éé :3. . ROE 0F CAT-30m AND QSMO‘ Li m LOCAL BLOOD FLOW REGUlATlON AND IN HEMORRHAGE. - ' .7237 . 3.... 'Thesi for'the Degree of pm f MICHIGAN STATE UNiVERSITYf DANiEL PHILIP RADAWSKI ...;.....:31:2...3‘. 1.. .3 a . r . .11“ % ,... 3-7-10}.- 133Cfi_: I 1' RARY \ 5.92m; 53:: State . Universi 1.7 1. This is to certify that the thesis entitled The Role of Cations and Osmolality in Local Blood Flow Regulation and in Hemorrhage presented by Daniel Philip Radawski has been accepted towards fulfillment l of the requirements for - Ph.D. Physiology degree in ’-fl D Major profeM Date Febrmx 24. 1971 031639 ABSTRACT ROLE OF CATIONS AND OSMOLALITY IN LOCAL BLOOD FLOW REGULATION AND IN HEMORRHAGE By Daniel Philip Radawski The role of the cations, potassium, magnesium and hy- drogen, and of osmolality in local blood flow regulation was . examined by inducing the local regulatory responses, active and reactive hyperemia, in skeletal and cardiac muscle while simultaneously measuring the cation concentration and osmo- lality of the venous effluent from these tissues. The effect of hemorrhage on coronary vascular resistance and the cation concentration and osmolality of arterial and coro- nary sinus blood was also studied. 9222;.glggg,§;gg Regulation Active and reactive hyperemia were studied in the skele- tal muscle (gracilis) and heart of the dog. Active hyperemia was produced by electrical stimulation of motor (skeletal muscle) or sympathetic (heart) nerves. Reactive hyperemia was induced by release of arterial occlusion. Venous osmo- lality and potassium ion concentration rose above control val- ues during active hyperemia in skeletal muscle and reactive Daniel Philip Radawski hyperemia in heart but did not change during reactive hypere- mia in skeletal muscle and active hyperemia in heart. The magnesium ion concentration rose in effluent blood from the skeletal muscle and heart during active hyperemia. The hy- drogen ion concentration rose in coronary sinus blood during active and reactive hyperemia. Osmolality and resistance levels observed in exercising skeletal (gracilis) muscle were compared to those in the resting gracilis during intra- arterial infusion of hyperosmotic solutions. Comparable in- creases in osmolality produced by intra-arterial infusion in the resting gracilis failed to produce similar decreases in resistance. Increases in the hydrogen ion concentration of the magnitude observed in local blood flow regulation in skeletal muscle (l,2,3,4) were produced locally in the rest- ing forelimb and the effects on segmental vascular resistance observed. Increases in hydrogen ion concentration failed to regularly affect muscle segmental resistances, but decreased skin small vessel and large vein resistances. These findings indicate that cations and osmolality play a role in active hyperemia of skeletal muscle and reactive hyperemia of the heart and that all of these factors do not operate in re- active hyperemia of skeletal muscle and active hyperemia of heart. Hemorrhage The effects of hemorrhage on coronary vascular resist- ance and on the cation concentration and osmolality of Daniel Philip Radawski arterial and coronary sinus blood were studied in 19 dogs. A blood volume equal to 2% body weight was removed over an average of nine minutes and the effects of hypovolemia were followed for 120 minutes. Coronary vascular resistance was unaffected over the first 45 minutes but then fell from 60 to 120 minutes. Systemic and coronary sinus hydrogen and magnesium ion concentrations and osmolality rose during hemorrhage, the rise in cation concentration and osmolality in the coronary sinus preceded the rise in arterial concen- trations. Coronary sinus oxygen tension and left ventricu- lar contractile force were also measured. Coronary sinus oxygen tension initially fell (0 to 15 min) and then pro- gressively rose over the remainder of the hemorrhage period and at 120 minutes was slightly but significantly greater than the control value. Contractile force increased; the increase beginning at 30 minutes. Thus, under these experi- mental conditions coronary vascular resistance decreases only late in hemorrhage. It seems likely that increased cation concentrations and osmolality play a role in the fall in coronary resistance. 1. Kontos, H.A., and J.L. Patterson, Jr. Carbon dioxide as a major factor in the productibn of reactive hyperemia in skeletal muscle. Clin. Sci. 27:14}, 1961+. — — 2. Ross, J., Jr., C. Kaiser and F. Klocke. Observations on the role of diminished oxygen tension in functional hyperemia of skeletal muscle. Circulation Res. 15:#73. 196A. Daniel Philip Radawski ‘ 3. Rudko, M. The role of certain chemicals in local regula- tion of blood flow (Ph.D. thesis). Norman University Oklahoma, 1966. 4. Scott, J., M. Rudko, D. Radawski, and F. Haddy. Role of osmolarity K+, H+, Mg++, and 02 in local blood flow regulation. 52-.i- Physiol. 218:338-45, 1970. ROLE OF CATIONS AND OSMOLALITY IN LOCAL BLOOD FLOW REGULATION AND IN HEMORRHAGE By Daniel Philip Radawski A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1971 DEDICATION To my wife, Mary Jo,and my parents Josephine and Vincent Radawski. ii ACKNOWLEDGMENTS The writer takes this Opportunity to express apprecia- tion to Drs. Francis J. Haddy and J.B. Scott for their encouragement and invaluable assistance during the course of these investigations. Appreciation is extended to the other members of the writer's Advisory Committee, Drs. R.M. Daugherty, Jr., J.M. Dabney and J. Hook, for their competent guidance. The writer also acknowledges the assistance of Mr. Booker Swindall, Mrs. Josephine Johnston and Judy Kortright. iii II. III. IV. TABLE OF CONTENTS INTRODUCTION . . . . . . . . . SURVEY OF THE LITERATURE . . 0 Action of Cation and Osmolal Increases in Resting Vascular Beds . Role of Cations and Osmolality Regulation and in Hemorrhage . Active hyperemia . . . . . Reactive hyperemia . . . . Hemorrhage . . . . . . . . METHODS O O O O O O O O O O 0 Local Blood Flow Regulation . Gracilis muscle . . . . . . Forelimb . . . . . . . . Coronary . . . . . . . . Hemorrhage . . . . . a m Analysis of Samples and Tre t e t 0 RESULTS 0 O O O O O O O O O 0 Local Blood Flow Regulation Gracilis muscle . Forelimb . . . . Coronary . . . . Hemorrhage . . . . O 0 g O O O O O O O O O O O C O I O O O 0 DISCUSSION . . . . . . . . Local Blood Flow Regulation Skeletal muscle . . . . . Cardiac muscle . . . . . Hemorrhage . . . . . . . . SUMMARY AND CONCLUSIONS . . e BIBLIOGRAPHY . . . . . . . . . iv in'LBcéi'Bioéd°Fiow O O O O O O O O O n O H. O O O O O O O O Seeeee creeeee .0... O O O O O O O O O O O O O O O O O O O O O O O O O O Table l. 3. A. 5. 7. LIST OF TABLES The average effects of nerve stimulation and a five minute period of ischemia on the constantly perfused gracilis muscle vasculature and the effluent blood osmolality and otassium and magnesium ion concentrations N=9) . . . . . . . . . . . Average effects of graded, local increases in brachial artery blood carbon dioxide on forelimb pressures (N=9) . . . . . . . . . Average effects of graded, local increases in brachial artery blood carbon dioxide on forelimb segmental skin and musCle vascular reSiStanceS (N=9) e e e e e e e e e e e e Effect of a 30 second period of left coronary artery occlusion on the left coronary vasculature and on the chemical composition of coronary sinus blood (N=ll) Average effects of a five minute period of left stellate ganglion stimulation on the left coronary vasculature and on the chemical composition of coronary sinus blOOd (N=10) e e e e e e e e e e e e e e e Average effects of systemic hemorrhage on systemic arterial (Sys.) and coronary (C.S.) concentrations of various chemical faCtorS (N=ll) e e e e e e e e e e e e nae Average myocardial uptake (+) or loss (-) of Mg, K, Na and Ca during pre- hemorrha e, hemorrhage and reinfusion PeriOdS N=10) e e e e e e e e e e e e e 0 Average effects of systemic hemorrhage and reinfusion on coronary sinus oxygen tension N39 e e eoe e e e e e e e e e e e e e Page A? 52 5A 56 58 65 67 68 Table Page 9. Average effects of intravenous norepinephrine inquion(N=9)eeeeeeeeeeeeeeee 70 vi Figure l. 2. 7. 9. LIST OF FIGURES Gracilis muscle preparation . . . . . ... Segmental resistance forelimb preparation . . . . . . . . . . . . . . . Extracorporeal lung-perfusion circuit. . . Open chest coronary sinus cannulation . . Average effects of a five minute period of faradic stimulation of the gracilis nerve on gracilis blood flow and resistance and on the osmolality and K+ and Mg++ in the venous effluent (N=9) . The average effects of a five minute period of gracilis artery occlusion on gracilis blood flow and rgsistance and on osmolality;K and Mg in 'the venous effluent (N=9). . . . . . . . . . . The effects of close arterial infusion of isosmotic sodium chloride and hyper- osmotic sodium chloride and dextrose solutions on gracilis blood flow and resistance and on the venous effluent 08m01alitY(N=9).o.o......... A plot of the percent change in vascular resistance as a function of the increase in venous osmolality during exercise and infusion of hyperosmotic solutions in the gracilis muscle. One and five refer to the first and fifth minutes of stimulation. Data was taken from figures five and seven. The effects of close arterial infusion of isosmotic sodium chloride and hyper- osmotic sodium chloride and dextrose solution on gracilis venous osmolality perfusion pressure and resistance (N=9) . . vii Page 23 29 31 34 no 43 45 48 Figure 10. ll. 12. Page The effects of graded, local increases in brachial artery carbon dioxide on pH, aortic and perfusion pressures, brachial and cephalic vein outflows and total forelimb resistance (N=9). . . . 51 The effects of removal of a blood volume equal to 2% of the body weight on left ventricular contractile force, aortic pressure, coronary sinus blood flow and coronary resistance (N=l9). . . . . . . . 60 The effects of reinfusion of shed blood on left ventricular contractile force, aortic pressure, coronary sinus blood flow and coronary resistance (N=9) . . . . 62 viii INTRODUCTION Of the many naturally occurring vasoactive chemicals the cations, potassium, magnesium and hydrogen and the physical property, osmolality, are of particular interest to study during states in which changes in blood vessel radius occur. The reasons for study are threefold. First, slight changes in cation concentrations and osmolality produce vasomotion (93). Second, changes of a vasoactive magnitude may readily occur since potassium and magnesium are the cations of greatest intracellular concentration and hydrogen is a major product of metabolism. Third, concentration changes of a vasoactive magnitude of one or more of the above chemicals are observed in the blood in various states asso- ciated with altered vascular resistance and/or systemic blood pressure (38). Local blood flow regulation and hypovolemic shock are two such states and this thesis will attempt to elucidate the role of these cations and plasma osmolality in the vascular resistance changes observed in these states. The local regulatory responses, active and reactive hy- peremia, have been well described (40). Most simply, active hyperemia is an increase in blood flow through tissues in which the metabolic rate has been increased. Reactive hy— peremia is an increase in blood flow in excess of control values resulting from release of arterial occlusion. It is evident that the changes in blood flow are produced by changes in vessel caliber; however, the mechanism or mechan- isms responsible for the caliber changes are uncertain. Three theories exist as to their cause. The capillaron hypothesis of Rodbard (5,82) contends that the changes in vessel caliber observed during local blood flow regulation result from passive vasomotion. It assumes that the capillary is a soft walled vessel whose diameter can be influenced by changes in tissue pressure and that the capillary is functionally enclosed in a capsule thus allowing tissue pressure to vary. Consequently in active hyperemia, according to this hypothesis, increased tissue pressure occurring during each muscular contraction expresses fluid from the extravascular compartment. With relaxation of the muscles, the extravascular pressure falls below intravascular pressure and conductance approaches maxi- mal values. As fluid reenters the extravascular compartment, transmural pressure and flow gradually return to the control levels. In reactive hyperemia arterial occlusion results in a drainage of fluid from vascular and extravascular compart- ments and extravascular pressure falls toward venous levels. On reopening the artery a positive transmural pressure fills the soft walled segment and conductance and flow increase to a maximum. As the intravascular pressure filters fluid into the extravascular compartment, the extravascular pressure begins to rise; as transmural pressure approaches zero the vessel partially collapses again and flow rate falls toward control level. Although this hypothesis is appealing, the assumptions required for it have not been proven in histiological or physiological studies. The capillary may not be a soft walled vessel as the theory assumes and tissue pressures may not vary in local blood flow regulation. In regard to the latter point studies by Haddy and Scott (39) in the kidney have shown that lymph vessel pressure, used as a measure of tissue pressure, does not vary greatly in