WIUHWilli(WIWWWNWHIIHIHHWIWI will l/IIU/ Willi/ll Ill Ill/WW This is to certify that the thesis entitled The Inter-Relationship Between Prostaglandin E1, Various Vasoactive Substances, and Macromolecular Permeability in the Canine Forelimb presented by Arthur Neil Gorman has been accepted towards fulfillment of the requirements for M. S . degree in Physiology Date August 8, 1979 0-7639 OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to book drop to remove this checkout from your record. THE INTER-RELATIONSHIP BETWEEN PROSTAGLANDIN E1. VARIOUS VASOACTIVE SUBSTANCES, AND MACROMOLECULAR PERMEABILITY IN THE CANINE FORELIMB By Arthur Neil Gorman A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1979 ABSTRACT THE INTER-RELATIONSHIP BETWEEN PROSTAGLANDIN E1, VARIOUS VASOACTIVE SUBSTANCES, AND MACROMOLECULAR PERMEABILITY IN THE CANINE FORELIMB By Arthur Neil Gorman The effects of PGE1, by itself and in combination with other vasoactive agents, on hemodynamics, lymph protein concentration, and weight, were examined in canine forelimbs perfused at constant inflow. PGEi (16 ug/min i.a.) produced profound vasodilation and caused marked increases in lymph protein concentration but failed to significantly increase lymph flow rate. Forelimb weights significantly increased owing to edema formation. Under similar conditions, this same dose of PGE1 infused with isoproterenol (3 ug/min), vasopressin (0.8 Pressor Units/min), angiotensin II (2 ug/ min), or methylprednisolone (15 ug/min) failed to increase lymph protein concentration relative to control. Serotonin (7 pg base/min i.a.) infused concomitantly with PGE; (16 ug/ min i.a.) produced increases in lymph protein concentration and limb weights similar to those achieved with PGE1 alone. The antagonism of the PGEl-induced increase in protein efflux by isoproterenol, vasopressin, angiotensin II, and methyl— prednisolone was independent of changes in blood flow, vas- cular pressures, or perfused surface area. Thus. like Arthur Neil Gorman histamine and bradykinin, PGE1 produces increases in micro- vascular permeability which can ve antagonized by other vasoactive agents. To my lovely wife, Anita, and to my wonderful parents; their love has lit my way to accomplishment and joy. ii ACKNOWLEDGMENTS Many people have contributed in many different ways to the success of this project. I would like to thank: Don Anderson, whose technical assistance and moral support were invaluable during our many months together; Jim Maciejko, for his assistance and patience in teaching me proper theory and technique; Rich Raymond, for convincing me to undertake this endeavor and for his encouragement along the way; Drs. C.C.Chou and T.E. Emerson for their services on the guidance committee; and Dr. George Grega, for his guidance, generosity, and kindness in helping me, not only in the preparation of this thesis, but in the finding of a new and promising future. Finally, I wish to express my gratitude to my wife, Anita, for her assistance in the typing of this thesis, but more importantly, for the love and understanding she has shown me during these long and difficult months. iii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . . . v LIST OF SYMBOLS AND ABBREVIATIONS. . . . . . . . . . vii INTRODUCTION . . . . . . . . . . . . . . . . . . . . l SURVEY OF THE LITERATURE . . . . . . . . . . . . . . h METHODS. . . . . . . . . . . . . . . . . . . . . . . 26 RESULTS. . . . . . . . . . . . . . . . . . . . . . . 30 DISCUSSION . . . . . . . . . . . . . . . . . . . . . 40 SUMMARY AND CONCLUSIONS. . . . . . . . . . . . . . . 50 APPENDICES . . . . . . . . . . . . . . . . . . . . . 52 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . 73 iv Table LIST OF TABLES Effects of PCB; (16 ug/min i.a., CF) on lymph flow and protein transport, lymph and plasma protein concentrations, vascular pressures, hematocrit, and limb weights . . . . . . Effects of PCB; (16 u min i.a., CF) and isoproterenol (3 u min i.a., CF) on lymph flow and protein transport, lymph and plasma protein concentrations, vascular pressures, and hematocrit . . . . . . . . . . . . . . Effects of PCB (16 ug/min i.a., CF) and vasopressin (0.8 Pressor Units/min i.a., CF) on lymph flow and protein transport, lymph and plasma protein concentrations, vascular pressures, hematocrit, and limb weights. . Effects of PCE1 (16 ug/min i.a., CF) and angiotensin II (2 ug/min i.a., CF) on lymph flow and protein transport, lymph and plasma protein concentrations, vascular pressures, hematocrit, and limb weights . . . . . . . Effects of PCB; (16 u min i.a., CF) and methylprednisolone 15 ug/min i.a., CF) on lymph flow and protein transport, lymph and plasma protein concentrations, vascular pressures, hematocrit, and limb weights. . . Effects of PCE1 (16 ug/min i.a., CF) and serotonin (7 us base/min i.a., CF) on lymph flow and protein transport, lymph and plasma protein concentrations, vascular pressures, hematocrit, and limb weights . . . . . . . . Effects of PCB; (16 ug/min i.a., CF) on lymph flow and protein transport, lymph and plasma protein concentrations, vascular pressures, hematocrit, and limb weights . . . . .-. . . Page 34 35 36 37 38 39 53 Table A-2 o A-3. A-6. Effects of PGE; (16 u isoproterenol (3 u and hematocrit . . . . . . . . Effects of PCB; (16 ug/min i.a., CF) and min i.a., CF) and min i.a., CF) on lymph flow and protein transport, lymph and plasma protein concentrations, vascular pressures, vasopressin (0.8 Pressor Units/min i.a., CF) on lymph flow and protein transport, lymph and plasma protein concentrations, vascular pressures, hematocrit, and limb weights. Effects of PGE1 (16 ug/min i.a., CF) and angiotensin II (2 ug/min i.a., CF) on lymph flow and protein transport, lymph and plasma protein concentrations, vascular pressures, hematocrit, and limb weights . Effects of PCB; (16 u methylprednisolone Effects of PCB; (16 ug/min i.a., CF) and serotonin (7 pg base/min i.a., CF) on lymph flow and protein transport, lymph and plasma protein concentrations, vascular pressures, hematocrit, and limb weights . vi min i.a., CF) and 15 ug/min i.a., CF) on lymph flow and protein transport, lymph and plasma protein concentrations, vascular pressures, hematocrit, and limb weights. Page 57 60 63 67 7O mmHg Mow. gm% um Hg ng ml min LIST OF SYMBOLS AND ABBREVIATIONS Millimeters of mercury pressure. Molecular weight. Grams per cent. Angstrom units. Micrometer. Microgram Nanogram Milliliter Minute Drug infusion into the vena cava. Drug infusion intra-arterially into the forelimb. Forelimb perfused at constant inflow. vii INTRODUCTION Although the issue has been under considerable debate, there is a great deal of evidence to indicate that prostaglandins, especially PGEl, are intimately involved in the inflammatory process (102). Numerous studies (11.12.19, 20.47.74) conducted in rats, and guinea pigs have demonstrat- ed that PGE; increases vascular permeability to macromole- cules. These studies have been conducted using the vital dye technique which has a number of methodological drawbacks which may have contributed to the unusual variability of re- sponse to PGE, found by some investigators (20). Using the more reliable hamster Cheek pouch prep— aration, Svensjo (95) and Joyner gt a1. (#5) have shown that PGE, increases the leakage of FITC-dextran from postcapil- lary venules by forming interendothelial cell venular gaps. However, they found PGE1 to be considerably less potent than histamine or bradykinin in producing FITC-dextran leakage sites. They and others (57,107,108) have also reported that PGE, potentiates the direct actions of histamine and brady- kinin on the microvascular membrane. To date, only a few studies have been conducted in the dog. Greenberg and Sparks (27), Daugherty (l3), and , Joyner (#3) have all failed to show that PGE; causes an 1 2 increase in microvascular permeability to macromolecules. However, these studies either did not measure protein fluxes directly, or did not study the effects of PGE; over a wide dose range. Recent work from our laboratory (58) has shown that a wide dose range of PGE; causes increases in lymph protein concentration and forelimb weights when infused locally intra-arterially into canine forelimbs perfused either naturally or at constant inflow. Lymph flow rate did not increase as expected. Following pretreatment with indo- methacin, PGE, produced increases in lymph protein concen- tration similar to those produced in the absence of indo- methacin, but now produced marked increases in lymph flow rate under both natural and constant inflow conditions. Based on these data in the dog and similar findings in other species, it appears that PGE1 is likely to have a role in inflammation and increases vascular permeability in a manner similar to that of histamine and bradykinin. Work from this laboratory (29.59.65) and others (98) has demon- strated that catecholamines possess the unique ability to antagonize the protein efflux produced by histamine and brady- kinin owing to a direct action on the microvascular membrane which counteracts that of histamine and bradykinin. This antagonism is independent of changes in blood flow, micro- vascular pressure and perfused surface area (29.51.65). It is now known that other agents possess the ability to antag- onize or potentiate the protein efflux produced by these agents (29.59.78). In this study, the effects of 3 catecholamines and a variety of other natural and synthetic agents were studied to determine if they antagonize or potentiate the direct actions of PGE, on the microvascular membrane. SURVEY OF THE LITERATURE The vascular system carries nutrients, hormones, electrolytes. gases. and