M.M uouuo c.3330 «M.M «M.M «N.M +N.M N.N Mecca M.n M.M M.N o.n N.N N.M N.M M.M N.M o.M N.N M.N M.N M.N M.M o.M N.M M.M M.M N.M n.m M.N o.n M.M N.M Nu oaouuv N.M o.n M.M N.M N.M aMououm MauoNN M.M M.M N.M N.M N.M N.M N.M N.M N.N M.M M.M N.M M.M M.M Note 3253. aMn.n +o.N N.N N.N M.N N.N M.N M.N M.N N.N N.N M.N N.N N.N Manna M.N N.N N.N N.N M.N N.N N.N N.N M.N N.N N.N o.N N.M m.— M.N N.N M.M n." N.M N.M N.M N.M o.N N.M N.M N.M N.M M.M N.N N.N N.M N.M N._ N.M N.M N.M M.M M.M N.M M.N N.M N.M N.M M.N M.N M.N M.N M.N N.N N.N N.N N.N o.m N.N N.N N.N M.N M.M N.N M.N n.n N.N N.N M.N M.N N.N M.N M.N N.N N.N Nu Mauuuv o.N N.N o.n N.N o.N M.N M.N N.N N.N M.N M.N M.N N.N N.N aMououm N.Moa aNaNM no.“ No.“ no.“ 8.“ 8..+. 8.“ 8.+ 8.+ o+ 3 9 o.+. ow oH “83 9.853. MsNM. «sac. Mano. no. Mo. No. No. No. Mo. No. Mo. Mo. Mo. Mo. «nuns me. no. No. No. Mo. Mo. No. Mo. Mo. Mo. _o. Mo. Mo. No. MM. no. Mo. Mo. no. no. No. No. No. No. No. No. No. No. no. No. No. .No. Mo. Mo. No. No. Mo. Mo. Mo. Mo. Mo. Mo. NM. NM. NM. MM. No. no. No. no. No. No. No. Mo. Ho. Mo. on. NM. NM. Mo. no. No. Mo. Mo. Mo. No. Mo. Mo. Mo. Mo. NcMa cMNNaO no. no. No. no. no. No. No. Na. Ma. No. Mo. Ma. No. _o. «Mam :on MNINM ONM oMN ooM oa om cN oo on oM on oN oM c oM- gnouauaav oaNa venom gamma 3.2.388: gouuaoo . v25.“ udOUII . Md ONN—nu. 64 mfl mfl mfl M+ M+ M+ M+ M+ M+ uouum unavawum MNNM MNNM NNM NNM NNM NNM NNM ..NNM NmM mamma noM moM moM NNM MNM NNM NNM NNM omM omM onM omM omM omM omM omM onM omM oMM OMM OMM omM NMM oMM oMM OMM oMM MNM mNM omM mmM mmM an NNM an mmM NMM onM omM onM mmM nnM an an omM Mum aaM «Mammoum ONM MMM NNM ONM MNM oNM mMM oNM ONM MOOMM MMMMMMMM OMaoMmNm NM NM NM NM. NH NH. NM NM. NM Mouuu MMMMaMMm MNM «NM MMM «NM «MM MoM «m o o mamas NM MM NM MM m a m o o NM NM NM NM MM MM MM M- o NM MM oM m m n m M- o MM MM N M M m N o c on NN MN NM MM MM NM 0 o Mmaauwv oN NM MM MM MM MM N o o MMNMmz MM MNaMMo co «M on mM oM m N o n- MmmMaaMaM oaMN flOHHflm dOHmnwflH HOHuflOU .AMIav mandammua umaaomw> van mwoawumwmmu uwaaomm> .waoam MOOHM .uaMNoa do «Mafia nuuom Mmmamuum NHHmusuma ouaw NHHmuoa Momsmau A.<.H .aNE\Moa M.Ov aaaaxhvmun Mo muouwwmll.M< manna 65 MN 2. MH MH M+ M+ M+ MH M..._.. uouuu 3353 N M N N N +N +N N N mamas N N N N N MM MM N N N N N N N N N N N N N N N N M N N N NM N N N N NM NM MM MM N N N N N N N N N NNN aaM M M M N N N N N N «Mammoum aMo> oMMNMNoo M“ NM NM NH NH NH NH N.+- MH ~33 338.: NM NM NM NM +NM «MN «NN NM NM magma N N N NM MM NM NM N N MM MM MM NN oN NN oN NM NM NM oN oN NM NM NM NM MM MM NM NM NM MM NM MM NM NM NM NM NM NM MN NN NN oN NM NM NNN aaM MM N NM NM NM NM NM N N unamuoum aMo> MMuaN aMMN oN NM NN NM NM N N o N- MmuusaMaM uaMN vow-09 GOHmflmflH HOHUOOO {I .NmsaMMaoo-.N< «MNNN 66 N+ Mu... NH NH NM NM... NH M+ M+ Menu 885: NM NM MM MM «NM «MN «NN NM NM magma NM MM NN MN NM NN NN MM NM N N N N N NM NN N N NM NM NM MM NM oN NN MM MM NM NM NM NM MM NM NM NM NM NM NM NM MN NN MN NN NM MM NmaMMN