1 EFFECT Q? Mm? 3:11;: Lgmgxg ca; ’ . . ‘ 7 . ; mm THE mm PEREJSEE) , : f 1 i WCHV‘AN STATE UNIVERSITY , '"*5{;fl;£976 VI ANN HAVELANI COLUNGSWORTH _ :_, 1 W'W'FT" ": ’1': f. "ft-151;”- "“ 1* a ’5 RY 6“ tin-:53 5 L filer!“ {'1 Michigan. State W Umve: é: Mi 1.»; a .~ --::.'» Y’IW‘N—u This is to certify that the thesis entitled EFFECT OF NERVE STIMULATION ON RELEASE OF ADENINE NUCLEOTIDES FROM THE RINGER—PERFUSED CANINE GRACILIS MUSCLE presented by Ann Haviland Collingsworth has been accepted towards fulfillment of the requirements for Ph.D. (“,ng in Physiology Wfl/ 3/ gig/A Major professor Date 8/13/76 l. EFFECT ADENINE 1 Exercise 0 dilation and an leChanism reSpo but it undoubte tent of the blC been PIOposed t hl’peremia, The the role 0f ade 1'1 Post-ocCluS: Perqued, Carri] artErially intl malyZed 3301‘ A m did not su except when in Heater). ATP ally into the kadient eluti D’Afi/ 6‘ 4 / ABSTRACT EFFECT OF NERVE STIMULATION ON RELEASE OF ADENINE NUCLEOTIDES FROM THE RINGER-PERFUSED CANINE GRACILIS MUSCLE BY Ann Haviland Collingsworth Exercise of skeletal muscle is associated with vascular dilation and an increase in muscle blood flow. The exact mechanism responsible for the vasodilation is uncertain, but it undoubtedly involves a change in the chemical environ— ment of the blood. Since ATP is a potent vasodilator it has been proposed that ATP is the chemical mediator of active hyperemia. The purpose of this study was to investigate the role of adenine nucleotides in exercise hyperemia and in post—occlusion flow changes in the isolated, Ringer— Perfused, canine gracilis muscle. ATP was infused intra— arterially into the gracilis muscle, and the effluent was analyzed for ATP content by the firefly—luciferase method. ATP did not survive passage through the muscle vasculature except when infused in large quantities (10 ug/ml or greater). ATP, ADP, and AMP were each infused intraarteri- ally into the muscle, and the effluent was analyzed by gradient elution ion exchange chromatography for breakdown products. ATP 5 to AMP on a sing to pass through Therefore, an a: :o detect nanog: duced by gracil caused no Chang the initiation Ilatiguing exerc Atwo minute oc siStent or sigr 1“” iSOPrOtex mine if it dile Ll Venous AMP c EIECiliS nerVe 0 '_4 CPS’ 1'6 me. A Curare did not preven Stm‘lation pa Ann Haviland Collingsworth products. ATP and ADP were both degraded almost entirely to AMP on a single pass through the muscle. AMP was able to pass through the muscle intact and with little uptake. Therefore, an assay was set up using the myokinase reaction to detect nanogram levels of AMP. Muscular exercise pro- duced by gracilis nerve stimulation at 5V, 2 cps, 1.6 msec caused no changes in venous AMP levels. This was true at the initiation of exercise, during a ten minute period of fatiguing exercise, and during the post—exercise period. A two minute occlusion of muscle perfusion produced no con— sistent or significant changes in venous AMP. The vasodi— lator isoproterenol was infused intraarterially to deter— mine if it dilated via AMP release. No significant changes in venous AMP occurred. Vasoconstriction produced by gracilis nerve stimulation at 6V, 6 cps, 1.6 msec or 20V, 10 cps, 1.6 msec was associated with increased outflow of AMP. A curare or gallamine blockade of muscle contraction did not prevent the AMP outflow which occurred with these stimulation parameters. Vasoconstriction produced by intraarterial infusion of epinephrine, norepinephrine, or vasopressin also significantly increased venous AMP levels, The maximal outflow was approximately 150 ng/ml. Phentol— amine abolished both the vasoconstriction and the AMP out- flow produced by nerve stimulation, epinephrine, and gr... norepinephrine . produce vasocon. outflow. It is probably do not (2) i50proteren l3) vasoconstri associated with this latter fin effluent AMP W5 Ann Haviland Collingsworth norepinephrine. Epinephrine concentrations which did not produce vasoconstriction also did not produce increased AMP outflow. It is concluded that (l) adenine nucleotides probably do not mediate active or reactive hyperemia; (2) isoproterenol does not dilate via release of AMP; and (3) vasoconstriction produced by a variety of agents is associated with increased AMP efflux. The significance of this latter finding is unknown. The source of the venous effluent AMP was not determined. EFFECT ADENINE EFFECT OF NERVE STIMULATION ON RELEASE OF ADENINE NUCLEOTIDES FROM THE RINGER—PERFUSED CANINE GRACILIS MUSCLE BY . .{H , '\ b“ .$0 0 Ann Haviland Collingsworth A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1976 £933.23 DEDICATION To my father, Thomas Haviland, and the loving memory of my mother, Rosemary. ii Iwish to guidance commil Scott, B. H. s, efforts in my 5 The author technical assi Davis for her was much nee de I wish to manl’ heurs he this thesis, for their Supp Finally, have been hell: to my life (1111 ACKNOWLEDGMENTS I wish to express appreciation to the members of my guidance committee: Drs. C. C. Chou, R. P. Pittman, J. B. Scott, B. H. Selleck, and C. H. Suelter for their time and efforts in my behalf. The author wishes to thank Mr. George Gamble for his technical assistance. Sincere thanks also go to Mrs. Amylou Davis for her help and advice in many situations where it was much needed. I wish to especially thank my husband, Carl, for the many hours he spent in preparing all the figures used in this thesis. I also wish to thank my father and brothers for their support and encouragement throughout my education. Finally, to the many persons in this department who have been helpful in many ways and have added bits of humor t0 my life during many trying moments I extend my thanks. LIST or TABLES. LIST OF FIGURE: LIST or ABBREV; DITRODUCTION. . L Active 20 Reacti lETHODsHHH. 10. 11. v 12. '_1 L1.) m .b- NH LA.) U'. . . . . Surgic Ion Eu AMP As l4c-zu Firef] ° Infusj Muscle Produ< ‘ Studie TABLE OF CONTENTS Page LISTOF TABLESOCO...IIO'OOOIIOO0.0000IOOOOIOIIDIOOOOO vii LISTOF FIGUMSOOOOOCIIODOIOO ..... COO-OOOOOOOOOOOU0.0 viii LIST OF ABBREVIATIONS................................ x INTRODUCTIONOOOCUOOOOOOOOOO'OIOOIOOOOIOIOOOOOIODOOIOI l LITERATUE REVIEWOOOOOOOOIOOI.....QO.III0.0.IIO...... 8 1. Active Hyperemia of Skeletal Muscle.......... 8 2. Reactive Hyperemia of Skeletal Muscle........ 55 METHOD80'..00.......I..OOIOOQOOOOOOI.CIOOOOOIOIOCO... 66 1. Surgical Techniques.......................... 66 2. Ion Exchange Chromatography.................. 67 3.AMPASSaYODOOOCOO.DOOOOOQOIOOOOO'COOOOQOOQOOI 78 4' l4C—ATP Purity CheckQOIIOOOOOIOOOOIOOIOIOOOO. 83 5. Firefly—Luciferase Assay for ATP............. 84 6. Infusion of Nucleotides into the Gracilis Muscle to Determine Uptake and Breakdown Products................................ .... 85 7. Studies of the Effect of Arterial Occlusion on Venous AMP................................ 87 8. Studies of the Effect of Nerve Stimulation on Venous AMP................................... 87 9. Studies of the Effect of Isoproterenol on Venous AMP................................... 88 10. Studies on the Effect of Nerve Stimulation on Venous AMP during Gallamine Infusion......... 89 ll. Studies on the Effect of Nerve Stimulation on Venous AMP during Curare Infusion............ 89 12. Studies on the Effect of Nerve Stimulation on Venous AMP during Phentolamine Infusion...... 90 13. Studies on the Effect of Norepinephrine and ADH Infusion on Venous AMP.. .......... ....... 91 iv TABLE OF CONTE' 14. Studi Venou 15. Studi sion 16. Studi Venou l7. Reage 18. Stati ESULTS ....... 1. ATP E Muscl 2. Break the C 3. Breal the C 4. Survi Grac: 5. Bffe< thr01 6. Surv; Grac: 7. Effe< 8' Effec 1.6 1 9- Effe« lO. Effe. duri; 11- Effe duri 12. Effe 0.5- 13~ Effe 1.6 Infu 14' Effe 15~ Effe duri l6~ Effe AMP, 17. ffe . AMP, f8° Effe i9- Effie 20' Rife Time TABLE OF CONTENTS——continued page 14. Studies on the Effect of Norepinephrine on Venous AMP during Phentolamine Infusion..... 92 15. Studies on the Effect of Epinephrine Infu- sion on Venous AMP. .... ... ........... 92 16. Studies on the Effect of Ouabain Infusion on Venous AMP.................................. 93 17. Reagents.................................... 93 18. Statistical Methods......................... 99 RESULTS-....OJOOIOOOIQOIOCCIOOICIIOOIDODOIOIOOOIOI... 101 1. ATP Survival on Passage through the Gracilis Muscle Vasculature.................. ........ 101 2. Breakdown Products of ATP on Passage through the Gracilis Muscle Vasculature............. 104 3. Breakdown Products of ADP on Passage through the Gracilis Muscle Vasculature............. 109 4. Survival of AMP on Passage through the Gracilis Muscle Vasculature;................ 109 5. Effect of Flow Rate on AMP during Passage through the Gracilis Muscle Vasculature..... 116 6. Survival of IMP on Passage through the Gracilis Muscle Vasculature................. 117 7. Effect of Occlusion on AMP Outflow.......... 120 8. Effect of Nerve Stimulation at 6V, 6 cps, 1.6 msec on AMP Outflow..................... 123 9. Effect of Isoproterenol on AMP Outflow...... 126 10. Effect of Nerve Stimulation on AMP Outflow during Gallamine Infusion................... 126 11. Effect of Nerve Stimulation on AMP Outflow during Curare Infusion...................... 132 12. Effect of Nerve Stimulation at 5V, 2 cps, 0.5 msec on AMP Outflow ......... ........... 132 13. Effect of Nerve Stimulation at 22V,10 cps, l. 6 msec on Venous AMP during Phentolamine Infusion............. ........ ............... 137 14. Effect of Norepinephrine on Venous AMP ...... 142 15. Effect of Norepinephrine on Venous AMP during Phentolamine Infusion................ 145 16. Effect of Epinephrine at High Dose on Venous AMP ..... ........ ........... . ...... ......... 145 17. Effect of Epinephrine at Low Dose on Venous AMP......... .............. ............. 150 18. Effect of ADH on Venous AMP. ....... .... ..... 150 19. Effect of Ouabain on Venous AMP ....... ...... 155 20. Effect of Cells and Increased Collection Time on Venous AMP... ............. .......... 155 TABLE OF CONTE 21. Relat and V DISCUSSION. . . . SUMMARY AND CC BIBLIOGRAPHY. . APPENDIX 0000.- Page TABLE OF CONTENTSe—continued 21. Relationship Between Perfusion Pressure and Venous AMP....II..IOIOOUOOIOOIICIOODOOO DISCUSSION.ocoonooncoooooo-ocoocooocaoouoooouonooaoo 163 SUMARYAND CONCLUSIONSODIOOIOOIO.IOOOCOOIOOOOOIUOI. 