macromolecules required by the cells throughout the body and it takes up the products of their metabolism for excretion. The actual exchange between the blood and tissues takes place in the capillaries and ven- ules and can occur by filtration, diffusion or micropino- cytosis. Fluid filtration is governed by physical forces and can be expressed by the following equation derived by Starling (91). F = k(PC - Pi - “p + vi). where F = the rate of fluid movement across the capillary wall; k = capillary filtration coefficient. This is a measure of the permeability of the microvascular wall to isotonic fluid. It is determined by the product of capillary permeability and surface area available for diffusion; Pc = capillary hydrostatic pressure; Pi = interstitial hydrostatic pressure; Hp = plasma colloid osmotic pressure; ti = interstitial colloid osmotic pressure. When the equation is positive. filtration of fluid occurs, and when it is negative, fluid reabsorption occurs. L, 5 Capillary hydrostatic pressure is determined by capillary blood volume and capillary compliance. Clough gt_gl. (8) has reported changes of only 0.1 pm in the diam- eter of capillaries in the cat mesentery during systole. This very low compliance is probably due to the incompres- sible nature of the surrounding gel matrix (21). Since there is little change in compliance, PC is determined primarily by capillary blood volume which is in turn determined by several physical factors affecting both inflow and outflow. These factors are related by the equation: Pc = (Pa - Pv) Rv + Pv. Ra + Rv where Pc = mean capillary hydrostatic pressure; Pa = mean arterial pressure; Pv = venous or outflow pressure: Rv = venous resistance to outflow; Ra = arterial resistance to inflow. An increase in arterial pressure. venous pressure, or venous resistance will increase PC. An increase in arte- rial resistance will lower Pc. Vascular resistances are re- lated to vessel caliber which is determined by active changes due to vascular smooth muscle activity, passive Changes (due to changes in transmural pressure). and blood viscosity. Interstitial fluid hydrostatic pressure is analagous to capillary hydrostatic pressure but is that pressure found in the interstitial spaces. It had been generally accepted that Pi was slightly positive (55) and would oppose fluid 6 filtration. Guyton (32), however, has suggested that inter- stitial fluid pressure is subatmospheric. thus enhancing fluid filtration. This issue remains to be resolved. Plasma colloid osmotic pressure is the pressure resulting from the concentration of dissolved protein in the blood. This oncotic pressure is estimated to be about 25 mmHg out of a total plasma osmotic pressure of 6.000 mmHg (3) but is of great importance for, unlike the electrolytes which contribute to the total osmotic pressure. plasma pro- teins do not diffuse readily into the interstitial spaces and are largely confined to the intravascular space. Plasma proteins are primarily a mixture of albumin (M.w. 69,000), globulins (M.w. 1no.ooo). and fibrinogen (M.W. 900.000). Approximately 65% of plasma colloid osmot- ic pressure is attributable to albumin and only 15% to globulins as the albumin molecule is only one-half the size of the golbulin molecule and is present in higher concentra- tions. These plasma proteins exert an oncotic pressure of about 19 mmHg. In addition. cations which bind to the nega— tively charged protein ions exert another 6 mmHg pressure. Similarly, interstitial colloid osmotic pressure is determined by the protein concentration of the interstitial fluid. Average total protein concentration of interstitial fluid is about 3 gm % and the colloid osmotic pressure about 10 mmHg (106). Protein concentrations and colloid osmotic pressures vary from one tissue to another. In skin and skeletal muscle the average protein concentration has been 7 found to be 2.0 gm % yielding an oncotic pressure of about 5 mmHg. In the liver. where discontinuous sinusoids allow for even greater filtration, protein concentrations of greater than 3.3 gm % and oncotic pressures of 16 mmHg have been reported. Discontinuous sinusoids which allow proteins and other large molecules to freely pass through large gaps. are also found in the bone marrow and the spleen. Two other types of capillaries have been distin- guished in studies using electron microscopy. Continuous capillaries are found in smooth, skeletal, and cardiac muscle, as well as in connective tissue and the central nervous system (4). They are characterized by an uninter- rupted endothelium and intercellular gaps #0 A wide. These intercellular junctions may correspond to the "small pore" system postulated by Landis and Pappenheimer (55). These pores represent only 0.1 - 0.2 per cent of the total capil- lary surface area. Peroxidase, a protein tracer of rela- tively low molecular weight (90,000), has been shown to pass rapidly through these clefts while ferriten (M.W. 500,000) does not (#9). Cerebral capillaries are impermeable to both peroxidase and ferriten reflecting the barrier function of the cerebral capillary wall (blood-brain barrier). Fenestrated capillaries, found in the renal glomeruli, endocrine glands. and the intestinal mucosa. have circular pores 1 pm in diameter which penetrate the endothelium. These pores are usually covered by a very thin diaphragm o and appear to be regularly spaced approximately 1300 A apart. 8 Tissue colloid osmotic pressure can be measured by different techniques. Measurements using implantable devices such as perforated capsules that theoretically equilibrate with the interstitial fluid may be inaccurate because of the possibility of contamination by plasma, or that the sampled fluid may not contain all osmotically active particles. A more common method, lymph fluid analysis. makes the assump- tion that the lymph accurately reflects the interstitial fluid contents. This method has been challenged, for changes in lymph concentration could occur as the lymph flows centrally from the terminal lymphatics to the larger lymph vessels. Lymph analysis may also not reflect protein concentration gradients that exist in the interstitium. Using dextran molecules of known molecular weight and size. Renkin and Garlick (80) found that concentrations were equal in lymph and interstitial fluid thus precluding the possibility of protein concentration gradients existing in the interstitium. Studies conducted by Garlick and Renkin (22) and Mayerson gt g1. (66) have shown that exchange only occurs at lymph nodes and not in the lymphatic trunks. Thus, if lymph is sampled before it reaches a node it should be a true reflec- tion of what is at the terminal lymphatic vessel. The lymph vascular system forms a "drainage system" as lymph is conducted from the lymphatic capillaries through successively larger vessels that ultimately empty into the venous system. The lymph is propelled centrally through one-way valves that only permit unidirectional flow. When 9 a lymph vessel is distended with fluid, contractile elements or smooth muscle cells, if present in the vessel wall. con- tract (31). In addition to the pumping caused by the intrin- sic contraction of the vessel walls. any external factor that compresses the lymph vessel can also contribute to the move- ment of lymph fluid. Such factors are muscle contraction, passive movements of parts of the body, arterial pulsations, and compression of the body tissues from the outside. Though usually ignored, a variety of vasoactive agents have the ability to contract or relax lymphatic vessels in vivo and in vitro. Diffusion is the most important means by which sub- stances are transferred between the plasma and interstitium. This process results from the random thermal movement of water molecules and dissoved particles. Hence, the great- er the concentration gradient across the membrane, the great- er the rate of diffusion. Fick's Law describes the process of diffusion: _Q§_._ . . dc dt “D A d... where g: = the amount of substance moved per unit time: D = the diffusion coefficient for a particular mole- cule. (This value is inversely proportional to the square root of the molecular weight of the particle.): A = the cross-sectional area of the capillary membrane: 10 = the concentration gradient across the cap— illary membrane. dc dx Thus, the amount of substance which diffuses per unit time is equal to the product of the diffusion coeffi- cient, the area of the capillary membrane. and the concen- tration gradient. Small molecules such as water and urea diffuse rapidly through capillary pores. Lipid-insoluble substances must also diffuse through these pores but due to the larger size of these molecules their diffusion is more restricted. With increasing size diffusion becomes much more difficult. Molecules with molecular weights of 60,000 or greater are almost completely impermeable. Substances which are lipid-soluble such as CO2 and 0. can diffuse di- rectly through the lipid membrane of the cell. Since these substances can diffuse across the entire capillary membrane. their rates of diffusion are several hundred times the rates of most lipid-insoluble molecules. Micropinocytosis (cytopempsis) is a relatively slow transport process that may be responsible for the movement of large lipid-insoluble molecules between plasma and inter- stitial fluid. This process takes place via vesicles which invaginate from the plasma-membrane and migrate across the cytOplasm from one surface of the endothelial cell to an- other where the contents are released. The actual passage of the vesicle across the cell may be passive. but the struc- tural reorganization of the membrane during invagination