NNMNNMaNMaM NM MM N NM NN MN MN MM MM aoMmuso Naoaa> oMMMMNoN NH MH NH MH MH MN... NH MH MH not» c.8283 NM NM NM NM NMM MNM «MM N N mamas NM NM NM MM MM NM NM N NM N N N N N MM oN N N MM MM NM NM NM N MM N N NM NM NM NM NM MM NM MM MM oM N NM NM MM NM NM NM NM NNN aaM N N N N NM NM NM N N ousmmuum aMo> MMMMUNNN oN NM oN NM NM N N o N- MmmusaMav aaMN UOHHUQ fiOHMSMdH HOHudOU .NosaMMaoo-.NM «MMNN 67 M... NH MH N+ N+ M+ M+ 2. MH not... 335% NM NM NM NM N MN +N MM NM mamas N N N N N M M MM oM NN NN NN NN MN N N NM NM NM NM N MM N N N NM NM N N N MM N N N NM NM M -N ooM MM N N N N N NM NM NM x w-Ma x aMa x NN aav N NM NM NM N N N MM MM uaNMNMNoN aMMN MNMoN MM MM MM M“ NM NM NM M-. M.- Moto 3383 M M M N Og «Ng «Mg N N @6006 N N M N N N NM N N N N N N N NM NN N N N N N N N MM MM N N N M M N M N N N N N N N N NM MM NM N N MNaNMN NNMNMMaNMaM N N N NM NM NM NN N N aoMMMNN Naoao> MNMNUMMN oN NM NN NM NM N N o N- NmouacMaM uaMN flown—mm dOHmawaH HOHuM—OU .NoaaMuaoo-.N< oMNNN d’ in 68 .053 ouuu ou ozuuamu M0.0 .w. NM I ._. .258 oumu ou 9,336.» g0.0 W NM I « NH NH NH NH M+ NH NH N-+. on not» 2353 M M M M N M M M M Mamas N M.N M.N N.N M.N N.N M.N N.N N.N N N N M N N N N N M N N M M M M N M N N.N N.N N.N N.N N.N M.N M.N M.N NM N NNM M M M N M M N M M x -Ma x aMa x NN aav M M M M M M M M M muaNuNMNoN aMo> NNMNM NMMN NH NH NM NM NM NH NM NM MH not... 3353 MN MN NN NN NM «NM «NM NN NM Mamas NM NM MN NM NM MM MM MN NN NN NN NN NN MN N N NM NM NM NM NM NM NM NM N NM NM NM NN NN NM NN NN NM NM NM NM-N NNM NN NN NM NM MM NM MM MN NN x -Na x aMa x NN aav NM NN NM N N N N NN NM NMNNMNMNNN «Mama: MMMNN NN NM NN NM NM N N N N- MmmuaaMaM «3MB vOHuom flOHmfide HOHuflOU .MmsaMMaoN-.N< «MNNN 69 N+ NH NH N+ N+ NH N+ N+ N+ Manna NMNMNNMN +NMM NNM «NMM «NMM MMM +NMM MMM NNM NNM mamas NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NMM NNM NNM NNM NNM NN NN NNM NNM NNM NNM NNM NNM NNM NNM NNM NN NN NN NNM NN NN NN NN NN NNM NNM NN NMM NNM NNM NNM NN NNM NNN aaM ouammuum NMM NMM NMM NNM NNM NNM NNM NMM NMM NooMN MNMMNMMM NMamMmNN NM NM NM NH NH NH MH NH NH not» M38283 +g +g +NI +MI «MI «MI «Ogl O O mawua N N N- N- M- N- NM- N N N- N- N- NM- NM- NM- NM- M N N M- N- NM- NM- NM- NM- M- N M- M- N- N- N- N- NM- M N N N N- N- N- NM- NM- N N MNaNMNM NM NM NM N N M N- M N MMNMos NM NNNNMN NN NM NN NM NM N N N N- NmauaaMav oaNN vOHme GOHMMMMGH HOHudOU Mumamca N.M.H .afia\woa MO unauaamawnouoa Maw .AMIav mmusmmmua Huaaumw> Maw Nouawumwmmu uwaaumm> .Naoam MOON: .uswwoa do anafiamuom Mmmamuon NHHmuauma ouau NHHmuoa M.M.M .aMaNNoa N.NM NMNMMNNNNM Mo NMMNMMN-.NM oMMNN M+ M+ N+ N+ N+ N+ _NH NM M+ uouuu NMNNNNMN M M N N N N N N M memos N N N MM NN NN NN N N N N N N N N N M M N N N N N N N N N N N N N N M N M N N N M N N M N N N ANN aaM M M NM M M M N N N ousNNoMN NMo> NMMNMNNN 0 MH MH MH MH MH Mu..