181 BIBLIOGRAPHY-cococonut-'00....ooo'ooonooo-ocooooo-Io 183 APPENDIXoooooooooooccon...coo-cocoo-ouooo-otootolooo 205 vi TABLE 1. Breakdown 2. Change in ATP on pa 3- Change in ADP On pa 4' Change if AMP on pa 5. Effect 05 throuqh 1 6‘ EffeCt Q: LIST OF TABLES TABLE Page 1. Breakdown products of ATP....................... 110 2. Change in concentration of the adenine ring of ATP on passage through the gracilis muscle...... 110 3. Change in concentration of the adenine ring of ADP on passage through the gracilis muscle...... 113 4. Change in concentration of the adenine ring of AMP on passage through the gracilis muscle...... 113 5. Effect of flow rate on AMP during passage through the gracilis muscle vasculature......... 116 6. Effect of ouabain on venous AMP................. 156 PETE 1.Gracilis 2. Gradient raphy at 3.Gradien1 Pattern 4.Gradien- Pattern adenosi: S'AMP sta d ATP sur muscle 1 Breakdo the gra ~ Breakdc through . Breakdc the gra 10' AMP Sur mUSCle muSCle LIST OF FIGURES FIGURE Page 1. Gracilis muscle preparation.................... 69 2. Gradient elution ion exchange column chromatog— raphy apparatus................................ 72 3. Gradient elution ion exchange chromatography pattern for ATP, ADP, AMP, and adenosine....... 74 4. Gradient elution ion exchange chromatography pattern for ATP, ADP, IMP, cAMP, AMP, and adenosineo'OCI.0.0.0.........OOOCOOOIOOOOCOOCOO 77 5. AMP standard curve............................. 82 6. ATP survival on passage through the gracilis muscle vasculature............................. 103 7. Breakdown products of ATP on passage through the gracilis muscle vasculature................ 106 8. Breakdown products of l4C—ATP on passage through the gracilis muscle vasculature........ 108 9. Breakdown products of ADP on passage through the gracilis muscle vasculature................ 112 10. AMP survival on passage through the gracilis muscle vasculature............................. 115 ll. IMP survival on passage through the gracilis muscle vasculature...... ......... .............. 119 l2. Effect of occlusion and nerve stimulation on Venous AMP ......... .... ......... . ..... ......... 122 13. Effect of nerve stimulation at 6V, 6 cps, 1.6 125 mSec on venous AMP............ ...... ........... 14. Effect of isoproterenol and nerve stimulation On venous AMP ......... . ................ . ....... 128 viii LIST OF FIGURE FIGURE 15. Effect ( during 5 m - . Effect < 1.6 mser 17. Effect 1 msec on lllSEC 0n 19. Effect 1.6 mse infusio zo-Effect 21~ Effect Phentol 21 Effect Am". - Effect AMP. , 24‘ Effect 21 Effect 26' Effect LIST OF FIGURES——continued FIGURE 15. l6. l7. 18. 19. 20. 21 22 23 24 25 26 Page Effect of nerve stimulation on venous AMP during gallamine infusion..................... 131 Effect of nerve stimulation at 20V, 10 cps, 1.6 msec on venous AMP during curare infusion. 134 Effect of nerve stimulation at 5V, 2 cps, 1.6 msec on venous AMP during curare infusion..... 136 Effect of nerve stimulation at 5V, 2 cps, 0.5 msec on venous AMP............................ 139 Effect of nerve stimulation at 22V, 10 cps, 1.6 msec on venous AMP during phentolamine infusion.O.......0IOO.......OOCOOIOOOOIIO‘DOOO 141 Effect of norepinephrine on venous AMP........ 144 Effect of norepinephrine on venous AMP during phentolamine infusion......................... 147 Effect of epinephrine at high dose on venous 149 Effect of epinephrine at low dose on venous 152 Effect of ADH on venous AMP................... 154 Effect of cells on venous AMP................. 159 Effect of decreased flow rate on venous AMP... 161 ix ATP = adenos ADP = adenos ;~ I l - adenos WP = Cyclic T-Ci = microc .0 “9 = nanogr 19 = kilog: 39‘ = millig 1‘9 = micrOQ XG = times 53 = Stande m = antid: P02 : Partia P - C02 ‘ part1, - Hg = mini] ATP ADP CAMP HCi c/m LIST OF ABBREVIATIONS adenosine triphosphate adenosine diphosphate adenosine monophosphate cyclic adenosine monophosphate microcurie counts per minuts nanogram kilogram milligram microgram times gravity standard error antidiuretic hormone partial pressure of oxygen partial pressure of carbon dioxide millimeters of mercury Exercise dilation and i isn responSib] been suggested the blood vess nucleotides a: lators, the p< compounds may sented in thi possibility. The regu intact organi muscle tone 1: and outside t ”I antagonist tr01 systems Smooth muscle seneral to m. i‘tesonistic 3f remote c0 L“‘Clllde bloo INTRODUCTION Exercise of skeletal muscle is associated with vascular dilation and increased muscle blood flow. The exact mechan— ism responsible for the vasodilation is uncertain. It has been suggested that a change in the chemical environment of the blood vessels may mediate flow changes. Since adenine nucleotides are present in most cells and are potent vasodi— lators, the possibility exists that one or more of these compounds may mediate exercise hyperemia. The studies pre— sented in this thesis were designed to investigate this possibility. The regulation of the peripheral circulation in the intact organism involves adjustments of vascular smooth muscle tone by factors originating from sites both within and outside the tissue the vessels permeate. Synergistic Or antagonistic interactions between remote and local con— trol systems combine to determine the level of vascular smooth muscle tone. The remote control system functions in general to maintain systemic pressure and is usually antagonistic to the local control system. The mechanisms 9f remote control have been fairly well identified and include blood—borne vasoactive hormones, such as epinephrine and angiotensi thetic vasodil attempt to mai in each tissue as yet been c] Local cor (1) active by} lation; and (. in the absenm ences. Activ. whiCh aCCOmpa example, by a 51°“ 0f arter ated with a t b515511 value u reactive hYpe Systemic VASC results in a This ability jespite a var tion. The W olar COHStri, Pressllre par TWO maj. actiVe and r and angiotensin, and sympathetic vasoconstrictor and sympa- thetic vasodilator fibers. The local control systems attempt to maintain a constant ratio of flow to metabolism in each tissue. The mechanisms of local control have not as yet been clearly identified. Local control of blood flow includes four phenomena: (1) active hyperemia; (2) reactive hyperemia; (3) autoregu- lation; and (4) the venous-arteriolar response. All occur in the absence of any external nervous or hormonal influ- ences. Active hyperemia refers to the increased blood flow which accompanies increases in metabolic rate produced, for example, by activation of skeletal muscle. Temporary occlu— sion of arterial inflow to a vascular bed is usually associ- ated with a transient increase in blood flow above the basal value upon release of the occlusion; this response is reactive hyperemia. Varying the perfusion pressure to most I systemic vascular beds over the range 70 to 200 mm Hg results in a less than proportionate change in blood fIOW. This ability to maintain a relatively constant blood flow despite a varying inflow perfusion pressure is autoregula— tion. The venous-arteriolar response refers to the arteri— olar constriction which results from increases in venous pressure particularly in the intestine and skeletal muscle. Two major theories have been proposed to explain active and reactive hyperemia. These are the myogenic l l l theory and the originated wit that arterial iug force by c relaxation. 'I by the smooth become an acce lUchida and B( the instabilii lead to depola stretch could thereby Produi evideuce exis explanatiOn f llominaga e t mechaniSmS ma :iSSue, as Su The meta blood flow is tissues Via a vasodilatOrs Gaskell (1871 suPply to ti: their VaScule mdence tha. theory and the metabolic theory. The myogenic hypothesis originated with the observation by‘Bayliss (Bayliss, 1902) that arterial vascular smooth muscle responded to a stretch- ing force by contraction and to a diminution of tension by relaxation. This myogenic activity in response to tension by the smooth muscle cells of small blood vessels has since become an accepted fact. Although its mechanism is unknown (Uchida and Bohr, 1969), distension in some way may increase the instability of smooth muscle cell membranes and thereby lead to depolarization (Wiedeman, 1957). For example, stretch could increase membrane permeability to sodium, thereby producing depolarization. However, considerable evidence exists that the myogenic theory cannot be the sole explanation for either active or reactive hyperemia (Tominaga et al., 1973b; Jones and Berne, 1964). Myogenic mechanisms may be overcome by metabolic demands of the tissue, as suggested by Lundvall et a1. (1967). The metabolic theory of local regulation proposes that blood flow is adjusted to meet metabolic demands of the tissues via alterations in the concentrations of one or more vasodilators in the tissue fluid surrounding the arterioles. Gaskell (1877) was the first to suggest that the blood Supply to tissues could be regulated by chemicals affecting their vascular tone. Roy and Brown (1879) found further evidence that blood vessels dilate or constrict in accordance local blood f1 blood vessels evidence in s from bioassay effluent. Se been publishe Scott, 1968; Anrep anc‘ from resting 2 the muscle. l tIaCting muscl found that pe' the vena cava diate fall in exerC151ng m appearEd to creased rap i with the blood flow requirements of the tissue. They sug- gested that oxygen or dilator metabolites might play a role in determining vessel diameter. Gaskell (1880) also sug— gested that intermediary metabolites might be involved in local blood flow regulation, since lactic acid painted on blood vessels produced dilation. Since these early studies, evidence in support of the metabolic theory has accumulated from bioassay studies and chemical analyses of venous effluent. Several excellent reviews of this subject have been published (Ross, 1971; Mellander, 1970; Haddy and Scott, 1968; Haddy and Scott, 1975). Anrep and von Saalfield (1935) reinfused venous blood from resting and contracting muscles intraarterially into the muscle. Only the venous blood from the actively con— tracting muscles caused vasodilation. Scott et a1. (1965) found that perfusion of the forelimb with