and exocytosis is regarded as an active, energy-consuming ll process. Although such a mechanism is known to exist, its quantitative importance in capillary and venular exchange is under considerable debate. It has been repeatedly suggested that the vesicles are of major importance for transport of large molecules (5.81.87) and may contribute to a "large pore" system (81) which permit plasma proteins. hormones and antibodies to pass freely into the interstitium. It has been hypothesized by Renkin's group (5.4#.79) that this mode of transport may play a primary role in changes in vascular permeability associated with inflammation. Increasing the permeability of the microvascular membrane will result in the escape of plasma protein from the vasculature to the interstitial fluid which will raise the oncotic pressure of the intersti- tium. The increased interstitial fluid oncotic pressure will enhance fluid movement out of the vasculature. Using micro- peroxidase tracer in mice, Simionescu gt_g;. (88) found that leakage of macromolecules is primarily restricted to venules 8 - 16 pm in diameter. Svensjo gt 31. (98) reported similar findings using the hamster Cheek pouch preparation. This procedure can be used to study changes in permeability in the microvasculature. FITC fluorescein-labeled dextran (M.W. 145.000) is administered intravenously and a portion of the cheek pouch is dissected and viewed microscopically (96). Leakage sites are counted, after topical application of various agents, as areas of fluorescence appear. The tissues can then be collected and examined via electron 12 microscopy. Histamine and bradykinin. two of the most im- portant edemogenic and vasoactive agents in the body, produced a dose-related increase in the number of leakage sites in venules 9 - 16 pm in diameter (99). No leakage sites were detected in the arterioles or the capillaries. However. Svensjo gt 31. and other investigators (82) do not believe that the increased macromolecular leakage occurs as a result of increased vesicular transfer. They propose that interendothelial gaps form as adjacent endothelial cells "round up" in response to these vasoactive agents (36). These gaps are believed to be formed by active contraction, presumably as a result of shortening of actinomyosin-like filaments within these cells (63). Numerous morphological studies have demonstrated these gaps in tissues exposed to histamine (6,60,61,62) and bradykinin (30.33.86). The gaps formed were from 0.08 pm to 1.9 pm in width. large enough to easily allow passage of macromolecules. In contrast, there is no direct evidence to support the claim of Carter. Joyner. and Renkin (5) that inflamma- tory mediators increase vesicular transfer. Morphological studies have been unable to substantiate this claim.. Since cytopempsis must be somewhat of an active process (81), it would be expected that interference with cellular metabolism would curtail vesicular transport. However, micropinocyto- sis has proven to be relatively resistant to oxygen lack and/Cr to inhibitors of cellular metabolism (92). Active processes are temperature sensitive and yet it has been 13 demonstrated (82) that marked cooling of the rat hindquar- ters failed to alter the increased Clearance of macromole- cules in response to elevations in venous pressure. Jennings and Florey (#2) observed no change in vesicular uptake of ferriten during cooling, whereas Rippe and Grega (82) have shown that cooling markedly reduced the increase in macro- molecular efflux produced by histamine in rats. Indeed, this effect was immediately reversible by rewarming the pre- paration to 370 C. Thus, at present. there is no conclusive evidence to support the hypothesis that cytopempsis plays a significant role in transcapillary movement. 0n the other hand. a significant amount of morphological data is avail- able to attest to the formation of venular gaps as a means by which protein efflux can occur during inflammation. Inflammation is Characterized by vasodilation. in- creased vascular permeability, pain. and migration of leuko- cytes into the inflamed area (56). The high concentration of plasma proteins in inflammatory exudates as compared with normal extravascular fluid (90) makes it certain that what- ever other changes are present. an increased permeability of the vessel wall to protein is an essential feature of inflammation. Histamine and bradykinin have been implicated in playing a major role in inflammation. It has been shown that local administration of histamine (l - 64 ug base/min) or bradykinin (0.8 - 10 ug/min) into the canine forelimb significantly increases net fluid filtration in skin and l4 skeletal muscle (28,35.52.59.65) resulting in significant increases in the weight and volume of the perfused limb. The increase in net fluid filtration is due to both an in- crease in microvascular pressure resulting from the vaso- dilation produced by these autocoids, as well as a decrease in the transmural oncotic pressure gradient. The decrease in the transmural oncotic pressure gradient is due to an enhanced rate of protein efflux into the interstitium (36) subsequent to an increase in vascular permeability to macro— molecules. The relative contributions of these pressure- dependent and pressure-independent effects are dose related. Low doses of histamine (e.g. 5 pg base/min) and bradykinin (e.g. 0.8 ug/min) infused into the naturally perfused fore- limb produce increases in lymph flow. small skin vein pres- sure (a minimun for capillary hydrostatic pressure), and limb weight (28,52). These same doses infused into forelimbs perfused at constant inflow still increase lymph flow greatly but small skin vein pressures are not altered and the weight gain is largely attenuated (28,59). Thus. under conditions where microvascular pressure is held constant, the edema is considerably reduced. High doses of histamine (e.g. 64 ug base/min) and bradykinin (e.g. 10 ug/min) increase blood flows and skin small vein pressures to the same levels as do the lower doses, yet the edema is three to four times greater with the higher doses. Under constant inflow conditions the same 15 large increases in limb weight are seen despite the fact that skin small vein pressures, and inferentially, capil- lary hydrostatic pressures . probably remain at control lev- els. Thus. with the higher doses of histamine and bradykinin. the pressure-independent effects of these autocoids are dom- inant in producing edema (28,52), whereas at the lower doses. the pressure-dependent increases in capillary hydrostatic pressure are important in the genesis of the edema formation. Other agents have also been found to contribute to the inflammatory response. Among them are the prostaglandins. Prostaglandins are potent vasoactive agents having a wide range of actions and are synthesized from 20 - carbon poly- unsaturated fatty acids. The effects of prostaglandins were first noted by Kurzrok and Lieb (54) in 1930 when they ob- served that uterine muscle strips would relax or contract when exposed to human semen. A few years later von Euler (103) reported that human and sheep seminal fluid extracts had po- tent stimulatory actions on smooth muscle and lowered arterial blood pressure in experimental animals. He demonstrated that the biological activity of the seminal fluid was associated with a lipid-soluble acid which he called prostaglandin. During the 1950's and early 1960's pioneering work by Berg- strom and Samuelsson (2) found that at least four major groups of prostaglandins existed: i.e., the E, F, D, and A groups. Two other groups. the thromboxanes (TXAa) (37) and prosta- cyclins (PGIg) (71) have recently been isolated. 16 The main precursor for these prostaglandins is arachidonic acid (5.8.11.14. eicosatrienoic acid) which has four double bonds and gives rise to prostaglandins with two double bonds. The arachidonic acid found in the cell mem— brane is derived from the diet. either from elongation and desaturation of the essential fatty acid linolenic acid found in vegetables. or from the arachidonic acid content of meats. Even very slight chemical or mechanical stimuli can activate the enzyme phospholipase A; (72) which releases arachidonic acid from membrane phospholipids. The enzyme complex known as cyclo-oxygenase or prostaglandin synthetase, which seems to be present in the membranes of all cells, converts the arachidonic acid into the unstable cyclic endoperoxide PGGZ. This substance is rapidly converted to yet another unstable cyclic endoperoxide, PGHé. At this point the biochemical pathways diverge. Depending on the tissue in question dif- ferent prostaglandins will be synthesized, probably due to the differential activity of certain enzymes in their re- spective tissues. PGEa. a vasodilator and bronchodilator is formed from PGHQ. as is PGFaa which is a venoconstrictor and bronchoconstrictor. It has been pointed out that there is a certain symmetry between the various prostaglandins. The PCB and PCP compounds have been shown to have Opposing ac- tions in most tissues (41). It has recently been shown in monkeys. chickens, and pigeons (94) that PGEa can be converted to PGFaa by the enzyme PGEQ 9-Keto Reductase. It has been post- ulated that PGFga may be converted to PGE2 as well, but as yet no evidence exists to support this theory. 