autoregulation, another type of local blood flow regulation. It seems that if tissue pressure changes in local regulation it would do so in the kidney, an organ which is anatomically encapsulated. The myogenic hypothesis (Bayliss response) states that an increase in vessel transmural pressure stretches vascular smooth muscle which, in some manner, stimulates the smooth muscle to contract while a decrease in transmural pressure causes the vascular smooth muscle to relax. Thus a passive effect (transmural pressure change) elicits an active change in vessel radius. This theory proposes that in active hypere-. mia, external compression of the vessels (increased extra- luminal pressure, decreased transmural pressure) caused by the contracting muscle would produce a relaxation of the vas- cular smooth muscle. In reactive hyperemia the decrease in intraluminal pressure subsequent to decreased perfusion of the vascular bed would also decrease transmural pressure re- sulting in relaxation of vascular smooth muscle. There is strong experimental support for the existence 4 of myogenic activity in blood vessels. Stimulation of vas- cular smooth muscle by passive stretch has been demonstra- ted by recording contractile responses of isolated vascular preparations in 12222 (50), by microscopic observation of terminal vascular beds (11A) and by studies of peripheral vascular functions in circulatory experiments (25). The chemical hypothesis states that changes in metab-— olism (e.g. active hyperemia) or blood flow (e.g. reactive hyperemia) are followed by alterations in the concentration of vasoactive chemicals in the tissue fluids surrounding the arterioles; these alterations result in active vasomotion that.adjustsflow to a level more commensurate to the rate of metabolism. For example, changes in metabolism caused by rhythmic contraction of skeletal muscle or increased contrac- tion of cardiac muscle decreases the concentration of oxygen and increases the concentration of vasodilator metabolites in the tissue fluids, resulting in active arteriolar dilation, which in turn increases blood flow to a level more compatible with the new level of metabolism. Similar changes follow a decrease in flow as in the reactive hyperemic response. Bioassay studiesof the exercise hyperemia at corresponding levels of venous hyperkalemia. With regard to hydrogen ion concentration changes, Ross 2F él.(83) showed that active hyperemia of the dog gastroe- nemious muscle produces a rise in flow to 173% of the control value with only a 0.05 unit fall in venous blood pH. Kontos £3 §;.(57) found that active hyperemia of the human forearm muscle may triple flow despite only a 0.03 unit fall in pH. In a typical experiment Gollwitzer-Meier (29) observed a 0.06 unit fall in pH of venous blood and a rise in flow to 370% of the control value after stimulating the canine gastroe- nemious muscle for three minutes. Rudko gt al.(87) reported that skeletal muscle contraction of the dog hindlimb may pro- duce a rise in hindlimb flow to 270% of the control value and a 0.07 unit fall in effluent blood pH. The role of osmolality in active hyperemia of skeletal muscle has only recently been studied. Mellander g3 3;.(70) 15 suggested that increased osmolality is the dominant factor in active hyperemia of skeletal muscle. They reported that active hyperemia in the feline lower leg resulted in a de- crease in vascular resistance to 20% of the control value and a 38 mOsm/Kg rise in venous plasma osmolality. They also found that intra-arterial infusion of hypertonic glucose or xylose solution into the resting skeletal muscle at rates producing similar changes in regional osmolality elicited a pattern of vascular response resembling active hyperemia. In addition, the extent of the evoked resistance vessel dila- tion was only slightly below that seen in exercise. In the human forearm and leg, Lundvall gt §;.(65,66) showed that exercise produced a fall in resistance to 15% of the control value and a 17 mOsm/Kg increase in venous plasma osmolality. Moreover, there was a positive relation- ship between the degree of hyperosmolality and the extent of the exercise hyperemia. Intra-arterial infusion of hyperos- motic solution into the resting muscle, in this study as in that of Mellander gt gl,,produced a dilation of resistance vessels, and the vascular resistance tended to decrease pro- gressively with increasing osmolality. Much less work has been done in defining the role of cations and osmolality in active hyperemia of cardiac muscle. However, active hyperemia of cardiac muscle appears to be associated with increases in the venous blood hydrogen ion concentration. Parker gt al.(78) found that coronary sinus pH decreased and lactate production increased in patients with l6 coronary artery diSease whose hearts were atrially paced. They also reported an increase in potassium efflux from the heart during atrial pacing (79). Sybers gt gl.(103) found that right cardioaccelerater nerve stimulation, which increased heart rate from 130 to 250 beats/minute, was associated with a brief transient loss of potassium followed by a gain in the canine myocardium during stimulation. Atrial stimulation led predominantly to a loss of myocardial potassium. Using an isolated, supported heart preparation Sarnoff gt g1.(89) found that paired stimulation of the ventricles produced a loss of potassium from the myocardium, but a continuous infusion of norepinephrine caused a gain of potassium by the myocardium. Driscol and Berne (18), however, did not find a measurable net change of potassium from the myocardium during increases in coronary blood flow induced by intracoro- nary epinephrine infusion. The role of magnesium and osmolal- ity in cardiac muscle active hyperemia has not been studied. 2. Reactive hyperemia Reactive hyperemia in skeletal muscle is associated with increases in venOus blood hydrogen ion concentration. In the human forearm Kontos (56) demonstrated that flow increaSed to 10 times the control value while the pH of venous blood fell 0.03 pH units. Kontos gt g;.(56) and Rudko gt gl.(87) found that following release of arterial occlusion flow doubled in the dog forelimb compared to control and venous pH was lowered 0.03 - 0.10 units. Although venous plasma potassium increases 5 to 23 17 minutes after cross clamping the aorta (2) it appears that potassium is not lost from skeletal muscle during short term arterial occlusion (115). Rudko (87) found that after four minutes of occlusion release of the inflow artery in the dog hindlimb increased flow but had no effect on potassium ion concentration in the venous effluent. There are not many studies concerning the effect of arterial occlusion on the concentration of cations and osmo- lality in the blood draining the heart. However, it has been reported that occlusion of the circumflex artery for 40 min- utes increases the myocardial efflux of potassium (49). Magnesium and hydrogen ions are also lost from the heart during prolonged occlusion (64). .5. Hemorrhage Most vascular beds initially respond to hemorrhage with an intense increase in resistance due both to passive and active decreases in vessel radius (41). The increased resis- tance usually wanes with time, however. The response of the coronary vascular bed is seemingly more variable with in- creases, decreases and no change in coronary resistance being reported. Perhaps part of the disparity among responses lies in the variety of techniques that have been used to measure coronary blood flow and the number of different procedures which have been employed. A Vowles gt g;.(109) and Frank gt g;.(26) measured coronary sinus blood flow directly via a cannula and observed a fall in coronary vascular resistance. Vowles and associates bled 18 dogs into a reservoir, maintained systemic pressure at 30 mm Hg for five minutes and made their measurements of pressure and coronary sinus flow. Frank gt gt. also lowered pressure to 30 mmHg by removal of 53 ml/Kg of blood, but this pressure was maintained for 4.5 hours before making measurements. The reported decrease in resistance in this latter study was from 1.4 to 1.1 mm Hg/ml/min. I Catchpole gt gl. (9), Edwards_gt.g;. (l9), Hackel gt gt. (37), and Horvath gt gt. (48), using the nitrous oxide tech- nique for measuring coronary blood flow, sampled blood from the coronary sinus and found either a fall or no change in coronary vascular resistance during hemorrhage. Catchpole and Edwards bled their animals to a systemic pressure of 30 mm Hg and maintained these pressures for 45 and 150 minutes, respectiVely. They observed a fall in coronary resistance. Hackel bled to a pressure of 45 mm Hg, maintained this pres- sure for 60 min and did not observe any change in coronary resistance. Horvath removed a volume of blood equal to4% of the dog's body weight over an average period of 14.3 minutes. Immediately after stapping bleeding (14.3 min) coronary resistance was below control (0.74 vs 1.51 mm Hg/ml/ min). After 74.3 minutes of hemorrhage a second flow deter- mination was made and coronary vascular resistance did not differ significantly from control values. Saperstein.gt_g;. (88) and Takacs gt_g;. (104, using Rb86 clearance techniques in rats, found a fall and no change, respectively in total coronary resistance 20 minutes after l9 removal of 23 ml/kg of blood from the rat. Saperstein ob- served a rise in coronary resistance (from 72 to 84 mm Hg/ ml/min) when 10 ml/Kg of blood was removed from another group of animals. Corday gt gl.(l2) using a photoelectric drOpmeter inter- posed in the anterior descending artery also reported an increase in resistance (from 13 to 20 mm Hg/ml/min) in tissue supplied by this artery when dogs were acutely bled to a systemic pressure of 45 mm Hg. Using electro-magnetic flow probes Schenk gt g;.(91) and Granata gt g;.(3l) reported an increase in coronary blood flow and coronary resistance, respectively. Schenk removed 15 ml/kg of blood rapidly from the dog and monitored left circumflex artery blood flow up to an hour after hemorrhage. Granata gt al.also measured left circumflex artery blood flow and made graded removals of 1500 ml of blood over an average of 106 minutes. They observed an initial transient increase in coronary resistance. This increase was followed by a fall in resistance at the end of the bleeding period. Opdyke and Foreman (76), employing a perfusion type flow- meter interposed in the anterior descending artery of dogs, noted a decrease in coronary resistance when dogs were bled to a pressure of 30 mm Hg. ‘ Systemic hemorrhage is reported to change the plasma osmolality and cation concentration. The effect of hemor- rhagic shock on increasing the hydrogen ion concentration is well documented (10). Zwemer and Scudder (117) reported that 20 hemorrhage produces a rise in arterial potassium ion concen- tration. Stainsby (100) found that hemorrhage which lowered the blood pressure of dogs to 35140 mm Hg increased plasma potassium from 3.5 to 4.5 meq/fi, and osmolality from 329 to 350 mOsm/Kg in a typical experiment after 50 minutes of hy- povolemia. Schwinghamer gt gl.(92) reported that removal of 25%rof the dog's estimated blood volume did not change arterial plasma potassium or osmolality after 240 minutes of hemorrhage, but increased plasma magnesium from 2.24 to 2.39 meQKP. More severe hemorrhage (removal of 50% of the dog's estimated blood volume) increased arterial plasma osmolality (from 306 to 312 mOsm/Kg) and potassium (from 3.8 to 4.2 meq/ j), and magnesium (from 2.22 to 2.31 meq/j) ion concentrations after 60 minutes of hyvaolemia. To reiterate, the purposes of this study were twofold: l) to examine the role of the potassium, magnesium, and hydro- gen ions and osmolality in active and reactive hyperemia of cardiac and skeletal muscle, and 2) to characterize the vas- cular resistance response of the coronary bed to hemorrhage and examine the possible role of the above named cations and osmolality in its response. Coronary sinus oxygen tension and left ventricular contractile force were also measured to further eluCidate the coronary resistance response in hemors rhage. In addition, the effect of intravenous norepinephrine infusions on coronary sinus oxygen tension before, during, and after hemorrhage was also determined. These aims were accomplished in the following manner. Venous cation 21 concentrations and osmolality from skeletal (gracilis) and cardiac muscle were determined before and during active and reactive hyperemia. Since increases in osmolality were ob- served during active hyperemia of skeletal muscle similar increases in osmolality were produced in the resting muscle and the resistance responses were compared to those seen in active hyperemia. The hydrogen ion has been shown to in- crease in local regulation of skeletal muscle (56,57,83,87). Consequently, this ion was studied by making local stepwise increases in the carbon dioxide content of blood perfusing the resting forelimb and observing its effect on segmental as well as total forelimb resistances. In hemorrhage coronary vascular resistance was calculated from recorded parameters while periodicallymeasuring arterial and coronary sinus cation concentrations and osmolality before and after removal of a volume of blood equal to 2% of the body weight from the animal. METHODS Mongrel dogs of both sexes weighing between 12 and 18 kg were anesthetized with sodium pentobarbital (30 mg/Kg, intravenously) and placed on positive pressure respiration. (Harvard Apparatus Co., model 607, Dover, Mass.) Following the surgical procedures described below, heparin sodium (5 ms/Kg) was administered intravenously to prevent coagu- lation. All blood pressures mentioned were continuously recorded via Statham low volume displacement pressure trans- ducers (Statham Laboratories, model P23Gb, Hato Rey, Puerto Rico.) which served as inputs into a direct writing oscillo- graph (Sanborn Co., model 60-1300, Boston, Mass.). A. £953; giggg 3193 Regulation 1. Gracilis muscle In this preparation the right gracilis muscle was ex- posed and freed from connective tissue. All blood vessels communicating with the gracilis except the major artery and vein were ligated. Heavy occlusive cord ligatures were placed at each end of the muscle to eliminate collateral flow. A short section (8-l2cm) of the gracilis nerve was, carefully freed from investing fascia and encircled with a loose Frigature (Figure 1)., One of two types of experiments was then performed. 