venous blood from the vena cava, hindlimb, or heart produced an almost imme— diate fall in forelimb vascular resistance. The time course and magnitude of the reSponse were similar between assay and donor organ. Vasodilation was produced by venous blood from both resting and exercising tissues, but the dilation was greater when the venous blood was from an exercising muscle. The vasoactive substance or substances appeared to be labile, as the activity of the blood de— creased rapidly with time. Selby et a1. (1964—65) found mdoubtedly re in the vasoac Other studies flou-to-met by either ne artery, vasod to-metabolism vascular resi Harris and Lo theory is a by Pre-capillary rate of metab found the tim a SubStance Stance must that venous blood from a previously ischemic bed was vasoac- tive in the hindlimb. The vasoactivity of venous blood undoubtedly reflects at least an equally great alteration in the vasoactivity of the tissue fluid in the donor organ. Other studies (Rudko et a1., 1965) have shown that if the flow-to-metabolism ratio in the canine hindlimb is reduced by either nerve stimulation or obstruction of the femoral artery, vasodilation results. This indicates that the flow- to—metabolism ratio may be an important determinant of vascular resistance, supporting the metabolic theory. Harris and Longnecker (1971) suggested that the metabolic theory is a better explanation than the myogenic theory for pre-capillary sphincter regulation in tissues with a high rate of metabolism, such as the heart. Owen et a1. (1975) found the time required for blood flow to return to control levels increased as a function of contraction frequency and exercise duration at both natural and constant flow. Therefore, it would appear that enough evidence exists to seriously consider the metabolic theory in explaining the mechanisms of active and reactive hyperemia. Much work has been done on the identification of the Specific mediator or mediators of the vasoactivity of venous blood. To qualify as a mediator of local regulation, a substance must satisfy several criteria. First, the sub— stance must of course be found normally in the tissue and in altered cor cut during the intraarteriall the range of 1 served during the concentra‘ with the loca While n criteria, se good candida tive hyperem' dioxide, hydI inorganic phc Of these canc‘ Will be discr Possibility Stances may Possible the or interacti The pur (1959) and Venous effl in altered concentration in the tissue fluid or venous efflu- ent during the vascular response. Second, when infused intraarterially, the substance must be vasoactive within the range of plasma effluent or tissue concentrations ob- served during local regulation. Third, the time course of the concentration changes of the substance must coincide with the local regulatory response. While no substance has unequivocally met all of these criteria, several substances are still considered to be good candidates for the role of mediator of active and reac— tive hyperemia. These substances include oxygen, carbon dioxide, hydrogen ions, potassium ions, adenine compounds, inorganic phosphate, and osmolality. Relevant data on each of these candidates regarding active and reactive hyperemia will be discussed in the Literature Review. One further possibility exists, in that more than one of these sub— stances may be altered at the same time. Therefore, it is possible that local regulation is due to the combined effect or interaction of changes in several chemicals. The purpose of the studies described in this thesis was to determine whether or not adenine nucleotides are chemical mediators participating in active and reactive hyperemia in canine skeletal muscle. Forrester and Lind (1969) and Chen et al. (1972b) found ATP released into the venous effluent from exercising skeletal muscle. However, Kontos et 31. change in ATP mscle. Ther necessary. formed in the eliminate normally occ et a1., 1974) Kontos et al. (1968a) and Dobson et al. (1971) reported no change in ATP in the venous blood draining exercising muscle. Therefore, further studies in this area appeared necessary. The experiments to be described were all per- formed in the artificially perfused gracilis muscle to eliminate the rapid breakdown of adenine compounds which normally occurs in whole blood and plasma (Collingsworth et al., 1974). potassium, ox adenine compo hyperemia in show whether tions of the of resting mu Participation hYPeremia is Potassit LITERATURE REVIEW 1. Active Hyperemia of Skeletal Muscle This section reviews the evidence for the roles of potassium, oxygen, carbon dioxide, hydrogen ions, osmolality, adenine compounds, and several other factors in active hyperemia in skeletal muscle. Studies are included which Show whether or not vasodilation occurs when the concentra— tions of these chemicals are altered in the arterial inflow of resting muscle. In addition, the evidence for the participation and interaction of these factors in exercise hyperemia is reviewed. Potassium. Many studies have been done to determine the effect of potassium on vascular resistance. Dawes (1941) was the first to study potassium in detail; he sug— gested that it might mediate exercise hyperemia. In adrena— lectomized whole cats or in the perfused hindlimbs of dogs and cats, small doses (5 mg) of potassium chloride infused intraarterially produced vasodilation whereas large doses (20 mg) produced vasoconstriction. Glover et al. (1962) confirmed the vasodilator action of potassium in humans. Emanuel et al. (1959) noted vasodilation in the arterioles 0f the dog forelimb with infusions of hyperosmotic potassium suggesting vascular dilation, re This effect Sympatholyti not affected et a1. (1963 "hen the nor: duced toward Overbec aetion was n tions. Info into the dog that Previot Salt infusic and not sole alSO found 1 Probably a t of isotonic solutions up to a concentration of eight meq per liter. Above this concentration vasoconstriction occurred in both arteries and arterioles. The vasodilation still occurred after denervation and adrenergic blockade with phentolamine, suggesting that potassium dilates via a direct action on vascular smooth muscle. During the potassium—induced vaso— dilation, responses to norepinephrine were diminished. This effect was also noted by Frohlich et al. (1962) with potassium infusion at concentrations too low to produce vasodilation. Konold et al. (1968a) also confirmed this sympatholytic effect of potassium; venous resistance was not affected by any of the potassium concentrations. Haddy et al. (1963) and Roth et al. (1969) found vasoconstriction when the normal potassium concentration in plasma was re- duced toward zero. Overbeck et a1. (1961) confirmed that the vasodilator action was not due to hypertonicity of the infusion solu— tions. Infusion of isotonic solutions of potassium salts into the dog forelimb produced vasodilation, indicating that previous vasodilation seen with hypertonic potassium salt infusions was indeed due at least in part to potassium and not solely to hypertonicity. Lowe and Thompson (1962) also found that the vasodilator action of potassium was Probably a direct one. The dilation produced by infusion 0f isotonic potassium chloride solutions into the human local nerves et al., 1968c surface of is chloride solu infusions had in exercise h V0111d enter t The mech has been stud (19723) found “nine gracil was blocked 0 Pump inhibitc action of pot ha+-K+-ATPase of the Vascu] (Binge Et al. chEmQES Prodi ProducEd an : gradual incrr 10 forearm was not blocked by atropine or a B-receptor blocking agent. Baetjer (1934) found that potassium—induced dilation was not affected by curare. With isolated bovine facial arteries Konold et al. (1968b) found that tetrodotoxin did not block potassium-induced vasodilation, suggesting that local nerves are not involved. In further studies (Konold et al., 1968c) these workers found that rinsing the external surface of isolated bovine facial arteries with potassium chloride solutions produced the same vasodilator effect as infusions had. This further supported a role for potassium in exercise hyperemia, as the released potassium presumably would enter the vessels from the tissue fluid. The mechanism of potassium—induced resistance changes has been studied by several investigators. Chen et al. (1972a) found that the normal response of the isolated canine gracilis muscle to both hyperkalemia and hypokalemia was blocked or reversed by preetreatment with the sodium Pump inhibitor ouabain. This suggested that the vasodilator action of potassium was related to stimulation of membrane Na+—K+—ATPase, resulting in hyperpolarization and relaxation of the vascular smooth mu5cle cell. In further studies (BraCe et al., 1973) the time course of the resistance changes produced by potassium was studied. Hyperkalemia Produced an initial decline in resistance, followed by a gradual increase in resistance to values above control levels in fiv changes in re cuter model 0 gested that t the electroge try constrict cellular sodi excellent re Gebert response of ‘ muscle cells vasodilator The effect of “red by means CeIltrations c Produced very arterial strj the Presence chloride in 1 a1ternate hy} latioh was 01 stimulate Cy ‘ prminced dile tent of the I somatiOn . r. 11 levels in five minutes. The time course closely paralleled changes in resting membrane potential predicted by a com— puter model of a vascular smooth muscle cell. It was sug- gested that the initial dilation was due to stimulation of the electrogenic sodium—potassium pump, whereas the second— ary constriction was due to the effect of changes in intra- cellular sodium on the pump. Anderson (1976) presents an excellent review of this subject. Gebert et al. (1969) investigated the mechanism of the response of isolated arteries to potassium. Depleting muscle cells of potassium by cooling did not prevent the vasodilator response to increased extracellular potassium. The effect of potassium on the membrane potential was meas— ured by means of the sucrose gap technique. Dilating con- centrations of potassium sulfate and potassium chloride Produced very small or no depolarization. 