17 Prostacyclin (PGIa) is formed from PGHa in the walls of arteries and veins. PGIQ, which has a half-life of only two to three minutes (72), increases platelet adenyl cyclase activity. This results in increased cyclic-AMP levels and inhibition of platelet aggregation. PGI2 prevents platelet aggregation in vivo and in vitro at concentrations of 1 ng/ml. Prostacyclin is also a vasodilator and is broken down rapidly into 6-oxo-PGF1a which has only weak anti-aggregatory activ- ity. The symmetry of the system is preserved by thromboxane Aa (TXAg). another product of PGHé. TXAa is found in the plate— lets and constricts blood vessels (37). It is metabolized to the inactive compound. thromboxane B3, in approximately 30 seconds. TXAa decreases adenyl cyclase activity which decreases cyclic-AMP levels and promotes platelet aggregation. Thus PGI2 and TXA; are in opposition to one another. It has been post- ulated by Moncada and Vane (72) that the delicate balance of these two prostaglandins is essential for proper homeostasis and that prostacyclin may one day be used in a clinical set- ting to prevent thrombosis. Dihomo-gamma-linolenic acid (8.11.14 eicosatrienoic acid) contains three double bonds and gives rise to the 1- series of prostaglandins. i.e. those prostaglandins contain- ing just a single double bond. The biochemical pathways in- volved here are similar to those of the 2-series discussed above. Cyclo-oxygenase converts dihomo-gamma-linolenic acid into the cyclic endoperoxide PGG, which rapidly converts to 18 PGHl. PGH; gives rise to PCB; and PGFla. The relationship between these two prostaglandins is similar to that of their 2-series counterparts. The 1-series prostaglandins are not as prevalent as the 2-series prostaglandins and their physiologic importance is largely unknown. This is not to say that they have no role. PGE; has been shown to have high anti-aggregatory activity (77) due to its ability to increase cyclic-AMP levels(25), and can inhibit aggregation of human platelets (53) at a dose of l ug/ml. If the precursor dihomo-gamma-linolenic acid is administered, significant amounts of PGE; are synthesized in sheep vesicular gland (84) and the microsomes of bovine vesic- ular gland (112). Dihomo-gamma—linolenic acid competes with arachidonic acid for the cyclo-oxygenase of human platelets (111) and thus may effect the production of the 2-series prostaglandins. In 1971 Vane (101) proposed that the anti-inflamma- tory action of aspirin arises from the inhibition of prosta- glandin synthesis. This view has been supported by reports that aspirin can inhibit prostaglandin synthesis in human platelets (89) and semen (9). It is now known that low concentrations of aspirin-like drugs inhibit cyclo-oxygenase (72), the first enzyme in the arachidonic acid cascade, and thus inhibit the formation of the endoperoxides and all their subsequent products. Aspirin-like drugs have analgesic, anti- pyretic and anti-inflammatory properties, thus their ability to inhibit prostaglandin synthesis fits well with the belief 19 that prostaglandins are involved in the inflammatory process. There is a great deal of evidence to implicate prostaglandins in inflammatory processes. Increased concen- trations of prostaglandins. mainly PGE's. have been recover- ed from inflammatory exudates in man (14, 26, 76). dog (1). and rat (109). Anaphalaxis (75). tissue ischemia (16. 67), mechanical stimulation (15, 23), chemical inflammatory agents (110). and scalding injuries (1) all release prostaglandins. especially PGE's. PGE; induces migration of leukocytes (48) in concentrations of 1 ug/ml or greater. In man. 100 mg of PGE3 causes pain when injected subdermally (10). PGE; also sensitizes pain receptors for subsequent infusions of hista- mine or bradykinin (l7): i.e. concentrations of these auto- coids which did not ellicit pain in untreated skin were very painful after treatment with PGEl. In cats and rabbits (70) PGE; induces a dose-dependent pyresis with doses up to 10 ug. Thus. prostaglandins, especially PGEI. reproduce many of the cardinal signs of inflammation. PGE, can also affect net fluid filtration. Weiner and Kaley (104) have shown that in the rat mesocecum prep- aration in vivo, topical applications of PGE; in doses of l to 10 ug/ml, produces a dilation of metarterioles, pre- capillary sphincters and venules concomitant with an aug- mentation of capillary blood flow leading to increased fluid filtration. However considerable debate exists among inves- tigators concerning the relative capacity to which PGE; is involved in the movement of fluid and macromolecules across 20 the microvascular membrane. The different experimental techniques employed. different PGE; doses and routes of administration, species variability as well as other factors. may have contributed to the conflicting data found in the literature. Many investigators have employed the vital dye technique which involves the injection of a vital dye (e.g. Evans Blue) into the systemic circulation of the experi- mental animal and the subsequent measurement of the amount of dye leakage at the site of administration of the prosta- glandin The injection site is usually skin. The dye is assumed to bind to circulating plasma proteins and will therefore be transported with filtered proteins. Changes in microvascular permeability are quantitated by either measuring the diameter of the dye-leakage in the area of application. or more precisely. by recovering the extra- vasated dye by extraction from the tissue and measuring the concentration of the dye spectrophotometrically. A modifi- cation of the method involves the intravenous injection of readio-actively labelled albumin and the subsequent measure- ment of the radioactivity of the extravasated protein at the site of prostaglandin injection. However. the vital dye technique has three major drawbacks. l) The extent to Which the dye binds to the plasma protein is variable and uncertain. 2) The concentrations of PGE1 at the site of leakage are not well controlled. Depending on the rate of penetration and diffusion of PGE; in the tissue, 21 concentration differences could occur. 3) The injected PGE; would only have short-lived effects owing to the rapid metabolism of the agent by degradation enzymes located in the tissue. Thus it is not surprising that there is a di- versity of opinion among investigators utilizing this tech- nique. In 1963. Horton, using the vital dye procedure. re- ported that PGE, increased permeability in guinea pig skin (40). Since that time. other investigators (11.12.19.20,47, 74) have also shown increases in vascular permeability in response to PGE; in both rat and guinea pig. Kaley (47) noted increases in permeability, especially in venules. while Panagides and Tolman (74) observed a direct relation- ship between the dose of PGE, used and the average diameter of the lesion. Another group of investigators utilizing the vital dye technique. have reported that while PGE, itself does not alter vascular permeability. it potentiates the effects of bradykinin and histamine (17.57.100.107,108). This potentiation can be inhibited by indomethacin pretreat- ment (7). Using the more reliable hamster Cheek pouch prep- aration (as described above). Svensjo (95) and Joyner, Svensjo and Arfors (45) have shown that PGEl. in doses ranging from 1 to 100 ng, increases the leakage of FITC- dextran from postcapillary venules in a manner similar to that of histamine and bradykinin: i.e. via formation of interendothelial venular gaps. The number of leakage sites 22 was linearly related to the amount of PGE, administered. but PGE; was found to be at least ten times less effective than bradykinin in producing FITC-dextran leakage sites. PGE; also potentiated the response to bradykinin (95). Some in- vestigators (108) have suggested that PGE; only increases protein efflux due to its ability to increase blood flow. However, when terbutaline, a B-agonist. was administered together with PGEI. arteriolar blood flow was increased to levels even greater than those achieved with PGE; alone. while the number of leakage sites decreased. Panagides and Tolman (74) also noted that isoproterenol. a B-agonist, in- hibited the PCB; evoked increase in permeability, although it too increases blood flow. Thus it appears that the in- creases in permeability attributable to PGE; are not due solely to increases in blood flow. The antagonism of the increased macromolecular permeability by B-agonist has been postulated to be due to the ability of the B-agonists to re- lax the contractile elements of the venular endothelial cells, and thus close the interendothelial gaps (97). The evidence discussed above suggesting a role for PGE, in inflammation has been from studies conducted in rats, guinea pigs, and hamsters. So far. the few studies conducted in dogs have not provided substantial evidence to support this contention. Rosenthale (83) injected PGE; into the dog knee joint and it became inflamed. Greenberg and Sparks (27) studied the effects of PGE; on the vascular resistance, capacitance and capillary filtration coefficient 23 in the isolated canine hindlimb by measuring the total venous outflow of the popliteal vein and the changes in pleythysomographic recordings in an enclosed hindlimb. They found that the intravenous administration of 0.01 to 10 ug/ min of PGE, increases blood flow and vascular volume which is evidence for an increase in vascular capacitance. PGE1 also increased the capillary filtration coefficient. indi- cating either an increase in capillary permeability or an increase in capillary surface area due to increased relaxa- tion of precapillary sphincters. They postulated that the predominant effect of PGE, infusion was a decreased pre- capillary sphincter tone (or increased relaxation), since there was no net filtration associated with the increased capillary filtration coefficient. and the relative changes in resistance and the capillary filtration coefficient were similar in magnitude to those observed during exercise when there is no increase in capillary permeability. Daugherty (13) studied the effects of PGE, on skin and skeletal mus- cle vascular beds in the dog forelimb and observed little effect on filtration. In these studies, PGE, (2 to 10 ug/min) infused into the brachial artery produced large increases in both skin and muscle blood flows whereas total vascular resistance in skin and muscle decreased. However, the small effect on filtration. as measured by forelimb weight, was postulated to be due to a proportional dilation of the pre- and post-capillary vascular segments. 