22 25 1' T0 ETINULITGl EMWN.MEW ------ FENOML VEIN “““ luu‘“ ' PLA ..« , ! Figure l. Gracilis muscle preparation. a. constant pressure In 14 animals the gracilis vein was cannulated and its outflow diverted into a small polyethylene reservoir. Blood from this reservoir was continuously returned to the animal_via the external jugular vein, by means of a pump, (Sigmamotor Inc., model T-6SH, Middleport, N.Y.). Blood flow through the muscle was periodically determined by weighing timed volumes on a top-loading semi-analytical balance (Mettler Co., model 416, Princeton, N.J.). Blood weight in grams was directly converted to milliliters. Abdominal aortic pressure was continuously recorded via a cannula in- serted up the left femoral artery. Blood samples (2-4ml) were periodically obtained from the venous cannula for deter- minations of plasma osmolality and magnesium and potassium ion concentrations. The experimental protocol included three steps (reactive and active hyperemia and solution infusion in the resting muscle). After an initial control period during which re- peated blood flow measurements demonstrated a stable flow, a sample of venous blood was taken and the gracilis artery was occluded with a non-traumatic clamp. The occlusion was maintained for five minutes. 0n release of the occlusion the venous outflow was sequentially diverted into a series of collection tubes for a 30 second period. Since this blood was subsequently analyzed it could not be returned to the animal. To maintain blood volume, the animal was given an equal volume of high molecular weight dextran in saline. 25 The sequential collection procedure precluded accurate quan- titation of the post-occlusion flow and hence made it diffig cult to assess the degree of reactive hyperemia. For this reason, after blood flow returned to the pre-occlusion level, the procedure was repeated but this time the venous outflow was collected in a single receptacle and weighed. At this juncture the gracilis nerve was sectioned and the distal and carefully fixed on platinum electrodes which were connected to a square wave stimulator (Grass Instruments, model S5, Quincy, Mass.). When gracilis blood flow became stable, a venous blood sample was taken and a five minute exercise period was simulated by faradic stimulation of the gracilis nerve (stimulation parameters were 6v, 0.06 millisec, 6/sec). Blood flow measurements were made during the first and fifth minute of stimulation and immediately on termina- tion of stimulation. Blood samples were taken after 10 seconds of stimulation and at one minute intervals thereafter throughout the stimulation period. The effect of local exogenous increases in plasma osmo- lality on gracilis blood flow were also determined. Hyper- osmotic solutions of sodium chloride and dextrose (900 mOsm/ Kg) were individually infused (Harvard apparatus, model 901, Dover, Mass.) directly into the gracilis artery at progres- sively increasing rates (0.38, 0.76, 1.91, 3.82 ml/min). Each rate was maintained for three minutes except the highest rate which was continued for six minutes. Blood flow was measured and venous blood sampled before starting the infusion 26 and during the last minute of each infusion rate. Isosmotic NaCl infused at the same rates served as a control. b. constant £12! In another series of nine animals the gracilis mus- cle was prepared as described above except the muscle was perfused at constant flow. This was accomplished by inter- posing a finger type blood pump (Sigmamotor Inc., model TMlO, Middleport, N.Y.) between the left femoral artery and the gracilis artery. The pump flow was set at a rate that produced a perfusion pressure approximately equal to aortic pressure. Except for minor differences the experimental protocol followed in these studies was the same as in the constant pressure series. Ischemia was produced by shutting off the blood pump. Since, under conditions of constant flow, the response to an ischemic period is manifest as a change_in pressure (reactive dilation) rather than a change in flow (reactive hyperemia) it was possible to simultaneously quan- titate the degree of the response and collect venous blood samples. Thus, ischemia was studied only one time in each animal. Changes in venous plasma osmolality comparable to those observed during faradic stimulation were achieved in the resting muscle by infusing the hypertonic test solutions at the two lowest infusion rates. In both constant pressure and constant flow experiments gracilis muscle blood flow was converted to milliliters per 100 grams of tissue and vascular resistance was calculated 27 by dividing the perfusion pressure by the flow per 100 grams of tissue and expressed in millimeters Hg per milliliter per minute per 100 grams of tissue. 2. Forelimb In this study the effects of graded, local hypercapnia on forelimb skin and muscle pressures, flows and resistances were studied in nine dogs. This was accomplished by passing blood from the dog's femoral artery through an isolated lung and perfusing it at a constant rate into the brachial artery while continuously measuring pressures at various points in the forelimb and periodically determining brachial and cephalic vein outflows. The carbon dioxide tension was progressively increased by ventilating the isolated lung with various gas mixtures. Skin of the right forelimb was circumferentially sec- tioned 3-5cm above the elbow. The right brachial artery, forelimb nerves, and the brachial and cephalic veins were isolated and the muscles'and remaining connective tissue sectioned by electro-cautery. The humerus was cut and the ends of the marrow cavities packed with bone wax. Blood entered the limb only through the brachial artery and re- turned only through the brachial and cephalic veins. The forelimb nerves (median, ulnar, radial, and musculocutaneous) were left intact and coated with an inert silicone spray to prevent drying. Intravascular pressures were measured via small-bore polyethylene tubes (P.E. 10-60) inserted into the following J‘lI-Ilyllll III'III“ 28 vessels: 1) skin small artery from the third superficial volar metacarpal artery on the undersurface of the paw; 2) muscle small artery from a vessel supplying a flexor muscle in the upper portion of the forelimb; 5) skin small vein from the second superficial dorsal metacarpal vein on the upper surface cf the paw; 4) muscle small vein from one of the deep vessels draining a flexor muscle in the middle portion of the forelimb;5) skin large vein from the cephalic vein via a side branch; 6) muscle large vein from the bra- chial vein via a side branch (Figure 2). The brachial and cephalic veins were partially transsected 3-5 cm downstream from the sites of large vein pressure measurement and the distal end of each vessel was cannulated with a short section of polyethylene tubing (P.E. 320). Outflow from both veins was directed into a reservoir maintained at constant volume with a variable speed pump (Holter Inc., model REl6l-110, Bridgeport, Pa.) which continuously returned blood to the animal via a cannulated jugular vein. Blood flow was deter- mined by timed collections of the two venous outflows. In this preparation the median cubital vein represents the major anastomotic channel between the brachial and cephalic veins. This vessel was ligated in all experiments so that the brachial venous flow was predominately from muscle where- as cephalic venous flow was predominately from skin (71). Although this approach does not accomplish functional isola- tion of skin and muscle blood flow, the degree of flow sepa- ration is sufficient to permit comparison of resistance 29 .oOfipmammcum nsfiaonom monopmflmon Hopsosmom .N onsmah gag wag-em) gang 13% s1. Iran—gunfigv 4.442ng 30...... 23) 431045 _ _ >654 . 4554mm . -. . \eewem 30...». Zw> 034de gm? mmDmmwmm umamwmn— Egb z_w> Q.._ 1.4.1055 134—2m Z_¥m 30 changes in the two parallel-coupled beds. Mean systemic arterial pressure was continuously monitored via a catheter inserted in the lower abdominal aorta. An extracorporeal lung-perfusion circuit, free of reservoirs, was then estab- lished between the right femoral artery and the right bra- chial artery (Figure5). Femoral artery blood was pumped (Sigmamotor, model T-6SH) into the pulmonary artery of an isolated right lung removed from another dog. The pulmonary venous effluent flowed through a cannula tied in the partial- ly preserved left atrium. The flowing blood was pumped at a constant rate into the brachial artery. Blood flow through the brachial artery was adjusted to a value which maintained the limb at a perfusion pressure equal to systemic arterial pressure during a five minute control period. However, this flow was then kept constant through the remainder of each experiment. Pulmonary artery and vein pressures were monitored in the isolated lung and maintained similar to the normal t3 ztzg values by varying the rate at which femoral arterial blood was pumped (Harvard Aparatus) into the lung. The isolated lung was ventilated with a large stroke (400-500 ml) at a fast rate (20/min) in order to achieve maximum changes in carbon dioxide concentration of the perfusing blood. The isolated lung was sequentially ventilated with room air, 20% 02-5% C02, 20% 02-75% 002, 20% 02-10% 002, 20% 02-15% 002, 20% 02-20%1C02. The arterial and venous pressures were continuously monitored while pH's and the venous outflows 51 va PA FR M Blood from I Pumo I lung Pump FemoroLAnery To Respirator Figure 3. Extracorporeal lung-perfusion circuit. 32 from the brachial and cephalic veins were measured just prior to termination of a ventilation period. A ventilation period was terminated when venous outflows reached a steady value (usually 2-3 minutes) and blood samples for'pH deter- mination were taken from the extracorporeal circuit between the lung and outflow pump. Total and segmental (large artery, small vessel and large vein) vascular resistances (mm Hg/ml/min) in muscle and skin were calculated by dividing pressure gradients (mm Hg) by appropriate blood flows (ml/min). The following resistances were calculated using the formulas indicated: Total Forelimb Resistance = PBA - PV EIC+F§ Total Skin or Muscle = P - P or P Resistance BA 0% B C B Large Artery Resistance = PBA - PSA or PMA c°rs Small Vessel Resistance = PSA or PMA - PSV or PMv c°rB Large Vein Resistance = PSV or PMV - PC or PB CFC or F3 where P = pressure, F = flow, BA = brachial artery, V = vein, (PC + PB) S = skin, M = muscle, C = cephalic vein, B = bra- -_27—_' chial vein, SA = skin small artery, MA = muscle small artery, SV = skin small vein, MV = muscle small vein. 55 5. Coronary In these studies the thoracic cavity was entered via a longitudinal incision in the fourth intercostal space (Figure 4). To more clearly expose the area under operation the left lung was carefully wrapped in an unfolded gauze sponge and draped over the dorsal end of the incision. The pericardium was incised and the atria and ventricules ex- posed. Investing fascia was removed from the left common coronary artery and the left sympathetic nerve trunk immedi- ately caudal to the stellate ganglion and loose ligatures I were placed around these structures. A specially shaped glass cannula was inserted into a cut appendage of the atrium, manipulated into the coronary sinus and tied securely in place. A short extra corporeal circuit composed of the glass cannula, rubber tubing, and a large plastic cannula which was inserted into the superior vena cava via the left exter- nal jugular vein directed coronary sinus blood back to the animal. A "Y" tube interposed in the extracorporeal circuit could be opened to the atmosphere while simultaneously occluding the circuit close to its entrance into the jugular vein. This procedure permitted diversion of coronary sinus blood for ascertaining coronary blood flow and removal of 4-6 ml blood samples. The samples of blood were assayed for osmolality, and the potassium, and magnesium ion concentration. In addition calcium and sodium ion concentrations were also determined. Since it was necessary to obtain anaerobic samples for pH determinations, these blood samples were 54 From coronary smus Figure 4. Open chest coronary sinus cannulation. 