- MH gH gH uouuo Muwvcmum 7 «MN «MN «MN «MM «NM «MM «gM Og Og mauve NM MN MN NN NN NN NM N N NN NN NN NN NN NN NN NM NM NM NN NN NN NN NM NM N N NN NN NN NN NN NN NM NM NM NN NN MN NN MN NM NN NM NM NNN aav NN NN NN MM NN MM NN NM NM NMNNNNMN aMo> MMmaN aMMN NN NM NN NM NM N N N N- MNoMNNNaN oaMN UOH-Nmm dOHmfimflH Hound—00 .NmaaMMaoo-.NN NMMNN 71 M+ NH. M+ M+ M+ M+ M+ M+ M+ Mouuo NNNNamuN «N «M «M «N «N «N «M 0M 0M mamas N N M N N N N NM MM M N.N M.N N.N M.N N.N M NM NM M M M M.o M.o M N oM oM M M N N N N N N N M M N.N M M M M NM NM MmaNMN NNMNcMaNMaN .N M.N M.N M.N M.N M.N M.N N N aoMNuso Naoao> uNMNnNmN MM. {H n“. n“. ifl EH Q“ mfl 1H uouum VMNVQMuN «NM «NM +MM +MM +MM «NM +MM M M Namoa MM oN MN MN MN MM MM M m NN NM NM M N N NM M M NN NN NN MN NN NM N M M M M M M M M N M M NN NN NN NM MM NN N N N NNN aav NM NM N NM MM MN NM M M ousmmmum aMm> McMnouuN 0M MM OM MM oM M N o M: AmmusaNav maNH wadhmm GOHmfiwdH HOHUQOU .- .NmsaMuaoN--.N< NMNMN NM+ NMMH NNN. NM+ NN+ MM“. N+ N+ N+ noun» NNNNNNMN «NMM +NNM +NNM «MMM «NMM NN NN MN MN mamas MN MM MN NM N N NM NM MM MMM MNM NMM NMN NNN NNN NN NM NM NNM NMM NNM NNN NNN MN MN NM MM NNM NNM MN NN NN NN MN NN NN M -N NNM NN NN MNM MN NN MM NN NM NM x -Ma x aMa x NN aaN NN NN NN NN NM NN NN MM MM NUNMMNMNNN «Mumps MMNON 2 NH NH MM MH NH NH MH NH NH .838 3383 7 «M «M «N «M .3 «M «M N N 2805 M N N N NM MM N NM MM M M M M.N M.N M.N N N N M M M M.N N.N N N N N M M M N N M N N N M M M M N N N N N NNaMNN NNMNaMaNNaM M N N M N N M NM NM soMNNNN Naoao> MMMMoNNN NN NM NN NM NM N N N N- AmouaaMaN oaMN vOHuflm flOHmMMHQH HOHufiOU .NuaaMuaoN--.N< oMNMN 73 .maNu cums on mzumaou M0.0 Iv. NM n + .033 cyan ou 0>Nuwauu 8.0 .w. NM u « NN+ NN+ NN+. NN+ NNN NMN NNN NH. NH. Nouuo NNMMNNMN MN +NN NN +MNM +NNM +NNM NM M M Nance M M M N N N N M.N M.N NN NNM NN NNM NN NNM NN M M NM MM NN NN NM NN N M.N N.N MN NN NN NN NM NM MM N N , MM-N NNM NN NM NN MN NM NN NN N.N N.N x M-Ma x aMa x NN aav NNN NNN NNN NNM NNM NNM NNM M M moamuNNNmN aMo> NNNNM NMMN NNM+ MNM+ NNM+ NNM+ NNM+ MNM+ NNM+ M+ M+ uouuu NNMNNMNN +NNN «NNN +NNN «MNM +NMN «NNN +NMN MM MM mange NN MN NN NN NM MM NM NM MM NMN NNN NNN NNN NNN NNN MN N N NNM NN NNM NNN NNN NNN NM NM NM NN NN MM MN NM NN NN NM NM NMuN NNM MMM NMM NNN NNN MN NMM NNM N N x M-Ma x aMa x NN aaN NNNM NMMM NNNM NNMM NNMM NMNM NNNM MM MM ooaMuNmeN NMMN MauoN NN NM NN NM NM N N N N- MmmuuaMaN maMN flown—0m dOHmamN—H HOHHGOU .NoaaMuaoN--.NM NMMMN 74 NMM MMM SH SH N+ N+ N+ NH N+ Mon-Mu 838$ MNM MNM MNM NNM NNM NNM NNM NNM NNM mamas NMM NMM NMM NMM NMM NNM NNM NNM NNM NNM NMM NMM NMM NNM NNM NNM NNM NNM NN NN NN NN NN NN NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NMM NMM NMM NMM NMM NMM NMM NMM NNM NNN aav NNNNNNNN NMM NMM NMM NMM NNM NNM NNM NNM NNM NooMN MMMNNMNN oMaouNNN NM NM NM NM MH MH NM NM NM N88 83.83 «MM «MM MN N M N N N N Nauoa NN NN NM MM N N N M N NN NM NM N N M N M- N M M N N N M N N- N NM MM N N N N N N N N- N- M- N- N N N N N MNaNNNN N N N N N M N N N MMNMNN NM NNNMNN NN NM NN NM NM N N N N- MmmuscMav uaMN UOMHOQ flown—MHGH HOHUGOU .Aolnv muuammuun umaaomd> can mouauumwmuu umaaumm> .wsbNm vooan .uMwNoR no NMENN quow vmm=muun MaudMumnoo OuaN MMHaooa vumamau A.