24 The above studies did not measure protein fluxes directly nor did they study the effects of a wide dose range of PGE1. It has been shown (18) that large amounts of PGE; can be inactivated in a single passage through the lungs and liver (39). Thus the doses used in the above studies may have been insufficient. The duration of the infusions were also very short. Recent work by Joyner (43) afforded a direct measure- ment of protein fluxes in the hindlimb of the dog. Subcutan- eous injections of 0.01 ug of PGE, failed to show increases in vascular permeability as ascertained from the protein concentration of collected lymph. Joyner proposed that PGEl enhanced transcapillary fluid movement. as evidenced by an increased lymph flow. primarily by its vasodilatory proper- ties. But Joyner himself raised the issue as to the accura- cy of the study. Lymph samples were collected at 30 minute intervals and, if the increase in permeability was of a short duration. as was likely with the low doses employed. the permeability effects could have been masked. Moreover. in the same study. when the vital dye technique was used in the dog, PGE, (10-20 ug administered intradermally) produced bluing in 71% of the trials. Recently. a systematic study of the effects of a wide dose range of PGE; on fluid filtration and macromolecu— lar efflux was conducted in our laboratory (58). Sixty min- ute local intra-arterial infusions of PGE, (2 to 16 ug/min) into canine forelimbs perfused either naturally or at constant 25 inflow produced profound vasodilation and increases in lymph total protein concentration. but only slight dose-independent increases in lymph flow. PGE1 (16 or 32 ug/min i.a.) also produced marked increases in forelimb weight owing to edema formation. Following pretreatment with indomethacin (5 mg/ kg, i.v.), PGE, produced vasodilation and increases in lymph total protein concentration similar to those produced in the absence of indomethacin, but now produced marked increases in lymph flow rate under both natural and constant inflow conditions. Based on these data in the dog and similar findings in other species, it appears that PGE; is likely to have a role in inflammation and increases vascular permeability in a manner similar to that of histamine and bradykinin. Work from this laboratory (29.59.65) and others (98) has demon- strated that catecholamines possess the unique ability to antagonize the protein efflux produced by histamine and bradykinin owing to a direct action on the microvascular membrane which counteracts that of histamine and bradykinin. This antagonism is independent of changes in blood flow, microvascular pressure and perfused surface area (29.51.65). It is now known that other agents possess the ability to an- tagonize or potentiate the protein efflux produced by these agents (29.59.78). In this study, the effects of catechol- amines and a variety of other natural and synthetic agents were studied to determine if they antagonize or potentiate the direct actions of PGE; on the microvascular membrane. METHODS Thirty-five mongrel dogs of either sex. having an average weight of 27 kilograms (range: 17—45 kg). were anesthesized with sodium pentobarbital (30 mg/kg) and venti- lated with room air using a Harvard respiratory pump. In these studies. the intact canine forelimb per- fused at constant inflow was used to collect skin lymph and measure lymph protein concentration. The surgical procedure consisted of using an electrocautery to make small incisions superficial to the brachial artery, cephalic vein (5 cm below the elbow), and second superficial dorsal metacarpal vein in the right forelimb. A side branch of the brachial artery. a lymph vessel. and a vein, respectively, were isolated. After administering 10.000 U.S.P. units of heparin intra- venously, these vessels were cannulated in an upstream di— rection with polyethylene tubing for monitoring brachial artery perfusion pressure, lymph collection, and small skin vein pressure. respectively. The side branch of the brachial artery was cannulated with PE-50 tubing while the small vein in the paw was cannulated with PE-60 tubing. The cannulated small vessel acts as an extension of the catheter, and thus the catheter measures pressure in the vessels to which the cannulated vessel connects (30, 33, 34. 86), which is 26 27 representative of all small venous vessels in the skin of the paw. The lymph vessels in the area of the cephalic vein below the elbow drain forelimb skin and paw (36.69). Two or three were usually tied centrally and one of them was cannulated distally with a 10 cm length of PE-10 tubing which had been beveled at the cannulating end. The walls of these vessels were quite substantial requiring that they be punctured with a 22 gauge needle prior to cannulation. The brachial artery was then isolated, tied off and transected about 5 cm proximal to the side branch which had been cannulated. Blood was obtained from a cannula inserted into the femoral artery and pumped at a controlled flow into the transected brachial artery. A Sigmamotor pumb (Model T68H. Sigmamotor Inc., Middleport, N.Y.) was used to keep inflow constant at a value which produced a perfusion pres- sure similar to aortic pressure. Aortic pressure was moni- tored via a cannula in the left carotid artery. All pressures were monitored with Statham pressure transducers (Model P23Gb, Statham Instruments. Inc., Oxnard, California). connected to a direct writing oscillograph (Model 7754A, Hewlett-Packard Co., Palo Alto, California). Lymph was collected at 10 minute intervals in mini- ature 0.3 ml graduated cylinders. constructed from plastic pipettes. Forelimb small skin vein pressure, aortic pressure. and brachial artery perfusion pressure were continuously mon— itored and recorded at the end of each 10 minute period. 28 After two consecutive control periods local (intra-arterial) administration of PGE, and/or other drugs was begun by in- fusing directly into the circuit behind the Sigmamotor pumb. Arterial blood samples (5 ml) for measuring hematocrit and plasma proteins were drawn from the cannula monitoring aortic pressure. Samples were taken five minutes before the local infusions of drugs began, as well as at 30 minute intervals throughout the experiment. The protein concentration of the lymph and plasma samples were analyzed by the modified Biuret reaction (51). The samples were read spectrophotmetrically in grams percent with an ACCU-STAT Blood Chemistry Analyzer (Clay Adams, Model 2000) which had been calibrated with samples known protein concentrations. Lymph protein trans- port was calculated as follows: Lymph Protein Transport = (10)(Lymph Flow Rate)(Lymph Protein Concentration). PGE1 was obtained from the Upjohn Company. Kalamazoo. Michigan (U-10136. Lot No. 12874-JHK-1020 and ll894-VOV-77). Stock solutions were prepared by dissolving 10 mg of PGE, into 10 ml of absolute ethanol. Appropriate dilutions were made up using normal saline as the solvent. and the PCB, was then in- fused into the animal at a rate of 16 ug/min for 60 minutes. The following drugs were also used in this study and prepared in normal saline: 1) isoproterenol hydrochloride. 3 ug/min infused for 60 minutes, Winthrop Laboratories: 2) vasopressin. 0.8 Pressor Units/min infused for 60 minutes. Calbiochem Co., Inc.: 3) angiotensin II, 2 ug/min infused for 60 minutes. Sigma Chemical Co.: 4) methylprednisolone 29 (Solu-Medrol). l5 ug/min infused for 60 minutes. Upjohn Co.: 5) serotonin creatinine sulfate. 7 us base/min infused for 60 minutes, Sigma Chemical Co.. All drugs were administered intra-arterially at a delivery rate of 0.2 ml/min with a Harvard infusion/Withdrawal pump. Control infusions of eth- anol alone. in the same preparation, produced no significant changes in any of the measured parameters. At the conclusion of the experiment the animals were sacrificed. The right and left forelimbs were then severed approximately 2 cm above the humeral condyle. and the brachi- alis, biceps, and triceps muscles were carefully dissected down to their tendons of insertions on the ulnar and radial tuberosities. Great care was taken to insure that the limbs were always severed at the same points on the humeral condyle. The limbs were then exsanguinated and weighed. Limb weights (experimental vs. contralateral control) were compared by using a paired t test. Control infusions of ethanol alone. in the same preparation, produced no significant changes in limb weights. thus neither the ethanol vehicle nor the trauma produced by surgery were responsible for the significant in- creases in limb weights seen in dogs with significantly in- creased lymph total protein concentrations. Moreover, when lymph total protein concentration did not significantly in- crease. the experimental and contralateral control limb weights were always very similar. Thus, this method is a valid means whereby changes in limb weights may be detected. All other data were statistically analyzed by Analysis of Variance (Ran- domized Complete Block Design) and the means compared to con- trol by the Least Significant