55 obtained directly from the rubber tubing with a syringe and needle. As blood taken for samples could not be returned to the animal an equal volume of high molecular weight dex- tran in saline was given to maintain blood volume constant. Ten second timed blood flows were periodically collected in graduated cylinders. Blood removed for flow determinations was immediately returned to the right femoral vein of the dog via a gravity fed reservoir. Abdominal aortic pressure was recorded via a cannula inserted up the right femoral artery. Coronary sinus pressure was also recorded via a cannula inserted retrogradely up the rubber tubing and through the mouth of the glass cannula. In some of the animals left ventricular contractile force was measured with a 120 ohm strain-gauge arch (J. L. Butterfield, P.0. Box 412, Charleston, S.C.) sutured to the surface of the left ventri- cle. Samples of coronary sinus blood were taken and then the left common coronary artery was completely occluded by tightening the loose ligature which encircled the vessel. The occlusion was maintained for 30 seconds. Upon release of the occlusion coronary sinus outflow was sequentially directed into a series of four sampling tubes and a sample for pH was obtained. The sequential collection procedure made it necessary to repeat the reactive hyperemic step in order to determine the effects of a 30 second ischemic period on coronary blood flow and resistance. When coronary flow again became stable, coronary sinus 56 blood samples were taken and a five minute exercise period was simulated by faradic stimulation (Grass Instruments) of the left sympathetic nerve trunk (average stimulation parameters were 20V, l3/sec, 0.127 millisec). An increase in systemic pressure from initial values was used to signify the beginning of the five minute exercise period. Blood flow measurements were made during the first, third, and fifth minutes of stimulation, and a pH sample was obtained at three minutes. Samples for cations and osmolality were obtained at 10 seconds and one, three and five minutes of stimulation. Blood flow and samples were also obtained after cessation of stimulation. Coronary vascular resistance (mm Hg/ml/min) was calcu- lated according to the formula: systemic pressure minus coronary sinus pressure divided by coronary blood flow. B. Hemorrhage This study employed the same preparation for studying coronary blood flow described previously and consisted of two series of experiments. In both series, dogs were bled a volume of blood equal to 2%>of their body weight via a cannula inserted into a femoral artery. The animals were kept hypovolemic for a two hour period after which time the shed blood was rapidly (less than 1 minute) reinfused into the dog via the gravity fed reservoir. Coronary sinus blood flow was measured and coronary resistance values were calcu- lated before and during the hypovolemic period and after reinfusion of shed blood. During hypovolemia, measurements 57 and calculations were made at 1,2,3,5,10,15,30,45,60,75,90, 105, and 120 minutes. In the first series of 10 animals coronary sinus and femoral artery blood samples were taken before, during and after the hypovolemic period. Arterial blood samples were taken at 5, 60 and 120 min of hemorrhage and 5 minutes after reinfusion of shed blood. Coronary sinus samples were taken at 2,5,15,30,60,90 and 120 minutes of hemorrhage and 5 min- utes after reinfusion of shed blood. The myocardial balance of each cation was determined during control and 5, 60 and 120 minutes of hemorrhage. Uptake or loss was computed according to the formula: arter- ial concentration minus venous concentration times blood flow. In the second series of nine dogs coronary sinus oxygen tension was monitored with an oxygen macroelectrode (Beckman Instruments Inc. Spinco Division, Palo Alto, Calif.) in- serted into the extracorporeal circuit. The macroelectrode was coupled to a Beckman model 160 physiological gas analyzer which had a direct readout on the Sanborn polygraph. Coro- nary sinus oxygen tension measurements were recorded. immedi- ‘ately before each blood flow measurement. In addition, norepinephrine was periodically infused intravenously in this. series of experiments while measuring coronary sinus oxygen tension and blood flow. The infusion rate of norepinephrine was adjusted to produce similar increases in systemic pres- sure and contractile force during each infusion period. In 58 this series of experiments all measurements were made for up to two hours after reinfusion of shed blood. In order to test the effects of the surgical procedure and time on coronary resistance and arterial and coronary sinus cation concentrations and osmolality sham experiments were run in four animals. Coronary sinus blood flow and arterial and coronary sinus blood samples were taken immedip ately after and at one and two hours after completion of the surgical procedure. C. Analysis 23‘. Samples g._n_d_ Treatment 9_f_ Pgtg. Blood pH was measured with an expanded_scale microelec- trode pH meter (Radiometer Inc., model 22, Copenhagen, Den- mark). Plasma magnesium and calcium ion concentrations were determined by atomic absorption spectrophotometry (Perkin Elmer Co., model 290B, Norwalk, Conn.). Plasma potassium and sodium ion concentrations were determined by flame photon- etry (Beckman Inc., model 105, Fullerton, Calif.), and plasma osmolality by the freezing point depression method (Advanced Instruments, model 31L, Watertown, Mass.). Statistical eval- uation was by the paired-replicate method of Wilcoxon (98). RESULTS A. Eggg;.B;ggg Flow Rggulation 1. Gracilis muscle a. constant pgessure Figure 5 shows that electrical stimulation of the gracilis nerve produced an eight fold decrease in vascular resistance within the first minute. This decrease in resis- tance was associated with a comparable increase in blood flow. At the fifth minute of stimulation blood flow was slightly higher and resistance slightly lower than at the first min- ute. Systemic pressure was not affected by the stimulation. Venous effluent from the gracilis, sampled ten seconds after beginning stimulation, showed an unchanged osmolality, a slight increase in magnesium ion concentration (1.91 to 1.98 meg/p, 0.01sPso.05). and a substantial increase in potassium concentration (3.6 to 4.8 meq/I, P50.01). The increased levels of magnesium and potassium were still present in the venous effluent at the end of the first minute of stimula- tion. In addition, at this time there was a slight rise in the plasma osmolality (294 to 303 mOsm/Kg. P30.01). Over the subsequent three minutes of stimulation the plasma concentra- tions of magnesium and potassium and the plasma osmolality 59 ACTIVE W "'9 7.0 r— so, “9.. 2.0 I’M—N L7 E Mumps 325~ Oemolohty means/“l3°°'o*"I‘I_"“"""'-‘--ee 2751: Systemic '50); IOO - J; . .12? 7°— \ Iran offing 5° ' 30 . IO .. t W '5 _ mug/wish! IOOg of tissue '\ 0 Figure 5. Average effects of a five minute period of faradic stimulation of the gracilis nerve on gracilis blood flow and resistance and on the osmolality and K+ and ngtt in the venous effluent (N=9). 41 progressively fell toward control. By the fifth minute of stimulation only venous plasma potassium ion concentration remained above the control level. Hence, at the time that resistance and flow responses to stimulation were maximal, venous plasma osmolality and magnesium ion concentration had returned to prestimulation levels. The average effects of a five minute period of ischemia on the gracilis vasculature and on the measured parameters in the venous effluent are shown in Figure 6.- The characteris- tic vascular response to a period of ischemia was observed in each animal, i.e. a decrease in resistance and an increase‘ in blood flow. The response, however, was not associated with measurable changes in the venous blood osmolality or magnesium and potassium ion concentrations. Intra-arterial infusions of isosmotic NaCl and hyperos- motic solutions of NaCl and dextrose were given in an attempt to further investigate the role of osmolality changes in the vascular response of skeletal muscle to stimulation. The aim of these studies was to produce osmolality changes in gracilis venous plasma similar to those which occurred during stimu- lation and to observe the effect on flow. With respect to changes in venous plasma osmolality, it is apparent from Figure 7 that on the average the 0.38 ml/min infusion rate of the hyperosmotic solutions exactly mimicked stimulation, that is, venous osmolality increased 9 mOsm/Kg (from 298 to 307 mOsm/Ks). Vascular resistance, however, fell to only 83% of the control level with NaCl (from 10.7 to 9.0 mm Hg/ml/ NE MESH“ "39 K" 7.0 moo/L 5.0 . ...... a 3.0 -—¢— “9.. 20 e. ________ a meq/L 4; v L71); .0003 Osmdfi, 325 )- "'°""°V|“ 300 5-..--." 4 275; . I50 I- System Pt”. I a "It‘ll-lg '00 .. ,L. Fm' ‘r ' III/nil/IOOO 50 .. 0' “m ................... .o “3., ............. i: “m I5 '1» ....... mHO/ml/win/ ............ -- qu 'm .---. ....... T 0 4' 7’ g?) ,‘5 20 25 30 Figure 6. The average effects of a five minute period of ' gracilis artery occlusion on gracilis bloo§+flow .and resistance and on osmolalityK+ and.Hg in the venous effluent (N59). "pm III“ 503.4 0.10% “I : II! J, ‘00 (— 4 Veeoue . .47 .47 r‘1'/ «Cornet/H 35° ‘ ’1'] . .17 300 533.42::" O--— “I 300“ I-IG 9"- “I one M “'00 me mm .0" 50 “3““ FIG! 30 (v “‘ «unmanned .0 - “:55“ often- 20 heist-Ice a--. mmwunuaumr I0 'm—4t====g:::_ IOOQdfieeue _ . . . . ° 3 e 9 l2 Is The (ninth) Figure 7. The effects of close arterial infusion of isosmotic sodium chloride and hyperosmotic sodium chloride and dextrose solutions on gracilis blood flow and fle‘sigatance and on the venous effluent osmolality 1.4 min/100 gm tissue) and 73%lof the control level with dextrose (from 14.9 to 11.1 mm Hg/ml/min/lOO gm tissue). At the same volume infusion rate isosmotic NaCl produced a fall in re- sistance to 88%lof the control level (from 16.4 to 14.6 mm Hg/ml/min/IOO gm tissue). Greater increases in the venous plasma osmolality produced by increasing the infusion rate of the hyperosmotic solutions were associated with further falls in resistance. The resistance at the first minute of the highest infusion rate however, was lower than at the sixth minute. Hyperosmotic (900 mOsm/Kg) polyethylene glycol, an inert molecule, was also infused in four of the animals. Similar infusion rates produced increases in plasma osmolality and decreases in resistance comparable to those observed with hyperosmotic NaCl and dextrose. In Figure 8 the percent change in vascular resistance is plotted as a function of the increase in venous osmolality during exercise and infusions of hyperosmotic solutions. It is apparent that the percent change in resistance for the same change in osmolality is much greater in the case of exercise. In fact, increasing venous osmolality by infusion to levels much greater than those observed in exercise still did not produce a comparable decrease in resistance. Figure 8 also shows that vascular resistance remained at the same level while venous osmolality fell toward the control value from the first to the fifth minutes of exercise. 45 . .oobou use 3.3 mean scum none» was .33 dogmas—25o no @3553 firfiu one Sonar. 23 on some." 3.3 use 25 .3035. magnum on» a." anagram, moans” headwaonfio noose» a.“ coaches." on» no sages?“ a no 3:333." good» a.“ omega pouches on» no no.3 < .w 93mg @essmoev £322.30 28:; c. .829: on om on 9. on ON 9 o q I11 q d a 1 mm on on 00. 293...... Roscoe 0.285%: x 228.12. .22 2.352»: e 3.0....qu o mm. mourns M (”MD 5° °/o) #6 b. constant £21 Table 1 shows that in the first 10 seconds of electrical stimulation of the gracilis nerve, perfusion pressure and hence vascular resistance decreased to almost 50%lof the control level. Perfusion pressure and vascular resistance continued to decrease to the fifth minute (Pi0.0l). By the tenth second of stimulation, osmolality and the mag- nesium and potassium ion concentrations in the venous efflu- ent were elevated. Maximal levels were reached by the third minute. From the third to the fifth minute of stimulation venous osmolality and potassium ion concentration fell slightly (Ps0.0l) from the three minute values while magne- sium ion concentration was not further affected. Table 1 also shows the average effects of a five minute period of ischemia. 0n reinstating flow, perfusion pressure and vascular resistance were well below control levels and gradually returned to control levels during the ensuing two minutes. As with reactive hyperemia at constant pressure, these changes in the gracilis resistance were not accompanied by measurable changes in venous plasma osmolality or magne- sium ion concentration. Hewever, unlike reactive hyperemia at constant pressure, potassium ion concentration rose slight- 1?. The effects of infusing hyperosmotic solutions on gra- cilis muscle vascular resistance at constant blood flow are shown in Figure 9. During hyperosmotic infusion into the resting muscle at a rate of 0.38 ml/min, average venous 47 .Honucoo on UmHmmEoo cons mo.onao.o .HOHucoo on Umnmmfioo cmnz Ho.o " m m VI m U .omm\m .ummflaaafi mo.o .> m on3 mumumamumm ceasefiseflumo .OOOaxas NH .OOOH\Hs OH 30am Uooamn scam sooamm QGOflumumaflo m>wuommm mcowumumawa m>wuo¢ ~O.H HO.H ~O.H OO.H OO.H so.~ OH.