<.H .aNa\mua w.0v aadaxmvunn mo ouuuwmmll.N¢ manna M+ NH M+ m+ M+ M...” NH M+ M+ uouuo vumvnmum MM NM NM MM MM NM NM MM MM menus NN NN NN NN NN NN NN NN NN NM NM MM MM NM MM NM MM NM N N N N N N N NM NM NM NM NM NM NM NM NM NM NM N N N NM NM NM NM MM NM NNN aav MM MM MM MM MM MM NM NM NM NMNNNNNN NMNN MMNaN NMMN n MN.)N MNM NNM NNM NMH NMM NM NM NH not» 335: «NNM NNM NNM NNM MNN «NN «NN MNM NMM magma NNN NNN NNN NNN NNM NNM NNM MNM NNM NNM NMM NN NN NN NN NM NNM NMM NN NN NN NN NN NN NN NNM NNM NNM NNM NMM NNM NN NN NN NMM NMM NNM NNM NMM NN NN NN NN NNM NNM NNN aev NMM NNM NN NN NN NN NN NNM NNM NNNNNNNN NNMNNMMNN NN NM NN NM NM N N N N- MmmuaaMaN aaMN vONumm Macaw-N65” Nouuaoo L .NoaaMucoN-.N< NMNNN Table A7.-Continued. Infusion Period Control 60 45 30 15 10 Time (minutes) Cephalic Vein Pressure (mm H8) 76 ~c l-c 61' <- eq |~¢ cu |~¢ MIN-t «1| <- co l-a MIN-r In Inn m 5 a v-i +| jfl standard error Brachial Vein Pressure (mm Hg) 25 25 25 20 20 17 15 12 12 means standard error jfl 77 M.M. m+ M.M. MN+ m+ q+ .N+ MN+ .N+ hon-Mm vumvamum NM NM NM NM NM NM NM NM NM mamas N N MM N N N N N N N N N N N N NM N N N N N N N N N M N NN NN NN NN NN NN NN NN NN MM MM MM MM MM MM MM MM MM MNNNNN NNMNNMaNMaN NM NM NM NM NM MM NM NM NM 3OMMMNN NaoNNN MNMMNNNN NH NH N-+. NH NH NH NH NH NH uouum 335$ NM NM MM MM MM MM MM NM MM mamas NN NN NM NN MN NN NN NN MN NM NM MM MM MM MM NM NM NM NM NM NM NM NM NM NM NN NN NM N N N N N N N N NM MM NM NM NM NM NM NM N MmamNN NNMNaMaNMaN MM NM MM MM MM MM MM NM NM soMNMNN NNNNNN NMMNMNNN NN NM NN NM NM N N N N- MmmuNaMaN oaMN vowhom 69—55de HOHHGOU .NaaaMuaoN-.N< NMNNN 78 N+ NH NH N+ N+ N+ M+ N+ M+ uouuo 3353 MM NM «NM +NM «N «N «N MM MM mamaa NN NN NM NN NN NN NM NN NN NM NM MM N N N M NM NM NM NM MM NM MM NM N MN NN N N N N N N N N N MM N NNM NM MM N N N N M N N « M-Ma « NMa x mm aav N N N N N M N N N NNNNNNMNNN NMNNNM Mauoa NH NH NH M.N... MH MH MH NH NH «8.8 3383 MM NM N N «N «N «M NM NM mamas NM NM NM NM N N M N N N N N N N N N N N M M M M M N N M M NM NM NM NM NM N N NN NN MM-N NNM NM MM MM N N N N NM MM « M-Ma x aMa « NN say NM N N N N M. N N N ouaMMNMNuN NMMN MuuoN NN NM NN NM NM N N N N- MmouaaMaN NaMN vOHHOm— flOHmflde HOHUGOU .NosaMuaoo-.N< NMNNN .oaNu ouou ou o>NumHou 36 W NM .- + $8.3 ouon ou o>NuoNou 36 H M n « 79 mm Q” NM NM NH N.._-. NM NM NH NH NH Note 3383 M M M M M M M M M Nance M N N N M M M M M N N.N M.N M.N M.N N.N N.N M.N N.N N N.N N.N N.N N.N N.N N.N N.N N.N M M M N N N N N N M -N NNM M M M M M M M M M x -Ma.x aMa « NN.aaN N N.N M.N N.N M.N M.N N.N N.N N.N «NNNMWMNNN NMNN NNMNM NMMN MM NM NM 3 N N o M- 33:55 03:. VOHHQM dOHmflmd—H HOHudoo .NmaaMuaoN-.N< NMMNN 80 N+ NH NH NH NH SH SH 2+ 2+ “8.3 335: «NNM «NNM «NMM «MMM «NNM «NNM «MMM NMM NMM Namma NNM NMM NNM NMM NNM NNM MMM NNM NNM NMM NNM NNM NNM NNM NMM NNM NN NN NNM NNM NNM