42K—labelled arterial strips did not show increased potassium efflux in the presence of dilating concentrations of potassium chloride in the external medium. Due to this evidence an alternate hypothesis for the mechanism of potassium vasodi— lation was offered. It was suggested that potassium might Stimulate cyclic AMP for the following reasons. Cyclic AMP produced dilation, and even after cold storage the ATP con— tent of the muscle cells was high enough for cyclic AMP formation. The phosphodiesterase inhibitor theophylline also produ sible that stimulation potassium. and frog. creased pot and for som tated by in However, the dilation see flllid potass hisher than tVIP-thirds o bl locally I Strated tha affect the crease in r tional Capi cases, Kje finding up during Cal f mildew .. 12 also produced vasodilation. Thus, it was considered pos- sible that cyclic AMP mediates potassium vasodilation. Baetjer (1934) was one of the first to report increased plasma potassium following muscular exercise in cats pro- duced by somatic nerve stimulation. Sympathetic nerve stimulation did not consistently produce increased plasma potassium. Fenn (1937) confirmed these results in the rat and frog. Kjellmer (1961), in cat calf muscles, noted in— creased potassium levels in venous plasma during exercise and for some time afterwards. The increase could be imi- tated by intraarterial infusions of isotonic potassium salts. However, the infusions only produced one—third of the vaso— dilation seen during exercise. Assuming that interstitial fluid potassium concentration during exercise would be higher than plasma potassium concentration, approximately two—thirds of exercise vasodilation could be accounted for by locally released potassium. In addition, it was demon— strated that both exercise and potassium infusion probably affect the same vascular sections, as rate of flow, in— crease in regional blood volume, and opening up of addi— tional capillaries were well correlated between the two cases. Kjellmer (1965) further confirmed these results, finding up to a 100% increase in venous plasma potassium during calf muscle contractions. Potassium infusions mimicked the vascular response to exercise: decreased flow 30 seconds. not return t Venous 001106 greater than Scott et al. gracilis mus< once with ext 0f exercise. Sium release dilation (Hi Potassium pr the Cat sole release and that in cat during a po Iespouse to Norepinephr‘ 13 accompanied by a proportionate increase in the capillary filtration coefficient and no changes in capillary perme— ability or capacitance vessel dilation. Laurell and Pernow (1966) found that the elevated plasma potassium levels following exercise in humans fell rapidly after the exercise, reaching control levels within 30 seconds. Other parameters, such as pH and lactate, did not return to normal levels as quickly. The arterio—deep venous concentration difference in potassium was much greater than the arterio—superficial venous difference. Scott et a1. (1970) found that in the dog hindlimb and gracilis muscle the venous potassium concentration rose at once with exercise and remained elevated during five minutes of exercise. In the cat gastrocnemius 20% of maximal potas— sium release was associated with 20 to 70% of maximal vaso— dilation (Hilton and Hudlicka, 1971). Further increases in potassium produced only small increases in blood flow. In the cat soleus there was no relationship between potassium release and exercise hyperemia. Kjellmer (1960) reported that in cat calf muscles no exercise hyperemia occurred during a potassium-induced vasoconstriction, although the response to injected vasodilators was not blocked. Norepinephrine—induced vasoconstriction did not prevent exercise hyperemia. Knochel and Schlein (1971) found that in canine gracilis muscles which had been potassium—depleted accompanied muscle with the vasodila dilation was Pressure to Portant role lFurther evid tion of exam 190110ng a s transient in crease occur tion Which a noted elevat “Scular con Slum remains ConfiIued h 14 by 25% the vasodilator response to exercise was much dimin- ished. A 10% depletion had no effect on exercise dilation (Anderson et al., 1972). Chen et al. (1972a) found that vasodilation still accompanied exercise after pre—treatment of the gracilis muscle with ouabain.> However, two changes were apparent in the vasodilator response. After ouabain the onset of the dilation was delayed, and the time required for perfusion pressure to reach a minimum stable level was prolonged. Since ouabain blocked the dilator response to hyperkalemia, it was suggested that potassium probably does have an im- portant role in the initiation of exercise hyperemia. Further evidence that potassium may cause the initial dila— tion of exercise was provided by Mohrman and Abbrecht (1973). Following a very brief tetanus in dog muscle, there was a transient increase in venous plasma potassium. The in— crease occurred rapidly enough to account for the vasodila- tion which immediately occurred. Tominaga et al. (1973a) noted elevated venous plasma potassium at the start of muscular contractions in the canine hindlimb. The potas- Sium remained elevated during an eight minute period of exercise. However, potassium levels rapidly returned to control following cessation of stimulation and showed no correlation with vascular resistance. These results were Confirmed by Morganroth et a1. (1974). Several sium cannot b response to level of pota kalemic durin skeletal musc Also, the mag greater than potassium a1 accounted fo occurred. the venous p slightly abo tion. The sour aPPears to be sive depletin exercise. st tilt plasma p Perfus'mg Wi ”93368 were 6°? red cell plasma. Kin magsium ll 15 Several workers have published data showing that potas— sium cannot be the important mediator of the maintained response to exercise. Anderson et al. (1972) changed the level of potassium in perfusing blood from normal to hypo— kalemic during the dilation of simulated exercise in canine skeletal muscle. No change in vascular resistance occurred. Also, the magnitude of exercise resistance changes were much greater than could be induced by local changes in plasma potassium alone. Only a 10% change in resistance could be accounted for by potassium, whereas a 32% resistance decrease occurred. Radawski et al. (1972b) found potassium levels in the venous plasma from the exercising gracilis muscle only slightly above arterial levels after two hours of stimula- tion. The source of the potassium efflux during exercise appears to be skeletal muscle. Sreter (1963) found progres— sive depletion of intramuscular potassium with prolonged exercise. Scott et al. (1970) found a rise in venous efflu— ent plasma potassium concentration during exercise while perfusing with red cell-free fluids. Also, potassium in- creases were exaggerated with constant flow. In addition, dog red cells have a potassium concentration similar to plasma. Kilburn (1966) observed no change in erythrocyte POtassium levels during exercise. Since both potassium and hydrogen ion levels were elevated in the venous plasma from else, would ' In addition, potassium wo potassium. fibers are t during exerc erre- betueen bloo reported the muscle decret that muscle : He Suggested blood flow. flow was dir smPtion ove (1953) showe tines Contrc Severn: Smetal mu: iihb increa 0qu“ Satu l6 exercising human muscles, it was suggested that the mechan— ism of the potassium increase was an exchange of hydrogen ions for muscle cell potassium ions. Hypoxia and acidosis, both of which are believed to occur during prolonged exer— cise, would increase potassium leakage from muscle fibers. In addition, a very small decrease (0.5%) in muscle fiber potassium would produce a large increase in extracellular potassium. The evidence therefore indicates that muscle fibers are the source of the potassium efflux observed during exercise. Oxygen. Several early studies suggested a connection between blood flow and tissue oxygen uptake. Verzar (1912) reported that decreasing the flow to resting cat skeletal muscle decreased oxygen uptake, although venous blood from that muscle still contained substantial amounts of oxygen. He suggested that oxygen consumption is limited by the blood flow. Pappenheimer (1941a) noted that muscle blood flow was directly proportional to its rate of oxygen con— Sumption over a considerable range. Hudlicka and Vrbova (1958) showed that oxygen consumption increased to five times control during muscle stimulation. Several workers have reported the effects of oxygen on Skeletal muscle vasculature. Blood flow in the canine hind— limb increased to three times the normal level as arterial Oxygen saturation declined from 100% to zero (Ross et al., 1962; ntting reported the dilation in content over had little e beds (Molnar strated that total-body v from muscle to 30 mm Hg showed extr tissue P02 r latter resul more sensiti Results from Similar. Th Oxygen may i Other 5 Melt dilates tion in the the constrit 902 remaine Studied the Pheres at r This proceé 4—4 17 1962; Attinger et al., 1967). Daugherty et al. (1967) reported that only P02 values below 40 mm Hg caused vaso- dilation in the canine hindlimb. Alterations of oxygen content over the range of normal arteriovenous differences had little effect on dog forelimb and hindlimb vascular beds (Molnar et al., 1962). Guyton et al. (1964) demon— strated that carbon monoxide or cyanide poisoning produced total-body vasodilation. Perfusing isolated small arteries from muscle with bloods in which P02 was decreased from 100 to 30 mm Hg caused progressive vasodilation. The arteries showed extreme conductance changes even in the normal tissue P02 range. Carrier et al. (1964) confirmed these latter results and found that smaller vessels were much more sensitive to decreases in P02 than larger ones. Results from constant flow and constant pressure series were similar. Therefore, these studies indicate that lack of oxygen may induce vasodilation in small arteries. Other studies further support the theory that oxygen lack dilates arterioles. Hyperoxia produced vasoconstric— tion in the canine hindlimb (Bachofen, 1971). The site of the constriction was the arterioles, as capillary and tissue PO2 remained constant (Duling, 1972). Reich et al. (1970) studied the effect of breathing 100% oxygen at three atmos— pheres at rest and after exercise on