Difference Test (93). RESULTS Table 1 In limbs perfused at constant inflow, intra-arter- ially infused PGE1 (l6 ug/min) produced a moderate increase in lymph total protein concentration (p<.01). Lymph flow rate increased slightly but not significantly (p>.05) by the 20 minute sample period and then waned. There was a tendency for a slight but insignificant (p>.05) increase in lymph protein transport. Limb weights were significantly greater (p<.01) in the experimental limbs when compared to the con- tralateral control limbs. Plasma protein concentrations were not Changed while the hematocrits were significantly increased (p<.01). Systemic arterial pressure increased significantly (p<.01) and remained elevated throughout the infusion period. Perfusion pressure decreased markedly (p<.Ol) and small skin vein pressure increased significantly but minimally (p<.01) toward the end of the infusion period. Table 2 Table 2 shows the effects of isoproterenol (3 ug/min. i.a.) infused concomitantly with PGE, (l6 ug/min, i.a.) into limbs perfused at constant inflow. Lymph flow rate. lymph protein concentration and lymph protein transport were un- changed. Systemic arterial pressure and small skin vein 30 31 pressure were not significantly altered. Perfusion pressure was markedly decreased (p<.01) for the duration of the infu- sion period. Plasma protein concentrations were not changed while the hematocrits were significantly increased (p<.01). Table 3 No changes in lymph flow rate or lymph protein trans- port were seen when vasopressin (0.8 Pressor Units/min, i.a.) was infused concurrently with PCB, (16 ug/min. i.a.) in fore- limbs perfused at constant inflow. Lymph total protein con- centration minimally decreased (p<.01) and then returned to control levels by the end of the infusion period. The weights of the experimental and control limbs were not significantly different. Systemic arterial pressure was significantly in- creased (p<.01) at the start of the infusion period and then waned, returning to control levels. Perfusion pressure and small skin vein pressure were unchanged. Plasma protein con- centrations and hematocrits were not significantly different from control. Table 4 In limbs perfused at constant inflow. angiotensin II (2 ug/min, i.a.) in combination with PCB, (16 ug/min. i.a.) did not change lymph flow rate, lymph total protein concen- tration or lymph protein transport. The weights of the per- fused limbs were moderately greater than those of the contra— lateral control limbs (p<.05). Systemic arterial pressure increased at the onset of the infusion period and gradually waned. Perfusion pressures significantly decreased (p<.01) and small skin vein pressure remained unchanged. Plasma 32 protein concentrations were not changed while the hematocrits were significantly increased (p<.01). Table 5 No changes in lymph flow rate. lymph total protein concentration or lymph protein transport were seen when methylprednisolone (15 ug/min, i.a., CF) was infused concur- rently with PCE1 (16 ug/min. i.a., CF). The weights of the experimental and control limbs were not significantly dif— ferent. Systemic arterial pressure increased (p<.01) while perfusion pressure decreased markedly (p<.01). Small skin vein pressure remained unchanged. Plasma protein concentra- tions were not Changed while the hematocrits increased sig- nificantly (p<.01). Table 6 Table 6 shows the effects of serotonin (7 HS base/ min. i.a.) infused concomitantly with PGE, (l6 ug/min, i.a.) into a limb perfused at constant inflow. Lymph flow rate in- creased slightly but not significantly (p>.05) by the 20 min- ute sample period and then waned. Lymph protein concentration increased significantly and remained elevated throughout the infusion period. There was a tendency for a slight but in- significant (p>.05) increase in lymph protein transport. The weights of the perfused limbs were markedly greater than those of the contralateral control limbs. Systemic arterial pressure decreased minimally but not significantly while perfusion pressure was moderately decreased. Small skin vein pressure increased moderately. Plasma protein 33 concentrations were unchanged while the hematocrits increased significantly. Thus, PGEI. and concomitant infusions of serotonin and PGE1, produced significant increases in both lymph pro- tein concentration and limb weight. 0f the other four agents, only angiotensin II. by producing a moderate increase in limb weight. significantly altered either lymph total protein con- centration or limb weight. Generally. systemic arterial pressure and hematocrit were significantly increased. 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AN. phcmmscha shewwhm0%hsss *s.m *0.0 sh.s *s.m 00.0 0.0 0.0 s.0 shcpchm Hcpca seeps Ashe oH\asv so. 00. no. 00. 00. so. so. so. cpcm scam amass Ammssv ssh ssh ssh ssh xmh sh 0h 0h ehsmmehm che> mwcsmvshhm 285 :00 *s0 #00 s00 *H0 :00 0Hh 0hh chsmmehmchthhhem $88 0hh mph mhh mph sap ohh 00h 00H ehsmmchm hchhcphs cheepmhm 00 00 00 00 00 0011 0 oh- UOHHmm SOMmSMHhH HOHPGOU Amucv .mpnwpmz papa chm .pphoopdEm: .mthmmmhm hmHsomm> .mhoppmhphmohoo hpmpohm mammam 0cm AQSSH .phommhmhp hpmpOhm chm Soap Shaka ho Amo ..m.p hp£\m00p w: Av hphopohmm chm Amo ..m.p :Ha\m: 0H0 "mom mo mpompmm .0 mapme DISCUSSION In forelimbs perfused at constant inflow (Table l). PGE, (l6 pg/min i.a.) infused for 60 minutes significantly increased lymph total protein concentration and limb weight. Small skin vein pressure remained unchanged relative to con- trol until gig 49, by which time lymph protein concentration had significantly increased. Thus the edema produced by PGE, in a constantly pump-perfused forelimb is not attributable to an increased microvascular pressure. but to an increased net fluid filtration subsequent to a decrease in the transmural colloid osmotic pressure gradient owing to an increase in microvascular permeability to macromolecules. This conclusion is supported by the work of Svensjo (95) and Joyner g1 g1. (45). These investigators have shown that PGE, increases the leakage of FITC-dextran (M.W. 145.000) from postcapillary venules in the hamster Cheek pouch prep- aration in a manner similar to that of histamine and brady- kinin (99): i.e. via formation of interendothelial cell ven- ular gaps. These gaps form in venules 9 to 16 um in diameter, owing to the active contraction of endothelial cell filaments. Thus. PGE, increases macromolecular efflux by increasing vas- cular permeability in the venular section of the microvascu- lature. 40 41 It has recently been suggested by Williams and Peck (108) that PGE; has no direct effect on permeability but simply increases blood flow and thereby potentiates the leak- age of macromolecules produced by histamine and bradykinin. However, in this study PGE; has been shown to increase protein efflux under constant inflow conditions which would not allow any increase in total blood flow. When the B-agonist isoproterenol (3 ug/min i.a.) was infused concomitantly with PGE; (Table 2), it failed to in- crease lymph total protein concentration. Joyner gt g1. (45) has shown that terbutaline, a B-agonist, markedly reduces the macromolecular leakage evoked by PGE; in the hamster cheek pouch preparation. The edemogenic effects of histamine (29. 65) and bradykinin (59) are also antagonized by isoproterenol. Although isoproterenol markedly increases nutritional flow (82), the simultaneous infusion of isoproterenol with these autocoids intra-arterially into the canine forelimb prevents the marked increase in protein efflux usually produced by histamine and bradykinin (59,65). Norepinephrine also antag- onizes the increase in microvascular permeability produced by these autocoids (29.59.65). This antagonism of the histamine and bradykinin protein efflux is completely independent of changes in blood flow, microvascular pressure. and perfused surface area (29.51.65). Instead, it reflects a direct action on the microvascular membrane which counteracts that produced by histamine and bradykinin. This conclusion is supported by morphological data which demonstrates that the marked increase 42 in venular leakage sites of FITC-dextran (M.W. 145,000) evoked by histamine and bradykinin is greatly reduced by the simultaneous application of catecholamines (98). Similarly, the antagonism of the PGEl-induced pro- tein efflux by the B-agonists is completely independent of changes in blood flow, microvascular pressure, and perfused surface area. Both PGE, and isoproterenol are potent vaso- dilators which are expected to increase surface area and. under constant inflow conditions, fail to decrease skin small vein pressure and, inferentially, microvascular pressure. Thus. as with histamine and bradykinin, isoproterenol and terbutaline may physiologically antagonize the action of PGE1. presumably by causing the relaxation of the actinomyosin-like filaments of the venular endothelial cells. thus closing the interendothelial gaps. It is interesting to note that, like histamine and bradykinin, PGE, only produces increases in macromolecular permeability in doses exceeding those necessary to produce significant vasodilation. Subcutaneous injections of PGE1 in doses as low as 10 ng/ml produce profound erythmea (26), and Daugherty (13) found that only 1-2 ug infused intra- arterially in the canine forelimb preparation produced maxi- mal vasodilation. Thus, as with some of the other ubiquitous prostaglandins. PGE, may have a physiological function, such as local regulation of blood flow. in addition to its role as a mediator of inflammation. 