NNM NNM NNM NNM NNM NNM NMM NMM NNM NNM NNM NNM NMM NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NMM NNN asv NNNNNNMN NNM NNM NNM NNM NNM NNM NNM NNM NMM NooMN MNMNNMNM oMaNMNNN NNH NMH NH NH NH MH MH NH NH «83 33%: «NN «MM «NN NM NM N N- N N mamas MN NM MN NM MM N N N N MN NN MN MM MM N N- N N NNM NNM NN NN NN NM N N N NN NN NN NM NM M M- N N NM N N N- NM- NM- NN- N M MNaNNNN NN NN NM MM MM N N N N MMNMNN NM NNNNMN NN NM NN NM NM N N N N- MNNNNNMaN maMN vOHumm QOHQSMGH HOHufiOU .Aoasv mousmmoun umaaumm> nan Nmucmumfimmu umaaowm> «macaw vooan «uswwma no mnaNNouom vomamuoa Nauamumsoo ouaN MHHwooM vomsmaw A.<.H .aNa\wua My oaNunamGNnmuoa wad A.<.H .aNa\woa w.0v nNnNMNumun mo muuommmll.w< macaw i M“ M+ M... M+ N-+. NH N+ M+ “83 838: «NM «MM «MM «MM «3 «.3 «NM NM NM canoe NN MN NN .NM NM NM NM NM NM NN MN NN MN NN NM NM NM NM NM NM NM NM NM NM MM NM NM NN NN NN NN NN, NN NN NM, MM NN NN NN NN NN NN NN MM MM NNN aaN NN NN NN NN NM NN NN N N NNNNNNNM NMNN MMNaN NMMN 1 MMM NMMfl NMM NMM NMH NNM NMH NH NH «eta 8353 8 «NMN «MMN «NNN «NMN «MMN «oMN «NMM MMM oMM momma NNN NMN NMN NNN NMN NMN NNN NNM NNM NNN NMN NNM NNM NNM NNM NNM NN NN NNN NNN NNN NNN NNN NNN NNN MNM NNM NMN NNN NNM NNM NNM NNM NNM NN NN NNN NNN NNN NNN NNN NNN NNM NMM NMM NNN aaN NNM NNM NNM NNM NNM NNM NNM NNM NNM NMNNNNNN NNMNNMNNN NN NM NN NM NM N N N N- MNNNNNMaN NaMN flown—mm GOHQMMMMMH HouuflOU .NosaMuaoN-.N< NMNNN MN MN NH M+ MH MH ¢+ Ml M+ uouuo M38233 «NN «MN «MN «MN «NN «0M «NN M M 239: NN NM NM NM NM NM NM N N MM MM MM MM MM NM NM M M MN MN NN NN NN NN NN MM NM MN NN NN NN NN NM NN N N NN NN MN NN NN NN NN N N NNN aaN NM NM NM NM MN MN MN N N NNNNNNNN aMo> MNMMNNNN 2 NM NM NM NH MM MM NH NH M.N «83 M5883 2. «NM «NM «NM «NM «NM «NM «NM N N Nance NN MN NN NN NN NN NN N N N N N N NM NM N M M NN NN NN NN NN NN NN NM MM NM MM NM NM NM N M N M NM NM NM NM NN NN NM N N NNN aav N N N N N N N N N NNNNNoNN NMN» NMMNMNoN NN NM NN NM NM N N N N- MNNNNNMaN oaMN Goa-HUN fiOHmflwdH HOHUQOU .NoaaMuuoN-.N< NMNNN 83 NH NH l I NH Mu... MH mfl M.N M+ N.M. uouuo M3253 m «MM «MM «NM «NM «MM «MM «MNM M N momma NM NM NM MM MM MM MM MM MM NM N N N N N NM N N MM NM NM MM MM NM MM NM NM N NM NM N NM MM NM N N NN NN NN NN NN NN NN NM MM MNaNNN NNMNNMaNMaN N N N N N N N N N NNMMMNN NNNNNN MNMNNNNN MH MH NH NH MM MH MH NH NH note 3883 «M «M «M «M «N «M «M MM 3 9.306 N N N N N N N NM NM N M N N N M M N N N N N N N N N NM NM N N N N N N N MM MM N N N N N N N NM NM MNaNNN NNMNNMaNMaN N N N N N N M N N NNMNNNN Naoao> NMMNNNNN NN NM NN NM NM N N N N- MNNNNNMaN maMN vowumm flOHmDudH HOHun—OU .NoaaMuaoN-.NN NMNNN N+ NH MH MM NH NH NH N+ NH N83 NNNNNNMN NM NM NM NM NM NM NM NM NM mamas NM NM NM NM MM NM MM MM NM MN NN NN NN MN NM NM MM MM NM NN NM NM NM NM NM MM MM NN NM NM NM MM NM NM NN NN MM-N NNM MM MM NM N N N N N N « M-Ma « aMa x NN aav MN NN NN NN NN NN MN MN MN ouaauNMNuN «Mona: MuuoN 4 MH NH NH NH NH NH MH NH NH uouuu NNNMESN no «NM «MM «MM «NM «MM «MM «NM NM NM mamas NN NN NN NN NN NN NN NM MM MN MN NN NN NN NN MN NM NM NM NM NM NM NM NN MM MM MM NN NN NN MN MN NN NN N N MM-N NNM NN MN NN MN NN NN NN N N « M-Ma « aNa.