calf blood flow in man. This procedure Significantly reduced both resting and post-exercis reducing air dilation onl distribution were oriente and had diam ters and art dilate. Dul extravascula similar. In small arteri Venous muscles wer minutes afte from resting steady furtr Venous oxyge to values e: tion within Venous oxyg Venous oxyg heavy work. exercise, 1' tissue (Per (1967), Gr‘ 44.4 18 post—exercise flows. Hutchins et a1. (1974) found that reducing air oxygen concentration to 18% produced a marked dilation only in terminal arterioles, metaarterioles, and distribution arterioles in rat muscle. All of these vessels were oriented transversely to the skeletal muscle fibers and had diameters of less than 40 u. Pre-capillary sphinc- ters and arterioles greater than 40 u in diameter did not dilate. Duling and Berna (1970; 1971) compared intra— and extravascular oxygen tensions in arterioles and found them similar. Intravascular P02 fell progressively from the small arteries to the end of the terminal arterioles. Venous oxygen changes during exercise in human leg muscles were recorded by Carlson and Pernow (1961). A few minutes after starting work venous oxygen saturation fell from resting levels of 53—81% to 22—42%. A slight but steady further decrease occurred with continued exercise. . Venous oxygen saturation increased rapidly after exercise to values exceeding resting levels, reaching 70-89% satura— tion within five minutes. By 15 minutes post-exercise, venous oxygen saturation had returned to resting levels. Venous oxygen saturation never fell below 21%, even during heavy work. Venous lactate and pyruvate levels rose during exercise, indicating increased anaerobic metabolism by the tissue (Pernow and Wahren, 1962). Welch and Stainsby (1967), Greenwood et al. (1965), and Barcroft et al. (1963) confirmed contraction saturation. (1973) found calf muscle correlation the canine h decreases in Conside sole mediate U935) found restricted f vasodilator venous blood higher venot Love (1955) Contraction: also found Calf muscle ranged from sdturation that was fi Were perfo: 44.4 19 confirmed these results, observing a relationship between contractiOn strength and amount of decline in venous oxygen saturation. Mohrman et al. (1973) and Mohrman and Sparks U973) found that in the low constant-flow perfused canine calf muscle resistance changes returned to control with approximately the same time course as oxygen following a brief tetanus. Tominaga et al. (1973a) found a significant correlation between venous P and vascular resistance in 02 the canine hindlimb; decreases in P were accompanied by 0 decreases in vascular resistance. 2 Considerable evidence exists that oxygen cannot be the sole mediator of exercise hyperemia. Anrep and von Saalfield (1935) found that blood collected during exercise with restricted flow in the canine gastrocnemius still had potent vasodilator activity even after reoxygenation. In addition, venous blood collected during rhythmic contractions had a higher venous oxygen saturation than control venous blood. Love (1955) confirmed this latter result during sustained contractions in the human forearm. Morganroth et al. (1974) also found increased venous oxygen saturation in canine calf muscle during the entire period of exercise, which ranged from 20 to 60 minutes. Following exercise, oxygen saturation rapidly returned to control with a half—time that was five—fold faster than resistance. These experiments were performed at constant flow, with exercise induced by dilation was cise. Scott forearm. R produced by blood from a increase of venous PO < creased flon mm Hg. Mus accompanied Venous P02 (1970) repc during exei with hypom hiEther in i 44.4 20 four twitches per second stimulation. Skinner and Costin (1971) reported that although hypoxemia produced vasodila— tion in the isolated gracilis muscle, the magnitude of the dilation was considerably less than that produced by exer— cise. Scott et a1. (1965) found the canine forelimb rela— tively insensitive to changes in oxygen tension over the range of the usual arteriovenous difference. Dornhorst and Whelan (1953) found that breathing 8% oxygen only slightly affected the recovery rate from exer— cise in humans. Likewise, Corcondilas et a1. (1964) found that breathing oxygen had no effect on the dilation produced by brief contractions of varying strengths in the human forearm. Ross et a1. (1964) compared increases in flow produced by exercise and by muscle perfusion with venous blood from a contracting donor muscle. An average flow increase of 173% was produced by exercise, with an average venous P02 of 25 mm Hg. Perfusion with venous blood in- creased flow by only 56%, with an average venous P of 23 0 mm Hg. Muscle exercise during venous blood perfusion was accompanied by an additional 143% flow increase, although venous PO only decreased an additional 3%. Scott et al. (1970) reported that perfusion pressure decreased more during exercise than when the canine hindlimb was perfused with hypoxemic blood, although the venous P0 was kept 2 higher in exercise by ventilation with 95% oxygen. woman et constant-fl twitch ret capillary o corrected v pre-exercis to 71% of Taken play some r cannot be t Althou smooth musc posed in t directly a1 necessary if is necessar states that Of release lated small ferent 0ny 1964) . Si] snggested ‘ Smooth mus Vasodilati Was a dire 44.4 21 Mohrman et a1. (1973) found that with free—flow or high constant—flow perfusion the vascular response to a brief twitch returned to control before the decrease in end- capillary oxygen tension returned to control. Stowe (1974) corrected venous P02 from exercising canine muscle to the preeexercise level, yet assay resistance still decreased to 71% of the exercise level. Taken together, these studies indicate that oxygen may play some role in exercise hyperemia. However, that oxygen cannot be the complete cause appears equally evident. Although the mechanism by which oxygen alters vascular smooth muscle tone is unknown, two theories have been pro- posed in this context. The first states that oxygen may directly alter vascular smooth muscle tone, since ATP is necessary for the contractile mechanism and adequate oxygen is necessary to maintain ATP stores. The second theory States that oxygen acts indirectly by determining the rate of release of some vasodilator substance. Perfusing iso— lated small arteries from muscle with bloods that had dif- ferent oxygen contents produced vasodilation (Guyton et al., 1964). Since no extravascular tissues were present, it was SUggested that decreases in oxygen directly alter vascular smooth muscle. Detar and Bohr (1968b) suggested that the vasodilation accompanying acute hypoxia in isolated arteries was a direct result of lack of sufficient oxygen to maintain ATP levels. tension deve ment of anae Bohr, l968a) canine hindl gen supply p of pure oxyg again sugges smooth muscl the consent: Sindies shm vascular Smc Other s vaSOdilation his (1968 smoth muse by ihCreaSe heme et a1 ADP, AMP, I exercisE in fund ihere from the do IhEIefore s of adenine SOWevEr I KC 22 ATP levels. After chronic hypoxia (6 to 8 hours) muscle tension development was much improved, suggesting develop- ment of anaerobic pathways for ATP synthesis (Detar and Bohr, 1968a). Hyperoxia-induced vasoconstriction in the canine hindlimb was not affected by wide variations in oxy— gen supply produced by different perfusion pressure rates of pure oxygen at three atmospheres (Bachofen, 1971). This again suggests a direct action of oxygen upon vascular smooth muscle, since the varying flows should have altered the concentratiOn of any other vasodilator substance. Studies should be done to determine if the ATP content of vascular smooth muscle actually declines during hypoxia. Other studies indicate that oxygen lack may produce vasodilation by release of some vasodilator substance. Honig (1968) suggested that oxygen sensitivity of vascular smooth muscle may be due to an inhibition of myosin ATPase by increases in inorganic phosphate and AMP during hypoxia. Berne et al. (1971) found increased total tissue levels of ADP, AMP, IMP, adenosine, inosine, and hypoxanthine during exercise in rat skeletal muscle. Dobson et al. (1971) found increased levels of adenosine in the venous effluent from the dog hindlimb during ischemic exercise. It was therefore suggested that hypoxia might induce the release of adenine compounds, which actually produced the dilation. However, Kontos et al. (1968a) found that dipyridamole did not affect thr danced the va he considered dilation by p smooth muscle enhance the \ nents of Kom pounds as me Several hypoxic vaso alone did no thereby ruli Shepherd et non P02 of his small < 9‘th does In it al. (197 thetic gang effect on t therEbY ten Vasodilatic mm uEEn perfO] “m hydrog< (i900) fou 23 not affect the vascular response to hypoxia. This drug en- hanced the vascular response to AMP and ATP. It should also be considered that if the AMP and/or adenosine produce dilation by production and accumulation within the vascular smooth muscle cells themselves, dipyridamole would not enhance the vascular response to hypoxia. Thus, the experi- ments of Kontos do not necessarily eliminate adenosine com— pounds as mediators of hypoxic dilation. Several factors have been ruled out as mediators of hypoxic vasodilation. Scott et a1. (1970) found that hypoxia alone did not cause increased venous potassium levels, thereby ruling this substance out as the mediator. Shepherd et a1. (1973) found that varying the bathing solu— tion P02 of isolated arteries from 680 to 0 mm Hg produced only small changes in arterial CAMP levels, suggesting that CAMP does not mediate oxygen's action on vessels. Hutchins et al. (1974) found that pharmacological blockade of sympa— thetic ganglia, d, B, or histaminergic receptors had no effect on the vascular response to mild systemic hypoxia, thereby tending to rule out nerves as mediators of hypoxic vasodilation. Hydrogen Ion and Carbon Dioxide. Many experiments have been performed to determine whether or not carbon dioxide and hydrogen ions mediate exercise hyperemia. Bayliss (1900) found that increased carbon dioxide concentration produced vas- Lactic acid confirmed th human hand. and Zsoter e 95 on either