~ mHO.H OO.H H\sms U Ezflmmcmmz O.m O.m s.m s.m m.m s.m ~.O O. . H\sms U o m U U U v m m Eswmmmuom NON mmm mmm mmm NON Oman sOHm esmm NON ms\Emos muaamaosmo O.HH O.O O.O s.O H.NH O.O m.m O.m ~.OH mOOH\cHs\Hs\mm as e e o e e e mocmpmflmmm. OHH OOH mm mm HNH on em mO OHH mm as U U U U U U munmmmnm coamzmumm .00m .00m .me .Umm HOHfiGOU .GHE .GHE .me HOHHGOU OOH OHH mO ms m O OH aflfiwnomH umom UGOHUMHDEHm .AOuzO .mcoflumuucmosoo coH Esflmmsmmz Usm EnammMpommxmwprHmHOEmo Uoon unmadmmm on» Usm mnsumasomm> edema: mHHflomuo UmmSMHmm wausmumcoo was so MHEmnomH mo UOOHmm muses: m>Hm m Use Gowumaszum m>umz mo muommmm mmmum>< one .H mamme INFUSIW RATE I'll/nil 0.38 0.76 "F J. .J. 4O - :2 m “n. —---'_-=::—=.... ____- _._-_-a mug/nanny 6F $—---------..--_--=3 L l I O 3 4 5 5 I i nus (mac) Figure 9. The effects of close arterial infusion of isosmotic sodium chloride and hyperomotic sodium chloride and dextrose solution on gracilis venous osmolality perfusion pressure and - resistance (ll-9). 9 49 osmolality rose from 294 to 310 mOsm/Ks with NaCl and from 298 to 313 mOsm/Kg with dextrose. Average vascular resistance fell from 9.7 to 8.7 mm Hg/ml/min/lOO gm tissue with NaCl and 9.1 to 7.8 mm Hg/ml/min/lOO gm tissue with dextrose. At an infusion rate of 0.76 ml/min average venous osmolality rose further to 336 and 337 mOsm/Kg with hyperosmotic NaCl and dextrose, respectively, and average vascular resistance decreased to 7.8 and 6.? mm Hg/ml/min/lOO gm tissue, re-a spectively. Isosmotic NaCl did not significantly change vas- cular resistance at those infusion rates. Thus, venous os- molality increased 41 mOsm/Kg with NaCl and 39 mOsm/Kg with dextrose while vascular resistance fell to 80%lof the control level with NaCl and 74% of the control level with dextrose. Exercise, on the other hand, produced a 21 mOsm/Kg increase in osmolality and a fall in resistance to 46%lof the control level (Table 1). In four animals, the resistance response was observed during simultaneous intra-arterial infusion of isosmotic KCl and 900 mOsm/Kg dextrose at rates which raised potassium ion concentration by h meq/f and osmolality by 36 mOsm/Kg. Resistance fell to 65%lof the control level. These values are to be compared to those seen with active hyperemia in the same four animals where potassium ion concentration rose 2.4 meg/1, osmolality rose 26 mOsm/Kg and resistance fell to 5h% of the control value. 2. Forelimb In order to investigate further the role of cations 50 in local blood flow regulation, the effect of local altera- tions in hydrogen ion concentration on forelimb vascular. resistances was studied. The forelimb was employed since it permitted study of segmented vascular resistance in both skin and muscle. The average effects of local increases in the carbon dioxide content of brachial artery blood on pH, aortic and perfusion pressures, brachial and cephalic vein outflows and total forelimb resistance are shown in Figure 10. Changing the ventilatory mixture in the extra- corporeal lung from 0% 002 (room air) to 5% 002 produced a fall in pH from 7.64 to 7.32. This increase in 602 and fall in pH was associated with a decrease in perfusion pres- sure from 142 to 118 mm Hg and total forelimb resistance from 1.2 to 1.0 mm Hg/ml/min. Changing from 0% to 5% 002 also produced a shift in forelimb blood flow. Brachial vein out- flow decreased from 61 to 57 ml/min while cephalic vein out- flow increased from 62 to 69 ml/min. Stepwise changes in the ventilatory mixture from 5 to 20% (:02 produced progres- sive changes similar to those just described. That is, pH, perfusion pressure and total forelimb resistance fell and there was a shift in blood flow from the brachial to the cephalic vein. Switching from 5 to 20% 002 produced changes which were similar in magnitude to those observed in chang- ing from O to 5%»002. Aortic pressure was not significantly affected by the local, graded increases in the carbon dioxide content of the blood perfusing the brachial artery. Table 2 shows the average effects of graded, local 78 P (use) 68 _, I ISO - Pressure A0,“, ml-ig ‘- —— §_ ___' mag Brachial artery 70 . Cephalic vein outflow Flow . 60 '- ml/mm Brachial vein outflow 50 .- Totol ! ’ Forelimb '-5 ‘ , RGSime MN m Hq/ml/min L . . 05 6 e 7'5 :8 :3 :0 % CO; Figure 10. The e ffects of graded, local increases in brachial artery carbon dioxide on pH, aortic and perfusion pressures, brachial and cephalic vein outflows and total forelilb resistance (Ii-9).. 52 Table 2. Average Effects of Graded, Local Increases in Carbon Dioxide on Forelimb_Pressures. SMA = small muscle artery, SSA = small skin artery, SMV = small muscle veins, SSV O small skin veins BV = brachial (muscle) vein, CV = cephalic (skins' vein, (N=9).. . Ventilatory Pressures mm Hg Mixture . SMA SSA SMV SSV BV CV Room air 95 96 12e6 1703 803 605 5% C02 81* 70* 10.6* 17.2 7.2* 6.6 7.5% 002 78* 65* 10.1* 16.6 7.1‘* 6.7 10% 002 77* 62*. 9.5" 16.2 7.7” 6.7 15% 002 72* 58* 9.3" 15.9 6.9" 7.3 20% 602 66* 51* 9.3* 15.9 i 6.6* 7.7 ’ * PS0.0§ when compared with control. 53 increases in carbon dioxide on forelimb pressures. All the pressures in the muscle vascular bed, that is, small muscle artery and vein and the brachial vein pressure, fell from control values while in the skin vascular bed only skin small artery pressure fell while skin small and cephalic vein pres- sure did not change. The average effects of graded, local increases in bra- chial artery blood carbon dioxide on forelimb segmental skin and muscle vascular resistance are shown in Table 3. The fall in total forelimb vascular resistance (Figure 10) ob- served on changing from 0% (room air) to 5%vCOZ resulted from a fall in total skin vascular resistance while total muscle vascular resistance was unaltered. The fall in total skin vascular resistance was due to a change in skin small vessel resistance while other skin and all muscle segmental vascular resistances were not significantly different from control values. Further stepwise increases (5 to 20% C02) in carbon dioxide ventilating the extracorporeal lung result«- 9d in.further decreases in skin total and small vessel vas- cular resistances. Moreover, ventilation of the extracor- poreal lung with mixtures containing 10, 15 and 20%lcarbon dioxide also resulted in slight falls in skin venous resis- tance from the control value. 3. Coronary The average effects of a 30 second period of left common artery occlusion on systemic arterial pressure and coronary sinus blood flow and coronary resistance in 11 dogs Table 3. Average Effects of Graded, Local Increases in Brachial Artery Blood Carbon Dioxide on Forelimb Segmental Skin and Muscle Vascular Resistances. TS = total skin, TM = total muscle, SA = skin artery, MA = muscle artery, SSV = skin small vessels, MSV = muscle small vessels, SV = skin veins, and MV = muscle veins (N=9). Ventilatori Resistances mm Hg/ml/min' Mixture TS TM SA MA ssv. MSV sv MV Room air 3.3 2.7 1.2 0.9 1.8 1.6 0.3 0.1 596002 2.1* 2.5 1.0 0.8 0.9* 1.6 0.3 0.1 7.5% C02 1.8* 2.5 0.9 0.8 0.8* 1.7 0.2 0.1 10% 002 1.4" 2.5 0.7 0.8 0.7" 1.6 0.1" 0.1 15% 002 106* 203 008 0.8 oe?‘ let} 002* 0e]- 20% C02 l.l+* 2.1 0.7 0.8 0.5* 1.2 0.1* 0.1 * PS0.05 when compared with control. 55 are shown in Table A. Nete that this short period of ische- mia produced almost a two and a half fold increase in coro- nary sinus blood flow at an average of six seconds after re- leasing the occlusion. At 27 seconds-after abruptly loosen- ing the ligature blood flow was still well above the control value. This increase in blood flow occurred despite a fall in arterial blood pressure and the increase in coronary sinus blood flow concomitant with the fall in systemic arte- rial pressure resulted in a large fall in left coronary vas- cular resistance. Table 4 also shows the average effects of a 30 second period of ischemia on the coronary sinus concentration of the various factors assayed. Cation concentrations and os- molality in samples which were collected beginning at an average of five seconds after releasing the occlusion were not significantly different from the concentrations in the control samples. At 10 seconds, however, there was a signi- ficant rise in hydrogen ion concentration (decrease in pH) and potassium ion concentration. At 15 seconds coronary sinus plasma osmolality also increased slightly and the rise in potassium ion concentration was maintained. Plasma os- molality and potassium ion concentration remained elevated above control values at 20 seconds after release of occlusion. Coronary sinus plasma calcium, magnesium and sodium ion con- centrations were not altered at this time. In six experiments left ventricular contractile force was continuously monitored during the control and reactive 56 .Houusoo ou anmmsoo sos3 Ho.o v mo eeH eeH meH meH meH H\oos as o . _ _ at m ce.O ee.O O.H sastsxmsss . . . . . mos oossumHmom mm H Hm H cm H om H om H sswmosmoz muosouoo o.v m.m m.m o.q m.m H\vos as o o H H o oHON o~H~ as sasts O O O O O 30H.“ om m so v om m N m H m sswnwwmom mssHm muosouou .u. .u. ee~.s nu- Hm.s so a m a mm as ousmmoum mssHm muosouou mM\EmOE mm as owom omom mmm mmm mmm huHHsHoEmo sun lo mm onsmmonm OHHHom one now com com Honusoo com com Houusoo ON mH OH m as a oHEosoahm MHsoHommm o>Huomom o>Huomom soHuHmomEou Uoon uncommom HoHsomo> .HHHuzv .Uoon msch muosouoo no soHuHmomEoo HoOHEosu on» so Usm ousuoHsomo> muososou anon on» so GOHmsHooo mucous humsouoo smog mo UoHuom Usooom on a mo muoommm .v Hands 57 hyperemic periods. In each experiment contractile force fell to an average low point of 11% of the control value at an average of 26 seconds after occluding and then rose to an average of 25% of the control value at six seconds after re- leasing the occlusion (36 seconds after occluding). From six to 27 seconds post ischemia contractile force rose in every experiment to a level which was below control in three experiments and above control values in the other three ex- periments. The average effects of a five minute period of left sympathetic nerve trunk stimulation on left coronary vascu- lature and the coronary sinus plasma cation concentration and osmolality are shown in Table 5. At 10 seconds after com-. mencing stimulation average systemic pressure rose from 87. to 102 mm Hg. This increase was maintained through the first and third minutes of stimulation but waned by the fifth min- ute of stimulation (PS0.05). Coronary sinus blood flow, like systemic pressure, rose above the control value at one minute after starting electrical stimulation and remained elevated through the third minute of stimulation but then fell slightly (Pgo.os) from the fourth to the fifth minuteof stimulation. Coronary vascular resistance fell from control at the first minute of active hyperemia and remained below control through the remainder of the stimulation periOd. Coronary sinus blood hydrogen ion concentration increased slightly from the control value in the third minute of elec- trical stimulation and magnesium ion concentration rose above Table 5. 58 Average Effects of a Five Minute Period of Left Stellate Ganglion Stimulation on the Left Coronary Vasculature and on the Chemical Composition of Coronary Sinus Blood (N=10). Control Stimulation 10 l 3 ’5 Sec min min min Aortic Pressure mm Hg 87 102‘at 112‘5L 116a 1063) Coronary Sinus Pressure mm Hg 4 6b 6b 7" 6‘1 Coronary Sinus Flow a; a min 91.3 __ 150 147 13234 Left Coronary Resistance a a ai mm Hg/ml/min 1.1 .__P 0.7 0.8 0.8 WOsmolality mOsmo 298 297 300 299 300 PH 1) 7.30 __ __ 7.27 __ Potassium ' W mEq/l 3.1 3.0 3.0 3.0 3.1 esium b mEq/l 1.74 1.75 1.74 L78 1.79 Calcium mEq/l 3.9 3.9 3.8 #.O; 3.8 Sodium P ' WI 142 142 142 143 143 a PS0.0l when compared to control. b 0.02 $P£0.05 when compared to control. 59 the control value in the fifth minute of active hyperemia. Plasma osmolality and potassium, calcium and sodium ion con- centrations did not change from control values during the five minute stimulation period. In six experiments left ventricular contractile force was recorded before and during active hyperemia. At 10 seconds after starting electrical stimulation contractile force rose an average of 16%lin four of five animals moni- tored. Contractile force continued to increase from 10 sec- onds to one minute in four of the five animals such that average contractile force was 23% above the control value. From one to three minutes of stimulation contractile force fell from the one minute values in four of five animals but still remained an average of 20%iabove the control value. Contractile force continued to fall towards control value in all animals from three to five minutes such that at five minutes contractile force was an average ’of 14% above the control value. B. Hemorrhage The average effects of removal of a blood volume equal to 2%lof the body weight in 19 dogs on left ventricular con- tractile force, aortic pressure, coronary sinus blood flow and coronary resistance are shown in Figure 11. Average time of bleeding was nine minutes. Systemic hemorrhage produced an immediate and marked fall in aortic pressure and coronary sinus blood flow. The fall in aortic pressure and coronary flow continued during the remainder of the bleed out period 60 I'l' ‘ in 3| 8 h V i ‘1 iii {a if! 3883888 Figure 11. The effects of removal of a blood volume equal to 2% of the body weight on left ventricular contractile force, aortic pressure, coronary sinus blood flow and coronary resistance (N319)e 61 and shortly after cessation of bleeding (15 min). From 15 to 120 minutes aortic pressure and coronary blood flow rose toward control values (P50.05). Left ventricular contractile force did not differ significantly from control values until 30 minutes into hemorrhage at which time it rose to 23%.above prehemorrhage value. Left ventricular contractile force then rose slightly more (P30.05) such that at 120 minutes of hemorrhage contractile force was 35%labove prehemorrhage value. Coronary vascular resistance was unchanged from con- trol until 60 minutes into hemorrhage at which time it fell (from 1.5 to 1.3 mm Hg/ml/min). Coronary resistance decreased further such that at 120 minutes of hemorrhage it was at a value of 1.1 mm Hg/ml/min (1353.05). Aortic pressure and coronary sinus blood flow increased from the 120 minute value at 5 minutes after reinfusion of shed blood while con- tractile force decreased (PS0.01) and coronary resistance was unchanged. The effects of reinfusion of shed blood on left ventri- cular contractile force, aortic pressure, coronary sinus blood flow and coronary resistance were followed for two hours in 9 of the 19 animals. The results are shown in Figure 12. As in the entire group of 19 animals hemorrhage produced a rise in contractile force, a transient fall in aortic pres- sure and sinus blood flow followed by a return of these param- eters towards control, and a fall in coronary resistance. Reinfusion of shed blood produced an immediate but transient fall in contractile force which was followed by a return of . mazv 0283.83 Sconce 28 no.3 use: use? once 5.3393 causes .ooaou oauoosauqoo nuasoaupnos can. we cognac." new 99322.3 1.3.1: ‘53:. a arm. oo— 92 o a sued no ocean no 3330 one .3" 9338 L . w a n6 . 