« NN aav _NN MN NN NN NN MN MM NM MN ouaMuNMNoN NMMN MNMNN NN NM NN NM NM N N N N- MoausaMaN oaMN HOHuflOU vowuom aonsmsH .NoaaMuaoN-.N< oMNuN .oaMMu ouou ou oZUoMou M0.0 W M I + 68.3 anon on mango» 36 w. M n « 5 M... M+ M+ M+ M+ M+ N+ ow... OH uouuo Muovsoum 8 «M «M «M «M «M «M M M M momma M M M N M N N M M N N M N M N N M M M N N N N M N M M N N N N N N NM M M MM-N NNM M M M M M M M N.N N.N « M-Na_« aMa « NM aaN N N N N N N N M M moaNMNMNmN «NNN oNuuM NMMN NN NM NN NM NM N N N N- MmuuaaMaN uaMN v0.“ Hum GOHmfimn—H Houufioo .NoaaMuaoN-.N< NMNNN 86 M+ M+ MH MH Mu... M.I_.. M+ M+ uouuo Mumvamum «MM «MM «MM «MM «MM «MM CM CM mamas N N NM MM NM MM N N NM NM NM NM NM NM NM NM NM MM NM NM MM MM N N NM MM MM MM NM MM NM NM NM NM NM NM NN MN NM NM NNN aav NM NM NM MM NM NM NM NM NNNNNNNN NMN» MMuaN NMMN NH NH NH NH NH NH NMM NH N83 M5353 MNM MNM NNM NNM NNM NNM NNM MNM mamas NNM NNM NNM NNM NNM NNM NNM NNM NN NNM NNM NNM NNM NNM NNM NNM NN NN NN NN NN NN NN NN NN NN NN NN NN NN NN NN NMM NMM NMM NMM NMM NNM NNM NNM NNN aav NNNNNNNN NNM NNM NNM NNM MNM NNM NNM NNM NooMN MNMNNNNM NMamuNNN NN NN NM NN NN NM N NM- 33:35 as; “OMN—0m dOHmflmflH HOHUMMOU .AMIaV uMuooumaon Maw mousmmoum umanomm> «mamoMa Mao MMEMM mo sowuwuuaouaoo aNououn «soMm MQSMM so mouacwa 0M you BOMMGN Monauoa um MENMouom onu ouaN MMMuNuouumnmuusN vomswna AcNB\Moa M.Mv aNaNMNvauM mo muuommu||.N< «Mama 87 N....... N.M N.M N.M N.N N.M N.M N.M Not» 3853 «M.M «M.M «M.M «M.M «N.M «N.M N.N N.N mamas N.N N.M N.N M.N N.M N.N N.N N.M N.M N.M N.M N.M N.M N.M N.N N.N N.M N.M N.M N.M N.M N.N N.N N.N N.N N.N N.N N.N N.N N.N N.M N.M M.M N.M N.M N.M N.N N.N N.N N.N MN NNNNNN N.M M.M M.M M.M N.N N.N N.M N.M NMNMNNN MmuoN MNaNM NM.“ NM.H NMN. MM .H NM.“ NN.“ NN.“ NN...N «83 2333 «MM. «NM. «NM. «NM. «NM. NM. NN. NN. Nance NM. NM. NM. MM. NM. MN. NN. NN. NN. NN. NN. MN. NM. NN. MN. MN. MM. NM. NN. NN. NM. MN. MN. MN. MN. NN. NN. NN. NN. MN. MN. MN. NN. MN. NN. MN. NN. NN. NN. NN. MNMa NMNMaN NN. MN. NN. NN. MN. NN. NN. NN. NNNM NNMM NNaNM NN NN NM NN NN NM N NM- NNNMNNMaN oaMN UOfihflm flOfimMMMMMH HOHUGOU .NaaaMuaoN-.NM NMNNN 88 .oBMu anon on 0>Nuuaou Mo.o.w_m I + .oaNu ouou ou obNuwMou Mo.o N.M I « uouuo unavamum momma mm NM mm uauooumamm M .H uouuo unavauum M.M momma N.M N.M M.M M.M N.M M.M N.M MN «EMMNN M.M aMououm uaNNMm OM on GM om ON CM 0 0M1 Ammundwav mafia vowuom sonnmaM Mouusoo .NoaaMuaoN-.N< NMNNN 89 MN MH MH M.._I. NH NH M+ ¢H uouuo vumvagm «MN «MN «MN «NN «MN «MN m N mamas MN NN NN NN NN NN N N NN NN NM NM NN NN MM MM MM MM NM NN NN NN NM NM NN NN NN NN MN NN N N NM NM NM NM MN NN N N NNN aav MN NM NN NN NN MN N N NMNNNNNN NMNN MMmaN NMMN NH NH NN. NH. MN NH MN NH uouuo NNNMNNMN «MMM «MMM «NMM «MMM «MMM «MMM MoM NoM mauve NNM NNM NNM NNM NNM