in the canin trols. Duli dimeter in from zero tc blood PH in for One hour 7'25! yet or SymPath-adr °Y8temic PC( Fleishx PE Changes < 7'0 t0 7.6 1 Carbon diox. increaSeS i the alkatlin hindlimb di strive Chan IeSponSe we "eh’ehted i 24 produced vasodilation in Ringer perfused frog extremities. Lactic acid had a similar vasodilator potency. Diji (1959) confirmed the vasodilator action of carbon dioxide in the human hand. Kester et al. (1952), Deal and Green (1954), and Zsoter et a1. (1961) found that large alterations in pH on either side of 7.4 produced considerable vasodilation in the canine hindlimb. Flows increased to 100% above con- trols. Duling (1973) reported an 18% increase in vessel diameter in the hamster cheek pouch as PCO was increased from zero to 32 mm Hg. Crawford et al. (1959) decreased blood pH in dogs by subjecting them to 20% carbon dioxide for one hour. It was calculated that blood pH declined to 7.25, yet no increase in flow occurred, perhaps due to the sympatho—adrenal discharge which accompanies elevation of systemic PC02' Fleishman et a1. (1957) studied the effects of small pH changes on vessel resistance. Blood pH was varied from 7.0 to 7.6 by hyperventilation and ventilation with 20% carbon dioxide. Small vessel resistance decreased with increases in hydrogen ions and increased with changes to the alkaline side. However, total resistance in the canine hindlimb did not change because of directionally opposite active Changes in artery resistance. The large vessel response was related to nervous connections, as nerve block prevented it. Therefore, in intact limbs, small changes in systemic arte once but migl Kontos e resistance dc tension from saline solut (on mm Hg. duced acidos of acid phos forearm Veno carbon dioxi panied by a at al. (1971 Ventilation him) VdSCula Radawsi in the dog 1 37 to 137 m, reSiStanCe ) (1974) demo; of blood pe arterial in MY Snail )hlnar et a iithough la 25 systemic arterial hydrogen ion would not alter total resist— ance but might alter blood flow distribution. Kontos et a1. (1967) reported that human forearm resistance decreased 43% on increasing venous carbon dioxide tension from 40 to 48 mm Hg by intrabrachial infusion of a saline solutiOn with a carbon dioxide tension greater than 600 mm Hg. In a further study, Kontos et al. (1968c) pro— duced acidosis by a different means. Intraarterial infusion of acid phosphate buffer solutions produced a fall in human forearm venous blood pH from 7.34 to 7.24 and a rise in carbon dioxide tension from 42 to 52 mm Hg. This was accom— panied by a 170% increase in forearm blood flow. Daugherty et a1. (1971) locally reduced femoral blood pH to 7.19 by ventilation with 30% carbon dioxide and observed a fall in limb vascular resistance. Radawski et al. (1972a) reported that at constant flow in the dog hindlimb increases in brachial artery PCO2 from 17 to 137 mm Hg produced a decrease in skin small vessel resistance but had little effect on muscle. Emerson et al. (1974) demonstrated that large stepwise decreases in the pH of blood perfusing dog gracilis muscle produced by intra— arterial infusion of isotonic lactic acid solutions produced only small stepwise decreases in muscle vascular resistance. Molnar et a1. (1962) obtained similar results. Therefore, although large changes in pH appear to alter total vascular - ,..__.. ‘ . resistance, 5 Gollwit: venous pH Che cnenius muscl (1) an initia phase of alk. of acidity. was a small Five to 20 s time when ve had returned the!) Occurre mattiy one In however, con acidity was longer Peric °hserved. '1 5nd intEnsit The last phe CIEaSe in b.) ml by t1 ShangeS had hirer eXerc; 26 resistance, small changes seem to have only a minor effect. Gollwitzer—Meier (1950) was one of the first to study Venous pH changes during exercise in the canine gastro- cnemius muscle. Three phases of pH change were observed: (1) an initial short phase of acidity; (2) a longer-lasting phase of alkalinity; and (3) a protracted, persistent phase of acidity. For example, during a ten second tetanus there was a small shift of pH to the acidic of short duration. Five to 20 seconds after the end of the contraction, at a time when venous outflow was reaching its maximum, the pH had returned to normal. A small pH shift to the alkaline then occurred, with a final return to resting values apprOxi- mately one minute after the contraction. The hyperemia, however, continued for several minutes. The third phase of acidity was not apparent with such a brief tetanus. With longer periods of tetany or exercise, all three phases were observed. The second and third phases varied in duration and intensity depending on how strenuous the exercise was. The last phase of acidity was never associated with an in— crease in blood flow. In fact, flow had often returned to normal by this time. It was concluded that venous pH changes had no relationship to blood flow, either during or after exercise in the preparation used. Tominaga et al. (1973a) obtained results confirming this conclusion. ( I i ) Carlson declined fr< load in hum; work. Scot' flow during muscle with (1964) had monitored p in the isol PH was only Stowe (1974 gracilis mu donor lung. decreaSed a ieVels of 7 rESiStanCe' of the Vent abolished 1 the alkal in reduced tht am' 131150 forearm ex. in VehOus . venous Carj 19.4 mm Hg 27 Carlson and Pernow (1961) reported that venous pH declined from 7.41 to 7.25 during leg exercise at maximal load in humans. The venous pH was 7.24 five minutes after work. Scott et a1. (1970) found a 270% increase in blood flow during sub—maximal exercise in the dog gastrocnemius muscle with only a 0.07 decline in venous pH. Ross et al. (1964) had obtained similar results. Radawski et al. (1972b) monitored pH changes during a two hour period of exercise in the isolated canine gracilis muscle. At two hours venous pH was only slightly below (7.30) control values (7.34). Stowe (1974) perfused the isolated, denervated canine gracilis muscle with blood pumped through an extracorporeal donor lung. The lung was ventilated with gas mixtures which decreased arterial blood pH. A pH decline from control levels of 7.41 to 7.06 produced only an 18% drop in vascular resistance. In addition, the increased vasodilator activity 1 of the venous effluent during steady—state exercise was not abolished by correcting the pH. Kontos (1971) found that the alkaline buffer amine, tromethamine, only slightly reduced the dilation accompanying exercise in the human fore— arm. Also, a three-fold flow increase which accompanied forearm exercise was associated with only a 5 mm Hg increase in venous blood PCOZ. Kontos et a1. (1966) found that venous carbon dioxide tension would have had to rise by 19.4 mm Hg to account for the dilation accompanying five minutes of 11 rise was onl that with re activity inc decrease. 5 minute) pro( activity. 1 acongenital 05 lactate : Yet their f1 ”Ohio and ( dioxide and locally, it tion Chinge lathitude t1 set Of or d that they In severe fixer In One at ‘11- (196 in? SOlUtiO the pH was damn diox hydrogen ic Elgniinant 28 minutes of mild exercise in the human forearm. The actual rise was only 4.5 mm Hg. Gebert and Friedman (1973) showed that with rapid contractions, although tissue hydrogen ion activity increased, the increase was preceded by a transient decrease. Slow stimulation frequencies (less than one per minute) produced only a decrease in tissue hydrogen ion activity. Patients with McArdle's disease, suffering from a congenital absence of phosphorylase, showed no evidence of lactate formation or change of pH in working muscles, yet their functional hyperemia was as great as normal (Tobin and Coleman, 1965). Therefore, although carbon dioxide and hydrogen ions have been shown to be vasoactive locally, it is questionable as to whether their concentra— tion changes during active hyperemia are of sufficient magnitude to be responsible for the dilation, either at on— set of or during sustained exercise. However, it is possible that they may play a role in the dilation of maximal, severe exercise. In one study on isolated bovine facial arteries Gebert et a1. (1969) found that increased PCO2 of the Tyrode bath— ing solution had no effect on the tone of the arteries if the pH was held constant. This indicates that increases in carbon dioxide may not dilate directly but via increases in hydrogen ions. However, Kontos et al. (1968b) found no Significant difference between the vascular response to hypercapnia acidosis in dioxide was that the pH in spite of crosses celI in this casn responsible Severa 1511 of acti. (1970) f0un1 n°h Prevent (1957) obse Persisted i CirCiliating ing lOCal F 0hServed th “109 with Mthush tt dilation wt to ehtirelh acreage p: 3:0“) (196; mm dilrim aree‘thing . 