5.5%... 030.333 .9. 2.6.30 m.— an 55>... Ber. too-n 350.0 09 av OISE 2:325 0.?22( co . n. .. 4lIIDIIIOIIIQ\\\\\\\\\\\\/m— oncosunxv [ll/lldli L 6.0 .>.._ I o? 63 force to a value which was not significantly different from the 120 minute value, but which was greater than control (pS0.0§). Contractile force gradually fell to control values in the latter stages of the reinfusion period. Aortic pressure rose from the 120 minute value (p50.05) immediately after reinfusion and attained a level which was not signifi- cantly different from control. Aortic pressure remained at this level until 240 minutes when it fell slightly (pso.os). Coronary sinus blood flow rose transiently from the 120 min- ute value immediately after reinfusion (pS0.05) but then fell gradually to a level which was significantly greater than the 120 minute value (p50.05) but did not differ from control. Coronary resistance was not affected by reinfusion of shed blood and remained below control throughout the reinfusion period. To test the effects of the surgical procedure and time on the measured parameters sham experiments were run in four animals. After performing the surgical procedures designated in Methods,measurements were made immediately after and one and two hours after completion of the surgery. None of the measured parameters were significantly different from control values at one hour. However, at two hours there was a fall in systemic pressure (from 93 to 76 mm Hg) and coronary sinus flow (from 61 to #7 ml/min).. There was no change in coronary resistance (1.5 vs 1.8 mm Hg/ml/min) or contractile force (29 vs 30 mm paper excursion). There alSo was no change in arterial (310 vs 310 mOsm/Kg) or coronary sinus (316 vs 312 51+ mOsm/Kg) osmolality, arterial (3.7 vs 3.6 meq/h) or coronary sinus (3.7 vs 3.6 meq/I) potassium, arterial (1.5h vs 1.53 meg/9) or coronary sinus (1.58 vs 1.56 meq/D) magnesium, arterial (7.39 vs 7.38 pH units) or coronary sinus (7.37 vs 7.36 pH units) hydrogen, arterial (151 vs 152 meg/1) or coronary sinus (15h vs 153 meg/9) sodium and arterial (4.4 vs h.4 meg/p) or coronary sinus (4.5 vs h.4 meg/f) calcium. The average effects of hemorrhage and reinfusion.on systemic arterial and coronary sinus cation concentration W and osmolality are shown in Table 6. Arterial pH increased slightly above control values at 5 minutes into hemorrhage but fell below control during the remainder of hypovolemia and after reinfusion of shed blood. Coronary sinus pH remained unchanged until 30 minutes into hemorrhage at which time it decreased. Coronary sinus pH.stayed below control during the remainder of the experiment. Both arterial and coronary sinus plasma osmolality rose during hemorrhage, the rise in sinus osmolality preceded the rise in arterial. Magnesium ion concentration also rose in the arterial and coronary sinus plasma during hemorrhage. As with osmolality the rise in coronary sinus plasma magnesium preceded the rise in arterial plasma magnesium ion concentration. Arterial potassium ion concentration rose slightly from control values at five minutes into hemorrhage while coronary sinus concen- tration of potassium fell slightly in the second and fifth minutes of hemorrhage. Neither arterial nor coronary sinus concentration of potassium differed significantly from the 65 .msam> mmmauuofimnmum on o>wumaon mo.o v a v Ho.o .msam> mmmnuuosmnoum ou w>wpmHmu mo.o W m w No.00 A .msas> omsnuuosmnoum on o>aumamn Ho.o_w mm om.s e.s ones sea m.m ~.m os~.~ om~.~ seam smom smH.s so~.s swmmewwem m.s m.s sea mes ~.m m.m oom.~ om~.~ nosm «mom sH~.s ss~.e ass ems es.s mes ~.m mH.~ swam use om om.s em.s mes Hes ~.m m.m oma.~ OH.~ seem ~om sm~.a eom.s use om m.s ass ~.m omo.~ neon .ns~.s gas on s.s Ned s.m use.” ssom ass ma e.s s.s Hes osfl s~.m ss.m mm.s em.s com com sm.s smm.s ass m s.s Hes ss.m mm.s Hem mm.s emmmfismgem s.s e.s med Hes m.m H.m mm.a em.s Hem mam ~m.s em.s emssusosmsmss .m.o .uam .m.o .mam .m.u .mam .m.u .mmm .m.u .mam .m.u .mam H\voE H\voa H\va H\me mx\HoEmOE mufiso ++mo 62 + ++mz Emo mm uneconoo was “.mmmv Hmauound oasoummm so ommnuuoaom owsmummm mo muoommm omeum>< J .AHanV.mHouomm Hmowsmsu maowum> mo muoHumnusmosoo “.m.uv macaw .w mqmdfi 66 control values during the remainder of the study. Arterial concentrations of sodium and calcium were not appreciable affected by hemorrhage or reinfusion of shed blood. Table 7 shows the average myocardial balance of the various cations studied. During the pre-hemorrhage period the myocardium lost potassium while there was no significant uptake or loss of the other cations studied. At 5 minutes after initiating hemorrhage the myocardial tissue took up potassium and lost sodium but there was no significant net flux of magnesium or calcium. During the remainder of hem- orrhage and after reinfusion the only significant net flux in the myocardium was a loss of sodium at 120 minutes of hemorrhage. Table 8 illustrates the average effects of systemic hemorrhage on coronary sinus oxygen tension in nine dogs. Hemorrhage produced a gradual fall in sinus oxygen tension which like systemic pressure and coronary sinus blood flow reached its nadir shortly after cessation of bleeding. Coro- nary sinus oxygen tension rose progressively from 15 to 45 minutes and thereafter sinus oxygen tension was unchanged. In the latter stages of hemorrhage oxygen tension was slight- ly but significantly (P50.05) above the control value. Re- infusing shed blood produced a rise in oxygen tension above the 120 minute value (PS0.05). This rise continued until 75 minutes after reinfusion at which time oxygen tension was not significantly different from the 120 minute value. The average effects of intravenous norepinephrine Table 7. Average Myocardial Uptake (+) or Lose (-) of Mg, K, 67 Na, and Ca During Prehemorrhage, Hemorrhage and Reinfusion Periods. (N=lO). ’ Mg++ x+ Na+ Ca++ meq/min meq/min meq/min meq/min Prehemorrhage -.OOlO8 -.0104Aa -.0886 -.00154 Hemorrhage -.ooo79 +.01407a -.579b -.00155 5 min - 60 min -.00119 +.00251 -.0651 -.00072 120 min -.OO3## +.00180 . -.12#9a +.00105‘ Reinfusion -.00576 +.OOO32 -.0511 +.00852 aHP$.Ol b .OlSPS.02 } \ 68 ma awe oma om awe m ma sflE om ma use moa Hm sHE m NH use me ma CHE om ma use N 0H use on ma use ms ma CHE H n Gas md ma :HE om .sofimsmsamm m sHE od 5a use ms «a use ONH m CHE m ma GHE om ea awe mad m saE m ma CHE ma ea sag om 0H :«E n ma use OH «H CHE ms HH use A scamsmssmm wmmsusoamm ma mmmnsso€mm Houusou m Asses N Asses N . Assess om macaw .U powwow om macaw .U powuom om macaw .U. possum .Amuzv .coamsos smmmxo macaw mumsouoo so cosmswsamm was mmssuHOEom oasmumMm mo muoommm ommno>m .m mqmde 69 infusions on systemic pressure, coronary flow and vascular resistance, contractile force and coronary sinus oxygen ten- sion are shown in Table 9. Norepinephrine was infused during the normovolemic period, and at 15 and 105 minutes after hemorrhage and at 15 and 105 minutes following reinfusion of shed blood. Each norepinephrine infusion increased systemic pressure, coronary blood flow and contractile force. Coro- nary vascular resistance decreased during norepinephrine in- fusion when the blood volume was normal and at 105 minutes after hemorrhage and reinfusion of shed blood, but was not changed by norepinephrine infusion at 15 minutes after hem- orrhage and reinfusion. Coronary sinus oxygen tension in- creased when norepinephrine was infused during normovolemia and 15 minutes after hemorrhage but was not changed during the norepinephrine infusions at 105 minutes after hemorrhage and at 15 and 105 minutes after reinfusion of shed blood. .msam> meow>onm on m>ausaos mo.o.w ms m.sH .m.o isms sass .om am messagesssesoz w. m.ss H.H as sh .. Houssou m. I. m s.sa o.d sass «sad see an messagessseuoz r. m.mH o.H as Hm .. Hosssou m H a m.~H .m.o .mos .ms .oe sm essussesssesoz o m.sa o.H on es .. Hospsou m T. .m.m H.H .ms imp ism ms messagesssesoz m m .s.ma .~.H .mma «mes .em as esssssessseuoz m m.sa m.H mm mm .. Houssoo m. sommmw GHE\HE\mmEE sHE\HE mass mmcmnu w sas\m: % wmhxfi QUGMumflmmm 30Hm mhflmmmhm TUMOR mmOQ u. mmsflm mo macaw .0 owsmumMm oaauosuusoo mmmum>¢ m. .Amflzv .SOHWflMGH OGHHSQQQfimmhoz m50§0>MHHGH HO mflOQMHm 00MHO>¢ em WQNGB DISCUSSION A. Local Blood Flow Regulation These studies show that active hyperemia in skeletal (gracilis) muscle is accompanied by increases in gracilis venous effluent potassium and magnesium ion concentrations and osmolality. However, the concentration of these factors is not measurably altered in the effluent blood from skeletal muscle during reactive hyperemia. Increases in venous efflu- ent osmolality similar to those seen in active hyperemia, produced by intra-arterial infusion of hyperosmotic NaCl or dextrose into the resting muscle failed to produce comparable decreases in vascular resistance. Increases in hydrogen ion concentration in the resting forelimb similar and even great- er in magnitude than those reported to occur in local regula- tion did not regularly affect muscle vascular resistance but decreased skin vascular resistance. Active hyperemia in cardiac muscle is associated with slight increases in coronary sinus plasma magnesium and hydrogen ion concentration. Reactive hyperemia in heart muscle is accompanied by moderate increases in potassium and hydrogen ion concentrations and osmolality in coronary sinus blood. 71 72 . 1. §§eletal muscle These studies lend more support to the hypothesis that the potassium ion is important in active hyperemia of skele- tal muscle. The magnitude of the increaSe in potassium ion concentration was comparable to the findings of others. Kilburn (53) showed that the exercising human forelimb venous plasma contained 0.7 meq/y more potassium than arter- ial plasma. In the dog Rudko gt al.(87) and Skinner at al. (96) demonstrated that active hyperemia which produced in- creases in blood flow to 270 and 353%lof the control value, respectively were associated with increases in venous po- tassium ion concentration of 0.8 and 0.95 meg/1 above their respective control values. Kjellmer (54) estimated that in cat skeletal muscle, the potassium released during exercise explains 25-65%rof the dilation, the percentage being the smallest when the dilation was the slightest. There are two possible sources of potassium in this vascular bed: 1) the contracting skeletal muscle cells or 2) cells in the vascular compartment (formed elements in plasma and vascular smooth muscle). Apparently, the skeletal muscle contributes the greatest portion of the potassium since a rise in venous effluent potassium concentration (average increment 0.39 meq/l) has been observed in the ca- nine gastrocnemius muscle during perfusion with red cell-free fluids (87.95). Increased magnesium ion concentration in the effluent plasma during active hyperemia has not been described 73 previously. The present studies do not indicate the source of this magnesium. 'In any event, the magnitude of increase was only 105%.of the control (1.91 to 2.0Q meg/fl) and disap- peared by the fifth minute of gracilis exercise. Even at constant flow, where hypermagnesemia was still present at five minutes, the maximum increase was only 112% of the con- trol value. Studies by Overbeck at él.(77) showed that a 13 mg/min infusion of isosmotic MgC12 into the resting fore- limb produced a 20% decrease in vascular resistance. Conse- quently, it is unlikely that magnesium, by itself, is re- sponsible for the active hyperemia of skeletal muscle but it may participate in the initiation of the response. The finding of an increased osmolality in exercise hy- peremia substantiates the work of others (65, 66, 70) but disagrees with these studies in terms of magnitude and time course. Mellander gt al.(70) found a larger increase in venous osmolality (up to 40 mOsm/Kg) which became more pronounced with time (up to 15 min). Lundvall gt al.