NNM NNM NNM NNM NMM NMM NMM NMM NNM NNM NNM NNM NNM NNM NNM NNM NMM NNM NNM NMM NNM NNM NNM NNM NNM NN NN NNM NNM NNM NNM NNM NNM NNM NNM NNN aav NNNNNNNN NNM NNM NNM NNM NNM NNM NMM NMM NooMN MNMNNMNM NNamuNNN NN NM NM NN NN NM N NM- NNNMNNNaN osMN UOH me MMOMGMMHGH HOHufiOU .AMIaV uNuuoumam; Mam mounmmoua uanomm> «NEMMMQ Mam MQEMM mo aONu Imuuaooaou aNououa «soMm MMEMM do mouaaaa OM you soMmaM Mousuos um MMmeuouuwuouuaN Momsmaw AGMB\Moa Mv onus odwunaoaanouon Mam AsNa\Moa M.0v aNaNMNMouM mo mucoMMMII.oM¢ mMan 90 N.M N.M N.N... N.M N.M N.N N.M N.M .Mouuu NNNMESN N.N N.N N.N M.N M.N M.N M.N o.N mamoa o.N M.M N.M M.M o.N N.N M.M M.M o.N M.N M.N M.N N.N M.N M.N M.N N.N M.N M.N M.N M.N N.N M.N M.N M.N M.N c.m M.M N.M M.M N.N M.N N.M N.M M.M N.M M.M N.M N.M M.M MN NamNNN M.N M.N M.N M.N M.N M.N N.M N.M afiuuoum Hmuoa nuahu 0H 0H ¢H CH 0H 0H OH OH uouuo vuuvamum M0. M0. M0. M0. MO. «N0. M0. M0. mauve M0. M0. M0. Mo. Mo. Mo. Mo. Mo. M0. M0. M0. Mo. M0. No. No. No. Mo. Mo. M0. M0. N0. M0. no. No. Mo. Mo. Mo. Mo. M0. M6. MO. N0. M0. M0. M0. Mo. M0. M0. M0. M0. Mafia oM\HEv Mo. Mo. N0. M0. Mo. No. M0. M0. mumm 30am smahq OM on OM OM 0N 0M 0 oMI Amouaawav GENE voNuom aonswnH Mouuaoo .NusaMMaoN-.NM< NMMNN 91 .238 ouou ou o>NumMou M0.0 .v- NM I + .95”. ouou 3 03333 8.0 .v- .M I « N+ M+ M+ uouuo M38283 «MM «NM NM 2306 on MM NM OM MM MM MM MM NM NM MM MM MM NM MM MM MM MM uNuuoudEmm M.“ M.M...- M.H uouuo Bacon: N.M M.M M.M undue M.M M.M M.M M.M M.M M.M N.M M.M M.M o.M N.M M.M N.N M.M N.M MN 2.3qu M.M N.M M.M dwououm namuflm OM on OM OM ON 0M 0 CM! Ammuaawav NEHB MONuom aONNnMaH Mouuaoo 63538-52 «Mama 92 NMN NMN MMH N+ MN MN N+ N+ NNNM» NNNNNNNN MNNM MNM NMM NNM «NN «NN NMM NMM Names NNM NN NN NN NN NN NN NN NMM NNM NNM NMM NNM NN NNM NNM NNM NNM NNM NNM NN NN NMM NMM NNM NNM NNM NMM NNM NNM NNM NNM NNM NNM NN NNM NN NN NNM NNM NNN aaN NMM NMM NMM NNM NN NN NMM NNM NNNNNNMN NNMNNNMNN M.N NH NM NM NM NM NM NH «83 335$ NNM «NNM NNM NNM MNM NNM NNM NNM canoe NMM NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NNM NMM NMM NMM NMM NMM NMM NMM NNM NNM NNM NNM NNM NNM NNM NNM NNM NNN aaN NNNNNNNN NNM NNM NNM NNM NNM NNM NMM NNM NNNMN MNMNNNNM NMauNNNN NN NN NM NN NN S N S- 332:8 25M. vowuom aonsmaH Mouuaoo .AMIaV uMuooumaon Mam mousmmoua NNMsumm> «NBNMMQ Mam snaMM mo aowuuuunooaoo aNououm «scam MMBMM no mounaNa 0M Mom soamaM unnumaoo um MENMouom «Mu ouaN MMMMMuouumlmuuaN MomnmdN AGNE\Moa M.Mv aMaNMNMmun mo mucoMMMII.MM< oMMMH 93 NNNM. NN.“ NN.H NN.H MN.H NN.“ N+ N+ «83 3338 «MM. «NM. «NM. «NM. +NN. MN. MN. MN. Manse MM. NM. MM. NN. NN. NN. MN. NN. MN. NN. NN. NN. NN. NM. NN. MN. NM. NM. NM. NM. NN. MN. MN. MN. NN. NN. NN. NN. NN. NN. MN. MN. NN. NM. .NN. NN. MN. MN. MN. MN. NNMa NMNMaN NN. NN. NN. NN. NN. MN. MN. MN. NMMM NNMM NNaNM .MH MH MH MH MM MM NH MM Hobo NNNMESN N NM NM N NM NM MM MM mauwa NM MM MM MM MM MM MM MM N N N N N N N N MM MM MM MM NM MM NM NM NM NM N N N NM MM NM MM MM MM MM MM NM MM MM NNN aaN N N N N N N N N NNNNNNMN NMNN MMMaN NMMN NN NN NM NN NN NM N NM- NNNMNNNaN uaMN UOHhflm MMOHmSmMMH HOHufiOU .NosaMuaoN-.MM< NMNNN Table A11.