29 hypercapnia with acidosis and that to hypercapnia without acidosis in the human forearm. A direct effect of carbon dioxide was thus indicated. It was mentioned, however, that the pH inside the smooth muscle cells may have declined in spite of bicarbonate infusion, since carbon dioxide crosses cell membranes far more freely than bicarbonate. In this case hydrogen ions, not carbon dioxide, would be responsible for the dilation. Several investigators have further studied the mechan— ism of action of changes in pH and PC02' Kontos et al. (1970) found that alpha and beta adrenergic blockade did not prevent hypercapnic vasodilation. Fleishman et al. (1957) observed that the vascular response to hydrogen ions persisted in the absence of central nervous connections, circulating and locally released catecholamines, and follow" ing local parasympathetic blockade. Molnar et al. (1962) observed that venous potassium levels increased in associ— ation with acidosis, persumably by release from erythrocytes. Although the rise in potassium could explain part of the dilation which occurred, it was not of sufficient magnitude to entirely explain it. Also, nitric acid, which did not increase plasma potassium, produced dilation. Lade and Brown (1963) also observed that skeletal muscle lost potas— Sium during hypercapnia induced by 30% carbon dioxide breathing in dogs. Contraction increased the rate of loss. Additional 5 adenine com; increased 0: Marshall an: tions of ten solutions '1; longed two I Continuous caused flow sion. Howe dilation pr in the Cani Read et al. of 1500 11105 in 01095, C increasing forelimb va flow was he Vasoflilatox 30 Additional studies should be done to determine if efflux of adenine compounds increases with decreases in pH. Osmolality. A large number of studies have shown that increased osmolality is associated with vasodilation. Marshall and Shepherd (1959) found that rapid local injec— tions of two ml of 10-20% sodium chloride or 25—50% dextrose solutions into the canine femoral artery produced a pro— longed two or three—fold increase in hindlimb blood flow. Continuous infusions of these substances for one minute caused flow increases which continued throughout the infu— sion. However, Gazitua et al. (1971) found that the initial dilation produced by hyperosmotic dextrose or sodium chloride in the canine forelimb waned during a ten minute infusion. Read et al. (1960) reported that rapid intravenous injections of 1500 mOsm/kg solutions produced peripheral vasodilation in dogs. Overbeck et al. (1961) demonstrated that locally increasing forelimb osmolality by 100 mOsm/kg decreased forelimb vascular resistance to 71% of the control when flow was held constant. Stainsby (1964) confirmed the vasodilator activity of various hyperosmotic solutions in the plasma—perfused gastrocnemius muscle. Stainsby and Fregly (1968) reported that decreases in resistance were Proportional to increases in plasma osmolality. Large mole- cular weight substances such as dextran were as effective as smaller molecules in reducing resistance. In mic: osmotic-ind1 precapillar} hyperosmoti< cat skeletal veins. The arteriolar ' However, Gel sucrose or isolated b0 Lundva to cat Skel Venous Osmo human forea creased blo deposits of rapidly Whe iSotOnic sc osmolality (Lundvall e that in hun :rose or Sc elity 10 tc ince 4 to l betWeen the Nile 0f res 31 In microscopic studies Gray (1971) localized hyper— osmotic-induced vascular changes to the arterioles and precapillary vessels. Gray et al. (1968) found that hyperosmotic infusions of up to 40 mOsm/kg above control in cat skeletal muscle dilated precapillary sphincters but not veins. The hyperosmolarity also decreased considerably the arteriolar vascular response to sympathetic stimulation. However, Gebert et al. (1969) found that hyperosmotic sucrose or sodium chloride solutions had little effect on isolated bovine facial arteries. Lundvall (1969) found that with hypertonic infusions to cat skeletal muscle, maximal dilation was reached when venous osmolality was raised by 40-100 mOsm/kg. In the human forearm osmolality increases of 15—20 mOsm/kg in— creased blood flow three to five times. Intramuscular deposits of Xe133 in resting muscle were cleared more rapidly when the tracer was dissolved in hypertonic than in isotonic solutions, indicating hyperemia when the hyper- osmolality involved primarily the extravascular compartment (Lundvall et al., 1969). Overbeck and Grega (1970) showed that in humans intrabrachial infusions of hypertonic dex— trose or sodium chloride increased cephalic venous osmol— ality 10 to 23 mOsm/kg and reduced forearm vascular resist— ance 4 to 14%. There was a positive linear correlation between the level Of initial vascular resistance and magni— tude of response to all hypertonic solutions. Stainsby and Barclay (19 start of a tion, indic in the init suggested t the osmotic interstitia addition, t tion of the of their in Mellan data on 051: of five to the Cat res ance associ 05mOlality, dollhced Wit glUCOSe Or 32 Barclay (1971) observed a 15—20 second lag between the start of a hyperosmotic infusion and the start of the dila— tion, indicating that hyperosmolality might not play a role in the initiation of active hyperemia. However, it was suggested that this delay might not apply to exercise, when the osmotically active substances could directly enter the interstitial space surrounding the resistance vessels. In addition, the delay may also have resulted from the dilu- tion of the osmotically active particles at the initiation of their infusion. Mellander et al. (1967) reported some of the earliest data on osmolality changes during exercise. Active hyperemia Of five to 15 minutes duration in the lower leg muscles of the cat resulted in a sustained 80% drop in vascular resist- ance associated with a 38 mOsm/kg rise in effluent plasma osmolality. The increase in osmolality became more pro— nounced with time. Intraarterial infusion of hypertonic glucose or xylose solutions at rates producing similar changes in regional osmolarity produced a vascular response pattern similar to that of exercise. Lundvall et al. (1969) and Mellander and Lundvall (1971) found similar results in the human forearm during exercise. Lundvall (1972) found that tissue fluid hyperosmolality during exercise was largely due to increases in sodium and lactate concentra— tions, based on analyses of venous plasma. Scott Although ve minute of e to normal 1: further. I ality by in blood flow. duce exerci firmed thes in venous c than during that althor eight minut corrElatiOr 80°“ returr and HUdlick and Venous (1972) folir longed, St} arteriwehc exerciSe ir i1974i four of exErcise 31‘ below Cc Smilar res 33 Scott et al. (1970) obtained different results. Although venous osmolality increased during the first minute of exercise in the canine hindlimb, it had returned to normal by the fifth minute whereas blood flow increased further. In addition, comparable increases in venous osmol— ality by infusions failed to produce similar increases in blood flow. Much larger increases still failed to repro— duce exercise hyperemia. Scott and Radawski (1971) con— firmed these results. They also observed a greater increase in venous osmolality with exercise during constant flow than during natural flow. Tominaga et al. (1973a) found that although venous osmolality remained elevated during eight minutes of exercise in the dog hindlimb, there was no correlation with vascular resistance changes. Osmolality soon returned to control levels following exercise. Hilton and Hudlicka (1971) found no relationship between exercise and venous osmolality in the cat gastrocnemius. Lundvall (1972) found a decline in venous osmolality during pro- longed, strenuous work. Radawski et al. (1972b) found no arteriovenous difference for osmolality after two hours of exercise in the canine gracilis muscle. Morganroth et al. (1974) found similar results after 20 and 60 minute periods Of exercise. During recovery, venous osmolality was actual— ly below control levels. Murray et al. (1974) also obtained similar results. In addition, they studied venous osmolality changes in lmOsm/l in concluded t account for sustained d tion to acc itY that ve ill during osmolality flow. It to role in the Lundvall (1 of the hype during heav be attrilout The me lation has relatiOn be HUSCle Was vein Strips Krebs Solut time SPOnta excitation dedium. Me (1971) four 34 changes in response to a one second tetanus, finding only a l mOsm/l increase for a 30% fall in resistance. It was thus concluded that changes in osmolality were too small to account for the dilation following brief tetanus or the sustained dilation of long exercise and in the wrong direc- tion to account for post—exercise hyperemia. The possibil~ ity that venous osmolality may be less than tissue osmolal- ity during exercise should also be considered, since outflow osmolality from the tissue may be diluted by non—nutritional flow. It was suggested that hyperosmolality might play a role in the first several minutes of exercise hyperemia. Lundvall (1972) reported that during light work in cats 40% of the hyperemia could be attributed to hyperosmolality; during heavy, short-term exercise 60% of the dilation could be attributed to hyperosmolality. The mechanism by which hyperosmolality induces vasodi- lation has been investigated by Arvill et al. (1969). A relation between cell volume and activity in vascular smooth muscle was found. Intracellular fluid volume of rat portal vein strips decreased 34% after 20 minutes of exposure to Krebs solution plus 150 mmoles/l of sucrose. During this time spontaneous muscle activity was inhibited. Immediate excitation was produced on return to a normal isoosmotic medium. Mellander et al. (1967) and Mellander and Lundvall (1971) found that hyperosmolality exerted negative chronotropic smooth muscl the potassir restored smr controls. might activl Sium concen‘ water from and relaxat m the adenine ”fury and s that intraa hindlimbs p 2 Y doubled effect was fifteenth t demonstrate longed thar (1954) four after inqu DObSon 8t 2 required u NIP Wire 25 «QSPeCtiVe: 35 chronotropic, dromotropic, and inotropic effects on vascular smooth muscle activity of the portal vein. A doubling of the potassium concentration of the hyperosmotic solution restored smooth muscle activity to a pattern similar to controls. It was therefore Suggested that hyperosmolality might actively dilate by increasing the intracellular potas- sium concentration subsequent to osmotic withdrawal of water from the cells, thereby producing hyperpolarization and relaxation. Other ion fluxes could also change. Adenine Compounds. The potent vasodilator capacity of the adenine compounds has been known since the studies of Drury and Szent—Gyorgyi (1929). Folkow et al. (1948) found that intraarterial injection of 0.1-0.2 Y of ATP into cat hindlimbs produced considerable vasodilation. Injection of 2 Y doubled the rate of flow. The threshold dose for the effect was 0.05—0.1 y, a potency about one-fifth to one- fifteenth that of acetylcholine. Sydow and Ahlquist (1950) demonstrated the vasodilator effect of AMP was more pro— longed than that of adenosine in the dog. Duff et al. (1954) found a three—fold increase in muscle blood flow after infusion of 16 pg ATP/min. into the human forearm. Dobson et al. (1971) found that the arterial blood levels required to elicit detectable vasodilation for ATP, ADP and 8 8 AMP were 28 x 10' M, 27 x 10‘8 M, and 31 x 10‘ M, respectively. Mg-ATP was a more powerful vasodilator than Na-ATP (Duf vasodilator small vesse Kjellmer an and venous caused no c were four t coronary b1 vasodilatic infusion in duced Oxyge tion, incre bodies, and tate. Forre: that ATP m; released f: studies by ATP did no- iOn leVel of are by “as 3 