(65,66) found lesser osmolal increases (range 1 to 28 mOsm/Kg) in blood draining the human forearm during exercise. These increases did not change or declined moderately with time, but were maintained above control through the exercise period. In the present study hyperosmolality (9 mOsm/Kg) observed in the first minute of exercise disappeared by the fifth minute whereas blood flow increased further. Moreover, in the studies of Mellander and Lundvall infusions of hyper- osmotic solutions into the arterial inflow of resting muscle 74 produced much greater decreases in vascular resistance for the same increment in venous osmolality than was found in the present study. The reason for the difference in active hyperemia and solution infusion between this study and those of Mellander and Lundvall is not clear. However, species differences, muscle types (red vs white), or differences due to technique might be responsible. When flow through the gracilis muscle was maintained constant in the present study, nerve stimu- lation produced a greater rise (26 mOsm/Kg) in the venous effluent osmolality than when flow was allowed to vary (9 mOsm/Kg). This is most likely due to dilution of a similar number of particles in a lesser volume. Thus, in the studies of others a restricted inflow might have elevated the osmolal response to nerve stimulation. The rise in osmolality may be due to increased pyruvate, lactate (107), phosphate (3) and perhaps adenine nucleotide (8) concentrations as well as the measured cations. While hydrogen ion concentration was not measured in the present study a number of investigators demonstrated that it increases in active hyperemia of skeletal muscle (29,83,87). The present study suggests that its role is minimal. In- creases in the venous pH in similar and even greater magni- tude in the resting forelimb did not regularly affect any muscle segmental resistance. However, since muscle vascular resistance remained unchanged while transmural pressure de- creased indicates that some slight active dilation must have 75 been balanced by passive dilation. The finding of a lack of an effect of an increased hydrogen ion concentration on skeletal muscle apparently conflicts with the findings of others (l4,l6,52,58,72,ll6). However, in many of the preparations employed (14,58,72) the increased hydrogen ion concentration was studied in a mixed vascular bed i.e. both skin and muscle, and the resultant changes were not separated. Thus increases in flow (decreases in resistance) may have resulted from increases in skin flow. In addition, employing CO2 to change hydrogen ion concentra- tion or infusing acid salts may affect the skeletal muscle vascular bed differently (Emerson, personal communication). That is, increased C02 produced locally, may not have any effect on vascular resistance, while a pH change of similar magnitude in the same vascular bed produced by local salt infusion may lower vascular resistance. The reason for this difference is not clear but may be due to relative ease with which CO2 traverses cell membranes. It is possible that the increased 002 may enter the red cell, react with water in the presence of carbonic anhydrase, and produce hydrogen ions which increase cell osmolality and thus attract water. The increased volume of the cell resulting from increased water would alter red cell size and may increase vascular resis- tance by increasing viscosity and obstructing capillaries. The increased viscosity may offset any dilation in the muscle vascular bed. However, since the skin vascular bed has pro- portionally more arteriovenous shunt vessels than muscle 76 (33) distortion of red cell size might not be sufficient to offset any direct dilation produced by the hydrogen ion. It is evident from the foregoing that exercise hyperemia results from multiple factors. In addition to those studied, the adenine nucleotides, the Kreb's intermediate metabolites and oxygen acting per se or via one or more of the previously mentioned vasoactive substances have been implicated. Venous blood oxygen tension has been shown to decrease during ex- ercise (69). However, Daugherty gt al.(14) observed only moderate decreases in forelimb vascular resistance when the oxygen tension of the perfusate was lowered to 30 mm Hg. Although some investigators (45) were unable to chemically detect adenine nucleotide changes during active hyperemia, Boyd and Forrester (8) found evidence from chemical analysis that ATP is released by exercising frog skeletal muscle in. gitgg. These latter researchers suggested a role for this agent in exercise vasodilation. A possible role of Kreb's intermediate metabolites has been suggested but not investi— gated (40). Moreover, a possible interaction of factors remain a possibility. Recently Skinner gt g1. (97), demonstrated that decreased oxygen, and increased potassium and osmolality interact to produce considerably greater vasodilation than that which occurred with any agent alone, or with combina- tions of two of the factors. In this study, lowering the arterial oxygen saturation from 95 to 41%, and increasing the osmolality from 301 to 352 mOsm/Kg and potassium from 77 4.0 to 6.9 meqle decreased resting gracilis vascular resis- tance to approximately 25%lof the control value. The present studies suggest that potassium, magnesium, and osmolality probably do not play a significant role in reactive hyperemia of skeletal muscle. Although venous effluent hydrogen ion concentration may increase during re- active hyperemia (56,87), data from the present study on its effect in the resting forelimb suggest only a minimal role for this ion. The finding of a slight increase in potassium ion concentration during reactive dilation (constant flow) suggest that potassium is, in fact, liberated in slight quantities. Maybe potassium in combination with oxygen and the Kreb's intermediate metabolites and the adenine nucleo- tides collectively contribute to the hyperemia. Perhaps relevant, and in support of the role of the chemicals measured in this study acting in local regulation of skeletal muscle, were the observations of Whang and wagner (113). They found that simulataneous venous occlusion and exercise in the human forearm produced increases in venous effluent magnesium, potassium and osmolal concentrations to 9.5, 19.2 and 8.5%labove their respective control values and a fall in pH from 7.35 to 7.21. 2. Cardiac muscle These studies demonstrate that the factors which play a role in active hyperemia of skeletal muscle (potassium, mag- nesium and osmolality) do not play a principal role in active hyperemia of cardiac muscle. Sympathetic nerve trunk 78 stimulation produced the expected increase in contractile force, systemic pressure, and coronary blood flow while decreasing coronary resistance. However, the increase in blood flow to 165%»of the control level was accompanied by only very slight increases in coronary sinus blood hydrogen ion concentration (from 7.30 to 7.27 pH units) and plasma magnesium (from 1.74 to 1.79 meg/9) ion concentration with no change in osmolality or in the other cation concentrations. When slightly larger increases in hydrogen ion concentration are made in the "resting" coronary bed little or no increase in coronary blood flow is reported (14). The increase in magnesium ion concentration occurred only in the fifth minute of exercise and thus could not account for the initial de- crease in coronary resistance. Again, the increase noted was insufficient, by itself, to account for the active hyperemic response. From these results, one must look to other factors,for an explanation of the coronary vascular response to exercise. Certainly oxygen acting per se or through adenosine and the adenine nucleotides (6) should be considered. Adenosine, is a potent vasodilator (6) and it (81) and its breakdown prod- note (6) appear in coronary sinus blood during anoxia. Based on this evidence, Berne (6) has formulated an hypothe- sis for the metabolic regulation of coronary blood flow. In this hypothesis the initiating factor is myocardial oxygen tension. Reduction in myocardial oxygen tension by hypoxemia, decreased coronary blood flow, or increased oxygen utilization 79 by the myocardial cells lead to the breakdown of heart muscle adenine nucleotides to adenosine. The adenosine diffuses out; of the myocardial cell, reaches the coronary arterioles via the interstitial fluid, and produces arteriolar dilation. Perhaps increased hydrogen and magnesium ion concentrations may contribute to .the response produced by adenine nucleo- tides to cause the active hyperemic response of cardiac muscle. Unlike reactive hyperemia in skeletal muscle, reactive hyperemia in cardiac muscle is associated with elevations of coronary sinus plasma potassium (0.9 meg/1), hydrogen (0.05 pH units) and osmolality (6 mOsm/Kg). The increased potassium, which was 1.3 times the control may account for a measurable portion 0f the increased blood flow since Katz and Linder (51) found this size increase in arterial plasma potassium increased coronary blood flow 52%. However, the method employed by them probably increased osmolality as well as potassium concentration, thus overestimating the effect of the potassium increase. Scott gt a1. (94) found that a (0.7 meg/min) infusion of KCl decreased coronary re- sistance to 83% of the control level. The hydrogen ion and cemolal increase taken alone, may not produce a sizable de- crease in resistance. In a study by Daugherty 23 a1. (14) increasing hydrogen ion concentration by 0.3 pH units resulted in only a 15% decrease in vascular resistance. Perhaps these three factors, taken collectively with oxygen, acting per se (75) or through adenosine and the 80 adenine nucleotides (6) may be responsible for the reactive hyperemic response of cardiac muscle. . Oxygen has been observed to fall to levels which may be vasoactive in reactive hyperemia of the heart (75). Recently Rubio et al. (86) indicated that in reactive hyperemia following 30-60 seconds of left coronary artery occlusion, the amount of adenosine calculated to be present in the in- terstitial fluid compartment of the heart is more than suffi- cient to account for the vasodilation observed. The observation of an increase in potassium, hydrogen and osmolality in reactive hyperemia might have a bearing on studies of electrolyte concentrations during atrial pacing in patients with coronary artery disease. In these studies (79) potassium is increased in the coronary sinus blood to a greater extent in patients with coronary artery disease than in patients who are normal. Perhaps coronary artery disease limits the amount that flow may increase in atrial pacing and introduces a condition resembling that produced in constant flow preparation which exaggerates the ion concentration in- creases. r B. Hemorrhage Removal of a blood volume equal to 2%»of the body weight results in a gradual fall in coronary vascular resistance. This fall is preceded by and associated with increases in arterial and coronary sinus osmolality and hydrogen and magnesium ion concentrations, and a rise in contractile force. Initially in hemorrhage there is a fall in coronary sinus 81 oxygen tension and an uptake of potassium by the myocardium. Later in hemorrhage coronary sinus oxygen tension increases. The lack of an initial change in coronary resistance and the fall in coronary resistance later in hemorrhage may be due to the algebraic sum of a number of factors. For example, there are at least five factors tending to increase vascular resistance. .Eirst, a transient increase in blood viscosity may occur in the dog due to contraction of the spleen (85). Second, angiotension and vasopressin may be released (106), (but apparently the magnitude of increase is insufficient to be responsible for much vasocontriction (42). Third, the fall in systemic pressure decreases transmural pressure and hence decreases vascular caliber.’ Fourth, there may be a direct effect of a sympathicoadrenal discharge to actively constrict the coronary vessels. The presence of alpha adrenergic re- ceptors in coronary blood vessels of the dog has been con- firmed by Ross and Mulder (84). They showed that cardiac sympathetic nerve stimulation after beta adrenergic blockade with propranolol decreased coronary flow. Feigl (23) demon- strated that these receptors can act reflexly through the baroreceptor mechanisms after beta adrenergic blockade. Perhaps chemoreceptor stimulation also contributes to the response. Fifth, late in hemorrhage the increased contrac- tile force may act to passively increase resistance by com- pression of the vessels. Early in hemorrhage these constricting influences are antagonized by the fall in oxygen tension and perhaps the 82 indirect effect of a sympathicoadrenal discharge. Later, increases in magnesium, hydrogen and osmolality and the decrease in calcium observed in this study acting in con- junction with a passive increase in radius due to the rise in systemic pressure toward control values predominate and decrease coronary resistance. A decrease in blood viscosity resulting from reabsorption of fluid may also be involved (10). No studies of intra-arterial infusions of magnesium, hydrogen or osmolality have been made in the coronary vas- cular bed during a time when blood volume is decreased and the concentrations of other factors kept at a control level. Consequently the relative contribution of each of these factors in the resistance decrease observed cannot be deter- mined. The finding of a fall in coronary vascular resistance late in hemorrhage agrees with the findings of many others but disagrees with some investigators. Vowles 93 al. (109), Frank 95 21. (26), Catchpole at al. (9), Hackel st 31. (37), Horvath gt a1. (48), Edwards gt a1. (20), Saperstein gt a1. (88), Takacs et al.