-Continued. Infusion Period Control -10 6O 50 4O 30 20 10 Time (minutes) 2.4 2.5 2.7 2.8 2.8 3.0 3.1 2.9 2.8 2.6 2.0 2.5 2.2 Lymph Total Protein 3.0 3.0 1.4 2.3 1.4 1.9 2.0 2.3 1.4 (grams Z) 3.4 3.1 3.6 2.0 1.9 1.9 2.4 3.2 3.3 3.3 2.9 3.1 1.9 2.9 3.3 2.0 2.7 2.3 3.2 2.8 3.6 3.7 4.2 1.4 1.5 94 « our) «5+4 2.4 2.9* 3.3* 3.2* . 3 4 3 2.0 1.9 1.2 means standard error @561th 00.... MMQ'MMQ wOmN'rcnw O 0 O O O O flquM-M¢fl~¢ MQHQ'QQ 000000 MQQGMQ Plasma Protein (grams Z) asp-I O O rr+1 NIN- M'+| I-‘N M+°I means standard error 9S .oENu ouou ou o>MuuMuu Mo.o.w m I + .oaNu ouou cu o>NuwMuu Mo.o.w a I « M+ M+ M+ uouuo unavauum OM MM MM mamas MM NM NM NM NM MM MM MM MM MM NM NM MM MM NM MM MM MM uNuoouoaom NM NM NM NM NN NM o NM- 33233 as: MoNuom :ONmnmaH Mouusoo \ .woadwudooII.MM< magma 96 5+ m“ NH N+ m+ mw 9+ N+ noun» cumuamum «NNN «NNN «mug «NNN «meg «5nd NNN mod magma NNN NNN NNN mfiu NNN mm“ mg“ mNH mou mm“ NNN mad an“ nag «NH n~_ NNN mm“ neg mag NMN neg mofi Nod mMH nMH NMN mMH mm” mm” om om NNN «NH NNN meg mNH on" NNN NH” Awm aav meg mhfl mm“ mug mmH on“ NNN moH unannoum aoqmamuam “H “M NM NM NH nH NH NH pots 338$ «MNH «MNH «MNM «NNN «NMN «NMN MNH Mgg wauma mag NMN NMN meg nMH mm“ NNN NNN «mg onfl um” NnN mad mofi NMN on“ NNN NNN mg“ mg“ NNN on" mNH NNN NNN NNN Fog nHN NNN on" NNN NNN mmfi mmfi on” mNH and on“ NH“ NNN ANN aav ousmmuum NN mm NNN nofi no“ NNN nod mod vooam HmfiumuMM oflauummm No on NM on Na NH o NN- Amuusuaav mafia UOHHQM fiOfimfimflH HOHUGOO H .Aolav uauooumams Nam amusmmwum umHsumm> .mamman can nuaua mo coaumuu aaouaou :auuoum .30Hm amaha do mmusafia No now sommcw uawumaoo um haamauuuumlmuuna vmmamaw AaNa\wua Mv ommn maausaoawmmuoa can Acfia\woa M.Nv afiawxzwmun mo muUNNNMII.NH< manna Table A12.-Continued. Infusion Period Control -10 20 30 4O 50 60 10 Time (minutes) 2.4 2.2 2.2 2.3 2.5 2.3 2.0 2.0 1.9 1.2 1.4 1.9 2.6 2.1 Lymph Total Protein 2.9 3.0 2.1 2.3 2.1 1.5 1.6 2.1 2.2 2.9 1.9 1.8 1.9 2.2 1.3 1.7 1.9 2.6 (grams Z) 2.3 1.9 1.9 2.0 2.7 2.4 1.9 1.9 2.5 1.7 2.1 2.1 2.4 2.8 2.0 1.8 97 2.2 2.2 2.3 2.4+ . . . 6 2.1 i 2.0 1.9 +.4 means standard error HQNw—‘Q lfld’fi’d'lfilfi Ommxoo~ HHMam NNMN NM NM NM Nm NN NH N NH- HmouNaHaN maHe UOHHUQ dOHmn—MGH HOHUGOU . voadwuaooll . N~¢ 3”an 99 .oaau anon on m>uumamu mo.o.w a I + .osau ouou ou m>aumaou Ho.o w.a I « M+ N+ N+ uouuo unavauum MMM +NM NM mawma on NM NM NM HM NM Mm NM MM HM NM NM NM MM MM OM OM mM uwuooumamm ON NM NM NM NN NH o NH: Ammusaaav mafia vofiuom aofimomaH Houuaoo .NaaaHuaoN--.NH< «Hana BIBLIOGRAPHY lo. BIBLIOGRAPHY Alabaster, V. A. and Y. S. Bakhle. Converting enzyme and brady- kininase in the lung. Cir. Res. Supp, II. 31:72-84, 1972. Allwood, M. J. and G. P. Lewis. Bradykinin and forearm blood flow. J. 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