43 The increase in macromolecular permeability produced by PGE, is considerably less than that produced by the other two autocoids. This has been noted by other investigators in the rat (102) and hamster (45). In the present study PGE; produced an average maximal increase in lymph total protein concentration of approximately 1 gm %, whereas high concen- trations of histamine and bradykinin increase the total pro- tein concentration of lymph to values approaching that of plasma (29.59). The more modest increases in vascular per- meability attained with large doses of PGE; suggest that the inability of other investigators (13.27) to demonstrate an effect of PGE1 on macromolecular permeability in the dog may be due to the dose studied. It has been shown that large amounts of PGE, can be inactivated in a single passage through the lungs and liver (18,39). In addition. these investigators failed to directly measure protein efflux. and therefore it is possible that the quantitatively smaller changes in fluid flux were not easily measurable with the techniques they em- ployed. However, results from numerous other studies (11,12. l9,20.45.74,95) in rats, guinea pigs. and hamsters. are con- sistent with the findings of this study. PGE, differs from histamine and bradykinin in yet another respect. Despite the increase in lymph total protein concentration, lymph flow rate (Table 1) does not significant- ly increase as it does with these other two autocoids (35,52. 64,65). Changes in lymph flow rate have traditionally been considered to reflect changes in transvascular fluid fluxes. 44 and increases in net fluid filtration generally produce roughly proportional increases in lymph flow rate. For in- stance. with low doses of bradykinin (0.8 ug base/min) in- fused locally intra-arterially into canine forelimbs perfused at constant inflow, a comparable increase in lymph total pro- tein concentration (e.g. 1 gm %) produced a 10 to 15-fold in- crease in lymph flow rate (59). In the present study, only a 3 to 6-fold increase in lymph flow rate was observed (Table 1). Since drainage of the limb was somehow impeded it would be ex— pected that for a comparable increase in lymph total protein concentration. the PCB, limbs would be more edematous than the low-dose bradykinin limbs. Indeed, the PCB; limbs were 60% heavier than the bradykinin limbs were, when comparable increases in lymph total protein concentration occurred (59). Following pretreatment with indomethacin (5 mg/kg, i.v.), PGE, (l6 ug/min) infused locally intra-arterially in- to canine forelimbs perfused at constant inflow (58) produced a significant increase in lymph total protein concentration similar to that produced in the absence of indomethacin, but under these same conditions produced a 10 to 15-fold increase in lymph flow rate as would be expected from the bradykinin data. Thus it appears that the PGE, causes the liberation of some endogenous substance which constricts the prenodal lymph vessels impeding drainage of the interstitium and compounding the edema. Since indomethacin blocks this constriction and restores normal lymph drainage. it is proposed that the endo- genous agent involved is another prostaglandin. It is inter- esting to speculate as to the identity of this agent. Both 45 thromboxane A2 and PGFaa can constrict vessels. and PGFaa has been shown to antagonize the increases in vascular per- meability elicited by PGE, (12.74). This agent may also have contributed to the unusual variability of the data found in the literature. Additional experimentation is needed to resolve these issues. Other substances which are released into the circula- tion during hemorrhage have been found to antagonize the protein efflux evoked by histamine and bradykinin (29.59.78). The simultaneous infusion of vasopressin (0.8 Pressor Units/ min i.a.) with PGE, (Table 3) prevented the increase in lymph total protein concentration and limb weights. as it prevented the increase in vascular permeability induced by histamine and bradykinin. However. angiotensin II (2 ug/min i.a.), which does not block the edemogenic effect of brady- kinin. does block the increase in lymph total protein con- centration produced by PGE, (Table 4). Apparently the 2 ug/min dose of angiotensin II was near the effective threshold dose for antagonizing the PGEl-induced protein efflux, as two of the seven dogs showed increases in lymph total protein concentration and limb weight. Hence. there was a slight but significant increase in limb weight for this group. However. lymph total protein concentration was not significantly increased indicating an inhibition of the protein efflux by angiotensin II. Low doses of methylprednisolone (l5 ug/min i.a.), a glucocorticoid, (Table 5) also block the edemogenic action 46 of PGE; although only a much larger dose (30 mg/kg i.v.) is effective in preventing the increases in macromolecular permeability elicited by histamine and bradykinin (29.78). Seeing that the more modest increases in vascular permeability elicited by PGE; were also more susceptible to inhibition by other vasoactive substances than histamine or bradykinin were, serotonin (7 ug base/min i.a.) was infused simultaneously with PGEI. This dose did not block the increase in lymph total protein concentration or limb weight. Lymph flow in- creased slightly but not significantly, to levels similar to those achieved when PGE, was infused alone. A higher dose of serotonin (15 ug base/min i.a.) does prevent the edemo- genic effects of histamine and bradykinin (29,78), and so it is likely that the dose of serotonin used in this study was simply insufficient to inhibit the actions of PGE1. Further experimentation is needed to determine whether serotonin. in higher concentrations. in fact inhibits the edemogenic effects of PGE1. The antagonism of the PGEl-induced protein efflux demonstrated in this study by a number of agents cannot be simply attributed to decreases in blood flow and/or perfused surface area. No changes in blood flow were possible as all experiments occurred under constant inflow conditions. Iso- proterenol increases perfused surface area (82) and yet this catecholamine antagonized the edemogenic actions of PGE1. Changes in vascular pressures were not responsible either. Methylprednisolone blocked the PCB, response even while per- fusion pressure was extremely low, while serotonin did not 4? block the development of edema with perfusion pressures that were relatively high. Vasopressin antagonized the PGE; effect but produced no Change in perfusion pressure. Skin small vein pressures and. inferentially microvascular pressures, were unchanged in all groups except for a small increase in the serotonin group due to this agent's ability to constrict skin small veins (24). Thus, it must be concluded that the antagonism of the PGEl-induced protein efflux by isoproterenol, vasopressin. and low doses of methylprednisolone represents a direct action of these agents on the microvascular membrane. The antagonism by angiotensin II may be due to a direct action on the microvascular membrane. or may be due to an indirect effect owing to its ability to release catecholamines from the adrenal medulla and adrenergic terminals (24). Local intra-arterial infusions of PGE1 (l6 ug/min), PGE, and vasopressin (0.8 Pressor Units/min), PGE; and angiotensin II (2 ug/min). and PGE; and methylprednisolone (15 ug/min) produced significant increases in systemic arterial pressure. Local intra-arterial infusions of PCB, and isoproterenol (3 ug/min) and PGE, and serotonin (7 ug base/min) produced no change in systemic arterial pressure. Under these conditions all groups showed significant increases in hematocrit except one. Vasopressin did not produce a significantly increased hematocrit. These results can best be explained if PGE, causes an increase in sympatho-adrenal activity. Others have shown that PGE1 causes a reflex in- crease in systemic arterial pressure and heart rate (50.73) 48 which can be blocked by pretreatment with ganglionic block- ing agents. PGE1 does not have a direct action on the adrenal medulla (68) and studies have shown that PGE; in- hibits the release of norepinephrine from adrenergic term- inals (38.46.85.105). The pressor response does not occur in dogs with their adrenal glands excluded from the circu— lation (50). Thus it is suggested that the increases in systemic arterial pressures and hematocrits may be due to catecholamine release from the adrenal medulla. However, we have no data to suggest what the stimulus for the sympatho- adrenal discharge might be as mean arterial pressure did not fall, and PGE, does not cause a release of adrenal catechol- amines. Histamine produces a similar vasodilation, and yet no increase in systemic arterial pressure occurs although it causes a direct release of catecholamines. Perhaps large doses of PGE; have a direct stimulatory effect on central vasomotor neurons which produce an increase in sympathetic outflow. Further experimentation is needed to resolve this issue. In general. the present study shows that PGE, in large doses increases macromolecular permeability in a manner similar to that of histamine and bradykinin. This data correlates well with morphological data available from Svensjo's laboratory. The increases in vascular permeability produced by PGE, are more modest than those produced by histamine and bradykinin, and are susceptible to antagonism by a greater number of other vasoactive agents. None of the 49 agents studied measurably potentiated the protein efflux produced by PGEI. Lymph flow rate failed to increase significantly despite the increase in vascular permeability induced by PGE1. Serotonin did not block the increased lymph total protein concentration produced by PGEl. and it too