(lo4). Schenk gt al. (91), and Opdyke and Foreman (76), using various methods and procedures all found a decrease in coronary vascular resistance or increased coronary blood flow in hypovolemic shock. Saperstein et a1. (88), also removed a lesser blood volume from another group ‘of rats and observed an increase in coronary resistance. An arterial pressure of 90‘: 30 mm Hg was observed in these 83 animals when 10 ml/Kg of blood was removed while a control group had an arterial pressure of 121 :.15 mm Hg. Perhaps the amount bled elicited baroreceptor reflexes which in- creased coronary vascular resistance directly by actively constricting the veSsels and indirectly by increasing extra- luminal pressure via increased contractile activity. Granata gt gl.(3l) observed an initial increase in coronary resistance in animals from which an average of 1500 m1 had been removed over 106 minutes. This rise began when arterial pressure was only slightly lowered. These authors attributed the rise in coronary resistance to alpha adrenergic stimula- tion, a decreased transmural pressure resulting from reduced systemic pressure and metabolic vasoconstriction subsequent' to decreased left ventricular work. Corday gt gl. (12) found a rise in coronary resistance in dogs which had been bled to a pressure of 45 mm Hg; presumably rather rapidly. These authors give insufficient information as to the time of bleeding. Consequently, evaluation of their results is diffi- cult. However, it is possible that decreased transmural pres- sure, alpha adrenergic stimulation and decreased metabolite concentration may all have participated in the response. Systemic pressure and coronary sinus flow decreased initially in hemorrhage. Systemic pressure is determined by cardiac output and total peripheral resistance. It is well known that hemorrhage reduces output by decreasing stroke volume due mainly to a reduction in filling pressure. The decreased systemic pressure reduced sinus flow as coronary 84 resistance was unchanged at this time. Systemic pressure and sinus flow increased toward control as compensatory mechanisms were activated. Systemic pressure and coronary sinus flow also decreased in four control animals while coronary resistance was unaf- fected. Like hemorrhage, the fall in pressure was probably due to a decrease in cardiac output. Output decreased due to a reduction in stroke volume. The fall in pressure in the face of an unaltered coronary vascular resistance also decreased sinus flow. The decreased pressure may have re- duced transmural pressure and hence coronary vessel radius, an effect which would increase coronary resistance. However, since resistance was unchanged, the passive decrease in radius must have been balanced by factors attempting to actively increase vessel radius. The failure of contractile force to change initially in hemorrhage may be the result of agonistic and antagonistic factors. There are at least four factors tending to reduce force. They are: 1) decreased filling pressure (due to the reduced blood volume [10]), 2) decreased aortic pressure, 3) decreased filling time (due to a probable increase in heart rate), and 4) decreased coronary flow. One through three act via the Starling mechanism. They were antagonized by a sympathicoadrenal discharge (41). The reduced coronary flow may have decreased oxygen delivery to the myocardium as evidenced by the decrease in sinus oxygen tension. In addi- tion, cation and osmolal concentrations may have been 85 increased at this time. Increased magnesium and hydrogen ion concentration reduce contractile force when introduced exogenously (61) while increased osmolality mayincrease. contractile force depending on the agent used to increase osmolality (27). The finding of a significantly increased myocardial contractile force later in hemorrhage was interesting Since many studies report a variable response or decrease in con- tractile force. Walton gt g;.(110), found a considerable variability of the changes in right ventricular force as a result of hemorrhage (average 3.9% of the body weight and arterial pressure approximately 38 mm Hg). The heart force, decreased markedly in about 60%lof the experiments, was not significantly affected in some and increased above control in others. Cooley and McIntosh (11) recorded myocardial force from the canine left ventricle after hemorrhage in three steps (equivalent to blood volume losses of 30939. #0-49, and 50-60%). There were only slight decreases in con- tractile force at each of the bleeding levels. Greenfield gt g1. (34) noted an increase in canine right ventricular contractile force until arterial pressure fell to 90 mm Hg. Below this level contractile force fell. Nutter gt gt. (74) produced hypovolemia by negative pres- sure in the lower body and did not show any significant al- teration in left ventricular contractility when they produced moderate hypovolemia. Entman (22) observed an increase in left ventricular contractility when dogs were bled a moderate 86 volume over a.30 minute period. The variation in response of myocardial contractility to hemorrhagic shock reported in the literature is perhaps due to the variability in the amount bled and in the time of bleeding. This may be evidenced by the study of walton et al. (110) who obtained a variety of responses in right ven- tricular force. They employed a variety of bleeding rates and bled various volumes (2.6-4.2%»body weight) and conse- quently obtained a widely different response in right ven- tricular myocardial force. Generally, in those experiments where small percentages of the'blood volume was removed (some of walton gt gl. (110), Nutter gt g;. (74), and Entmann (22)) a rise Or no change in contractile force was observed, thus indicating a predominance of sympathicoadrenal activity or balance of the sympathicoadrenal system with the Starling mechanism and coronary flow. In those experiments in which. a greater percentage of volume was removed (some of walton _e_t a__:_L. (110), Cooley and McIntosh (11), Greenfield gt g. (3#)) decreases in contractile force were observed, thus indicating a predominance of the Starling mechanism and re- duced coronary blood flow. The findings of increased magnesium and hydrogen ion concentrations and osmolality in the systemic plasma and blood agree with the studies of Stainsby (100) and Schwingg hamer (92). The finding of an initial decrease in arterial hydrogen ion concentration is interesting and cannot be readily 87 explained. A reduced hydrogen ion concentration has been observed in hemorrhaged animals which are ventilating spon- taneously (10), but the hydrogen ion concentration of arti- ficially ventilated animals has been observed to increase (7.102). Arterial and coronary sinus plasma potassium ion concen- tration changes, and myocardial flux of potassium before and F during hemorrhage are also of interest. Under normal con- ditions there was a loss of potassium from the myocardium ) (arterial concentrations less than coronary sinus). At five minutes of hemorrhage, there was an increase in arterial ion concentration and decrease in coronary sinus potassium ion concentration, thus producing a net uptake of potassium by the myocardium. Recently Todd gt g1. (108) has shown that systemic epinephrine infusion produces an initial rise in arterial plasma potassium concentration. WOrk by others (30,60) demonstrated that potassium is initially liberated from the liver and later by skeletal muscle during high CO2 breathing and that release from the former is blocked by phenoxybenzamine, an alpha adrenergic blocking agent. Werk in isolated rabbit hearts (99) has shown that in the presence of an elevated potassium and adrenaline, hearts took up po- _tassium whereas they lost potassium when adrenaline was added to a bath with a low potassium ion concentration. The lack of a rise in arterial plasma potassium later in hemorrhage may be due to the amount of blood removed from the animal. Schwinghamer gt gl. (92) observed a rise in_ 88 potassium with 50% blood loss, but no rise in potassium with 25%lblood loss. Again, the stimulus for this increase may be catecholamine in origin. The effect of norepinephrine infusion on coronary sinus oxygen tension deserves special mention. Norepinephrine in- fusion rates were adjusted to produce increases in systemic pressure, contractile force and coronary blood flow. The effects of these infusions on coronary sinus oxygen tension, an indirect estimate of myocardial tissue oxygen tension (assuming the ratio of nutriative to non nutriative blood flow through the myocardium does not change) was then studied. Prior to and early (15 min) in hemorrhage intravenous norep- inephrine infusion produced an increase in coronary sinus oxygen tension and hence, perhaps, tissue oxygen tension. This would indicate that proportionately mere oxygen was being delivered to the tissue by the increased blood flow than was being used. Later in hemorrhage and after reinfu- sion of shed blood however, norepinephrine did not change. the coronary sinus oxygen tension. This finding would indi- cate that the oxygen delivery to utilization ratio of the tissue was not altered by norepinephrine. The ability of intravenously administered norepinephrine to increase aortic pressure and coronary blood flow and decrease coronary resistance in hemorrhage is well docu- mented (7,10,102). However, to the author's knowledge only one study concerning the effect of norepinephrine on coronary venous oxygen in hemorrhage has been performed. Frank gt g;. 89 (26) showed that coronary sinus oxygen saturation increased from 14 to 32%»in dogs which received intravenously adminis- tered. norepinephrine at 4.5 hours after removing 53 cc/Kg of blood. SUMMARY AND CONCLUSIONS The purposes of this study were two fold: l) to examine the role of potassium, magnesium, hydrogen and osmolality in local blood flow regulation (active and reactive hyperemia) of skeletal and cardiac muscle, and 2) to characterize the resistance response of the coronary vascular bed to hemorbe rhagaamd.examine the role of the above named cations and osmolality in its response. _I_._o_gg_1_ M Flow Regulation 8 Active and reactive hyperemia were studied in the skele- tal muscle (gracilis) and heart of the dog. Active hyperemia was produced by electrical stimulation of motor (skeletal muscle) or sympathetic (heart) nerves. Reactive hyperemia was.induced by release of short term arterial occlusion. Venous osmolality and potassium ion concentration rose during active-hyperemia in skeletal muscle and reactive hyperemia in heart but these parameters did not change during reactive hyperemia in skeletal muscle and active hyperemia in heart. The magnesium ion concentration rose in effluent blood from the skeletal muscle and heart during active hyperemia. The hydrogen ion concentration rose in coronary sinus blood during active.and reactive hyperemia. Osmolality and resis- tance levels observed in exercising skeletal (gracilis) 9O 91 muscle were compared to those in the resting gracilis during intra-arterial infusion of hyperosmotic solutions. Comparable increases in osmolality produced by intra-arterial infusion of hyperosmotic NaCl or dextrose in the resting gracilis failed to produce similar decreases in resistance. Increases in the hydrogen ion concentration of the magnitude observed in local blood flow regulation in skeletal muscle (56,83,87, j 95) were produced locally in the resting forelimb and the 4 effects on segmental vascular resistance observed. Increases in hydrogen ion concentration failed to regularly affect muscle segmental resistances, but decreased skin small vessel and venous resistances. These findings indicate that cations and osmolality play a role in active hyperemia of skeletal muscle and reactive hyperemia of the heart and that all of these factors do not operate in reactive hyperemia_of skele- tal muscle and active hyperemia of heart. Hemorrhgge The effects of hemorrhage on coronary vascular resis- tance and on the cation concentration and osmolality of arterial and coronary sinus blood were studied in 19 dogs. A blood volume equal to 2%lbody weight was removed over an average of nine minutes and the effects of hypovolemia were followed for 120 minutes. Coronary vascular resistance was unaffected over the first 45 minutes but then fell from 60 to 120 minutes. Systemic and coronary sinus hydrogen and magnesium ion concentrations and osmolality rose during hemorrhage, the rise in cation concentrations and osmolality 92 in the coronary sinus preceded the rise in arterial concen- trations, Coronary sinus oxygen tension and left ventricular contractile force were also measured. Coronary sinus oxygen tension initially fell (0 to 15 min) and then progressively rose over the remainder of the hemorrhage period and at 120 minutes was slightly but significantly greater than the control value. Contractile force increased; the increase beginning at 30 minutes. Thus, under these experimental conditions coronary vascular resistance decreases only late in hemorrhage. It seems likely that increased cation con- centrations and osmolality play a role in the fall in coro- nary resistance. BIBLIOGRAPHY r- , - ‘1 3. 9. 5. 9. 10. BIBLIOGRAPHY Baetjer, A.M. 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