failed to significantly increase lymph flow rate. Local intra-arterial infusions of PGE, (l6 ug/min) produced significant increases in systemic arterial pressures and hematocrits. suggesting that PGE, causes an increase in sympatho-adrenal activity, perhaps via a direct action on central vasomotor neurons. SUMMARY AND CONCLUSIONS The canine forelimb perfused at a constant inflow was used to examine the effects of local intra-arterial infu- sions of PGE1, and to determine if catecholamines and other vasoactive agents antagonized or potentiated the actions of PGE; on the microvascular membrane. The results indicate that large doses of PCB, (16 ug/min i.a.) produced profound vasodilation and increased macromolecular permeability by mechanisms similar to that of histamine and bradykinin. as evidenced by marked increases in lymph protein concentration and forelimb weight owing to edema formation. This data correlates well with morphological data from other labora- tories. Lymph flow rate did not Change relative to control. The increases in lymph protein concentration and forelimb weight elicited by PGE, are more modest than those produced by histamine and bradykinin and are susceptible to inhibition by isoproterenol, vasopressin, angiotensin II. and methylprednisolone. The antagonism of the PGEl-induced pro- tein efflux by these agents is independent of changes in blood flow and vascular pressures, and may represent a direct action of these agents on the microvascular membrane. In contrast to the other four agents. concomitant infusions of serotonin and PGE1 produced increases in lymph 50 51 protein concentration, lymph flow rate, and forelimb weight similar to those achieved with PGE, alone. Thus serotonin failed to antagonize the PGEl-induced protein efflux. None of the agents studies potentiated the protein efflux pro- duced by PGEI. Local intra-arterial infusion of PGE, pro- duced significant increases in systemic arterial pressures and hematocrits, suggesting that PGE, causes an increase in sympatho-adrenal activity. In summary, these results in- dicate that PGE, may function as a mediator of inflammation in the dog, and that some natural or synthetic chemical agents can antagonize its effect on microvascular permeability. 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 A1 A2 A3 A4 A5 O\U‘t «PU N l-‘ A6 52 H a a h h h H o .mmh (Mop xma 0h 0H NH sh as 0H 0H sh 0H ma 0h 0h ha sh 0h sh 0h 0H ah oh oh 0H 0h 0H 0H 0h oh 0h sh 0H 0H 0H 0H 0h sh «H as 0h 0h mp 0H 0h 0h hp hp 0h 0h 0h 0h 0H 0h 0h 0h 0h mp 0h 0h 0H NH 0h pp 0 0 0 m m s s 0 *mm * 0 *mm *WM *0m #00 OHH moa 00 001 001 00 00 00 mph mph 00 00 00 00 00 00 mop 00H 00 00 00 00 00 00 00h ohh 00 00 00 00 0s 0: 00h 00H 00 00 00 00 00 00 00h 00h 00 00 00 00 00 00 00 00H 00 00 00 00 00 00 ohh 00h my 0 0 s s s s 0 a sash shah shah shad sons toms 00h 00H 00h 00h 0hp opp mph ssh 0Hh 0hh 00H 00H 00h 00H map was 0hh 00H 00h 00h 00h 00h 00h 00h 00h 00h 00H 00h 00h 00h 00H 00h 00h 00h 00h 00h 00h 00a 00a 00h 00h 00H 00h 00h 00h 00h 00H 00h 00h 00h 00h 00h 00h 00h 00H 00H 00h 00H 00 00 00 on 00 oh 0 oh- .004.”th COHmSHCH HOHPCOU hohhm chmphmpm mgmfi + Ammssv mhsmmmhm :pm>.HHmEm hpxm hOhhm chmchMpm whame +l Awmssv mhsmmmhm hopmSphmm hohhm chmchmpm mhMmE + Ammssv thmmmhm amphmph< opEmpmzm Amuhv .mpnmpms papa 0cm .pphoopmem: .mmhsmmmhm hmasomm> .mhoppmhphmohoo hpmpohg mammam 0:0 ahead .phommhmhp hpmpohm 0cm Soap Shaka ho Amo ..m.p hpfi\wa 0H0 hmum mo mpomppm .H< manna 54 on. p0. 00. 00.p op.0 0o.p 0p. 00. 0m. 00. 0o.p 00.0 00.0 p0.p 00. .mm. 0 .0 00.0 00.0 00.0 00.0p 00.0 00. 00. 00.p 00.p 00.0 00.0 00.0 00. 00. p0. 00. 00. p0. 00. p0. 00. 00. 00. 00. on. 00. 00.p oo.p 00. 00. 00. 00. p0. 00. 00. 00. 00. 00. 00. 0p. 0p. 0p. 0p. 00. sp. 00. p0. 0m. 00. 0s. 00. 00. 00. 0o.p 00. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 *0.0 l .0 0.0 p.0 0.0 0.0 0.0 0.0 .0.0 0.0 0.0 p.w 0.0 men 0.0 0.0 0.0 p.0 0.0 0.0 0.0 0.0 p.0 0.0 0.0 p.0 0.0 p.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 p.0 0.0 0.0 0.0 0.0 0.0 0.0 0.p 0.p 0.p 0.p 0.0 s.p 0.0 p.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 po. po. po. 00. 00. 00. oo. oo. 00. 00. mo. 00. so. 00. 00. po. 00. 00. 00. mp. 00. 0p. 00. 0o. 00. 0o. 00. 0p. op. p0. po. p0. p0. po. po. p0. po. p0. po. po. 00. po. 00. 00. 0o. 00. po. 00. p0. po. p0. po. po. po. po. po. po. po. p0. po. po. po. po. po. po. po. 00. 00. 00. 00. 0o. 0o. 00 00 00 00 00 op o op- UOflhmm SOHmSMQH HOHPGOU hohhm chapsmpm mamma +I Epe Sass phonmCMhe hpmpohm neezp hohhm 0hmchmpm mamms + as s00 hpmpOhm Hmpos cheap hOhhm chmchmpm i mamms + pups ofi\psv mpmm 30pm cheap .ocssppcco .p< spews 55 0 0 p a. m .13 mm mm 0m 0n n: m: 00 0: 00 0: s0 00 0m 00 00 00 N0 mm mm mm mm 00 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 p.0 0.0 0.0 0.0 p.0 0.0 0.0 0.0 p.0 0.0 0.0 0.0 0.0 p.0 0.0 0.0 p.0 0.0 pm on 00 on, 00 op o op- vophmm COpmsth HthcOu hOhhm chmthpm mhmme + pphoopdfimm hohhm chmthpm whame + as s00 hpmpohm memmpm .oessppcco .p< epnee hams map 0o hohhm chaphMpm n hams m0 0mmmthXm 0p00 mspp ohms op m>ppmamh 00. v max + pcwpms papa pohphoo awhmpmpwhphoo op m>ppmpmh H0. v mutt mapp ohms op m>pp0pmh H0. v mu: 56 It * VVMCDVVOCDVWODp QWWNI—IOONM u-i NMNNN mohmhmmppm papthphmnxm 0.0N hohhm Uhmuhmpm n n.0mm whame + . 0 0.p00 0.000 0.000 0.000 pmswv 0.p00 hopmsmhp has 00 hmppm 0.0ps pampcs espp wmmmmmm .ocssppsco .p0 cpnea 57 N H N H H H m 3 NHl. NH HH HH HH NH NH “H “H OH lWH WH DH WH WH #H OH OH OH OH O NH mm OM m m m m m m 0H 5 NH HH HH HH HH HH HH HH MH MH NH HH HH HH NH NH op op 0p pp pp 0 0 s #00 #00 #50 #00 *mm #00 AHH #HH 00 00 0m 00 (WW1 OW OOH WNH WOH mOH WHH OHH OHH mm OHH OHH 00 00 00 00 CO 00 OMH ONH on w: on on 00 m: 00 om mm mm mm 00 00 00 mNH mNH m m m m m m m m OWH OHM DNH OMH MMH NNH NMH MMH NéH 03H 03H .WWH 03H .lWMH 03H OOH mmH OOH OOH OmH NdH OJH OMH OMH OOH OOH OOH OOH NWH de 53H NdH mNH ONH OMH MNH ONH BHH OHH OHH WHH WHH OHH mOH MOH OOH MNH “NH OM Oml ON on ON OH O OHI 0ophmm :OpmSp:H Hohp:oo hohhm 0h00:0pm 0:0me H pmmssv thmmmhm :pm> pp08m :pxm hohhm 0h00:0pm 0:0m8 H Ammssv thmmmhm :opmSMhmm hohhm 0h00:0pm mflm $8 + pmmssv mhsmmmhm p0phmph< opampmhm opus .pphoop0sm: 0:0 .mmhsmmmhm h0HSom0> .m:opp0hp:mo:oo :pmpohm 0Em0HQ 0:0 :mshp .phomm:0hp :pmpohm 0:0 30pm :mezp :o Amo ..0.p :pa\w1 mv Ho:mhmpohmomp 0:0 Amo ..0.p :pS\w1 0H0 pmwm mo mpomppm .N< mHQ0B 58 00.0 RH.0 AN.0 0H.0 mm.0 00.0 0H.0 mm.0 mN.0 :m.0 00.0 00.0 m0.0 00.0 Nm.0 00.0 NN.0 03.0 3N.0 WN.0 NN.0 mN.0 00.0 NN.0 mH.0 NH.0 0H.0 BH.0 dm.0 0H.0 HN.0 0H.0 0N.0 mm.0 mN.0 30.0 00. 00.H mm.0 Nu.0 30.0 NH.H 00.H 0N.H m0.H N0.N 0N.H #0.N MN.0 :N.0 NN.0 mm.0 HN.0 3N.0 mm.0 NN.0 N.0 N.0 N.0 N.0 m.0 m.0 m.0 m.0 d.m :.N m.N :.N :.N m.N m.N :.N N.N 0.N :.N mam N.N m.N W.N N.N w.H m.H 0.H m.H m.H 0.H H.N m.H m.m m.m 0.m N.m 0.0 0.0 m.m 0.0 m.N m.N 0.N d.N :.N H.N m.H n.H m.N :.N N.N m.N H.N :.N m.N N.N 00. 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OO. ppogmcmge :HOpopN OOEOH O.O O.O O.O O.O N.O N.O N.O O.O Hounm OHOOOOPO H N.O O.O *H.O N.O 0.0 O.N O.N N.N OOOOO N.N O.N .mwO O.N O.N N.N O.N H.O O.O 0.0 O.O N.O N.O O.N O.N O.N O.O H.O O.O O.O 0.0 O.N O.N O.N HO OOO O.O N.O O.O 0.0 H.O O.N O.N O.N N.O 0.0 N.O N.O O.O O.O O.O O.O cproam Hmpoa OmaOH HO. HO. HO. HO. NO. OO. HO. OO. nongm OOOOOOOO H OO. OO. OO. OO. OO. NO. NO. HO. OOOOO OO. NO. NO. HO. NO. HO. HO. HO. OO. OO. HO. MO. NO. OO. OO. HO. NO. NO. OO. O. OH. NO. HO. HO. NO. HO. OO. NO. NO. HO. HO. HO. AcHa OH\HaO OO. OO. OO. OO. OO. OO. HO. HO. OOOO son OOOOH Om! OO1 OO Om! ON OH O OH- OOHHmm GOHOSHCH HoHPCOO .OOOOHHOOO .OO OHOOO same may %0 Hohnm vnmocmpm H came mm vmmmmpmxw MPOO panmz QEHH HoHPCoo HOHmPMHmnpcoo op m>HHOHmH Ho. V mu** 72 waHP ohms op m>HHmHmH no. v max OEH» ohms 0H m>HHmHmH Ho. v mu* m.mm N.Om O.Nmm Hounm Ohmuqmpm H ** . .mmm m.mm mcwme O.NO w.me .O 0.00 0.000 0.000 0.00 O.NOO 0.000 AOSOO O.HN 0.0MN c.mHm :onsmcH CH8 ow Hmpmm O.HN O.HOO O.ONO HOOHOB neHH mocmhmmmHa przmsHHwQMm Hohpcoo m N Hounm Onmcsmpm I NNO mm m:OOS + NO ON Nm mm OO HO om OO NM mm HHHoopmsmm 0.0 0.0 0.0 Honum Onwoqmpm H m.m N.O 0.0 mamme 0.0 m.O O.N Om O.N mm O. N. . o N.O 0.0 H.O Q OOO N.N m.N N.N :Hmponm OSOOHm ow om ow. om 0N 0H 0 OH: OoHHmm QOHOSHQH Honpcoo .OOOOHOOOO .OO OHOOO B IBLIOGRAPHY BIBLIOGRAPHY Anggard, E. And C.-E. Jonsson. Efflux of prostagland- ins in lymph from scalded tissue. Acta Physiol. Bergstrom, S. and B. Samuelsson. Prostaglandins: Proceedings of 2nd Nobel Symposium. Almquist and Wiksell. Stockholm; Interscience Publishers, New York. 196?. Berne, R.M. and M.N. Levy. Cardiovascular Physiology. C.V. Mosby Co., St. Louis. 1977. Bloom, W. and D.W. Fawcett. A Textbook of Higtology. W.B. 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B;. E, and F3“ on superficial hand veins in man. Brit. J. Pharm. 44:374. 1972. Crunkhorn. P. and A.L. Willis. Cutaneous reactions to intradermal prostaglandins. Epit. J. Pharm. “1649-56. 19710 Crunkhorn. P. and A.L. Willis. Interaction between prostaglandins E and F given intradermally in the rat. Brit. J. Pharm. 41:507-512. 1971. Daugherty. R.M. Effects of i.v. and i.a. prostagland- in E; on dog forelimb skin and muscle blood flow. Am. J. Physiol. 220:392-396. 1971. Dipasquale, G.. C. Rassaert. R. Richter. P. Welaj and L. Tripp. Influence of prostaglandins E, and Fan on the inflammatory process. Prostaglandins. 3:741-757. 1953. Edmonds. J.F., E. Berry and J.H. Wyllie. Release of prostaglandins caused by distension of the lungs. Brit. J. surge 56 3622-623, 1969. Edwards. Jr. W.G.. C.G. Strong and J.G. Hunt. A vaso- depressor lipid resembling prostaglandin E, in the renal venous blood of hypertensive patients. J. Lab. Clin. Med. 74:389-399. 1969. Ferreira. S.H. 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