ug/g the intrac the firefl 36 Na-ATP (Duff et al., 1954). Frohlich (1963) localized the vasodilator effect of the three adenine nucleotides to small vessels, particularly the arterioles, in the dog. Kjellmer and Odelram (1965) found dilation of both arterial and venous vessels with ATP infusion in cats. The ATP caused no change in capillary permeability. ATP and ADP were four times as potent as AMP and adenosine in increasing coronary blood flow (wolf and Berne, 1956). In addition to vasodilation, Raberger et al. (1973) found that adenosine infusion into the canine hindlimb was associated with re- duced oxygen consumption, increased carbon dioxide produc— tion, increased uptake of glucose, glycerol, and ketone bodies, and increased release of free fatty acids and lac— tate . Forrester (1966) provided the first direct evidence that ATP may be involved in active hyperemia. He found ATP released from active frog skeletal muscle in vitro. Further studies by Boyd and Forrester (1968) demonstrated that the ATP did not come from muscle cell damage, as the potassium ion level in the plasma did not increase. The mean output of ATP by the muscles for a 30 minute period of stimulation was 8 ug/gm muscle, amounting to a loss of about 1-2% of the intracellular ATP. Forrester and Lind (1969), using the firefly extract analysis for ATP, identified ATP in human plasma, both in resting and exercising subjects. Venous ATP 1 above restii passing thr< tration was blood flow . ing contrac the contrac Forrester ( no ATP coul from an occ effluent f: This ATP /n ATP/9111 Wet chOunt the Cludefl that ucl/Hlin.) t< Hamillion (: Plasma fro} mortgage i Perqued. Siderably differenCe Chen ent (hiring Control A'] 37 Venous ATP levels of exercising subjects rose consistently above resting values, indicating addition of ATP to blood passing through the muscle bed. The mean plasma ATP concen— tration was 500 ng/ml at rest. The pattern of forearm blood flow and the ATP output were similar, increasing dur- ing contraction, rising to a peak value immediately after the contraction, and subsiding slowly to control values. Forrester (1972) found that with a more refined technique no ATP could be identified in the venous effluent plasma from an occluded forearm without exercise. In the venous effluent from occluded, exercising muscle, 0.033 to 1.0 nmole ATP/m1 of plasma was observed, amounting to 1.5 ug ATP/gm wet weight of forearm muscle. After taking into account the amount of ATP lost by degradation, it was con- cluded that ATP was released in sufficient quantities (15 ug/min.) to mediate active hyperemia. Forrester and Hamilton (1975) found increased levels of ATP in the venous plasma from exercising cat soleus muscle. No significant increase in ATP occurred when the muscle was artificially perfused. In this latter case control ATP levels were con— siderably higher than in the blood perfused muscle, the difference being 0.85 uM. Chen et al. (1972b) also found ATP in the venous efflu— ent during active hyperemia in canine skeletal muscle. Control ATP levels were 206 ng/ml plasma in the femoral artery and hyperemia, 450 ng/ml r from 52 to with increa were still Parkii nucleotide: exercise it severity 0 exercise, MP to app Abood et a during dep Scott Cising Can the kidney Same IESpc and AMP bu in genes the left l ing a rapj tor frOm 1 biOasSay : 38 artery and 165 ng/ml in the femoral vein. During active hyperemia, femoral venous ATP levels rose to approximately 450 ng/ml plasma. Femoral venous AMP levels also increased from 52 to 188 ng/ml plasma. Increased flow correlated with increased ATP levels. Post-tetany ATP and AMP levels were still considerably elevated above control levels. Parkinson (1973) found that levels of all three adenine nucleotides rose in the venous plasma following a rowing exercise in humans. The rise was proportionate to the severity of the exercise. AMP appeared only during severe exercise. ATP and ADP rose to approximately 500 ng/ml and AMP to approximately 300 ng/ml during severe exercise. Abood et al. (1962) reported efflux of ATP from frog muscle during depolarization. Scott et al. (1965) found that the blood from the exer— cising canine hindlimb dilated the forelimb but constricted the kidney vasculature. They further demonstrated that the same responses could be produced by injection of adenosine and AMP but not ATP or other substances. With two kidneys in series ATP injected into the left renal artery dilated the left kidney but constricted the right kidney, indicat- ing a rapid breakdown of ATP or a release of a vasoconstric- tor from the donor kidney. Scott et al. (1971), in further bioassay studies in which lag time was considerably decreased and sensitivity increased, found that hindlimb blood durint afall in k. resistance : limb constr indicate th active hype degradation Brashe in venous 1: mill exerci Amy was c the rapid h Whole blood Hilton il9l Cat gastrm effluent a1 Sensitive 1 between ti] :ESPOnsibL Wham ti its and Ve hyperemia dilatmn a adrenal di LQCal Venc 39 blood during nerve stimulation at 6 cps or tetany produced a fall in kidney resistance. On stopping tetany kidney resistance rose. Stimulation plus occlusion in the hind— limb constricted the kidney. Therefore, these studies indicate that ATP or ADP may indeed be released during active hyperemia in skeletal muscle but also that their degradation in the blood is probably very rapid. Brashear et al. (1968) found no detectable increases in venous plasma ATP or ADP following five minutes of tread— mill exercise in humans. However, the sensitivity of their assay was only 0.2 mg%, and they used only cold to prevent the rapid breakdown of these compounds which occurs in whole blood and plasma (Collingsworth et al., 1974). Hilton (1962) found no ATP released during contractions in cat gastrocnemius muscle. This was true both of venous effluent and tissue fluid. The fluorometric assay used was sensitive enough to detect 100 ng/ml of ATP. ATP breakdown between time of collection and time of assay may have been responsible for these negative results. Kjellmer and Odelram (1965) suggested that since ATP dilates both arter— ies and veins, it would be unlikely to mediate exercise hyperemia involving the whole organism, where arteriolar dilation and venous constriction occur due to sympatho— adrenal discharge. However, Nagle et al. (1968) showed that local venous resistance decreases with active hyperemia. Dobson et a venous plas tions in th were used t the isolati balthing med 30 minutes enough to ( et al. (19' or followii Therefore, iuss, it i adenine nu Consi may mediat seVeral 0t adenOSine the hYpoxi tion that peerSate hearts. I produCts : the Subst- resultant :lmes mob 40 Dobson et a1. (1971) detected no adenine nucleotides in the venous plasma following five minutes of ischemic contrac- tions in the dog hindlimb. However, no adequate measures were used to prevent adenine nucleotide breakdown during the isolation of the plasma. No ATP was detected in the bathing medium of the isolated frog sartorius muscle after 30 minutes of contraction; the assay used was sensitive enough to detect 1 x 10“8 M ATP concentrations. Bockman et al. (1975b) found no increase in venous plasma ATP during or following tetanic contraction in dog or rat hindlimbs. Therefore, since we have both negative and positive find— ings, it is obviously controversial as to whether or not adenine nucleotides mediate active hyperemia. Considerable evidence exists indicating that adenosine may mediate blood flow changes in skeletal muscle and several other tissues. Jacob and Berne (1961) proposed that adenosine may be the mediator of coronary vasodilation in the hypoxic heart. This proposal is based on the observa— tion that inosine and hypoxanthine were present in the Perfusate and coronary sinus blood of severely hypoxic hearts. Due to the rapid breakdown of adenosine to these products in the body, it was suggested that adenosine was the substance released by hypoxic myocardial cells, with resultant vasodilation. Berne (1963) found that 3 to 27 times more moles of inosine and hypoxanthine were released from the hy] flow when i] nary artery arterial My heart. Ther decreased 0 adenine nuc diffuse out the interst creased cor the oarygen of adenine ism would 1 the heart.s In su} ddenoSine ' iiOns of C1 adenosine into the s The nemal was approx ThESe reSu levelS Cot .\ ‘ roper 0X} 41 from the hypoxic heart than were required to double coronary flow when infused as moles of adenosine into the left coro- nary artery. These substances were not detected in the arterial blood or in venous blood from a well—oxygenated heart. Therefore, Berne proposed that during hypoxia the decreased oxygen tension caused breakdown of heart muscle adenine nucleotides to adenosine. The adenosine would then diffuse out of the cells and reach the coronary arteries via the interstitial fluid, with resultant dilation. The in— creased coronary blood flow which would occur would raise the oxygen tension, thereby reducing the rate of degradation of adenine nucleotides to adenosine. This feedback mechan- ism would thus provide a way to adjust coronary flow to meet the heart‘s oxygen needs. In support of Berne's theory Imai et al. (1964) found adenosine in underperfused rabbit myocardium under condi— tions of complete ischemia. Rubio and Berne (1969) found adenosine released by the normal myocardium continuously into the surrounding interstitial fluid (pericardial fluid). The normal adenosine concentration of the pericardial fluid was approximately 10.9 x 10—7 M, presumably a basal level. TheSe results indicate that interstitial fluid adenosine levels could regulate coronary blood flow to maintain a PrOper oxygen balance. Dobsor fold in thy tion. Vent fold, resp. 0.7 nmole/. muscle. 3. tissue 1MP ischemic 5 et al. (19 10 and 25 not after after Cont below Cont adenosine Qt 511- (19 ing Contra Tomin Venous pla the ‘109 hi min-/loo g fOllowed i fOUnd that inorganic during e)“ :3 this WE 42 Dobson et al. (1971) found adenosine to increase five- fold in the venous plasma following ischemic muscle contrac— tion. Venous inosine and hypoxanthine increased 22 and 270- fold, respectively. Tissue adenosine levels increased from 0.7 nmole/gm to 1.5 nmole/gm in ischemic, contracting muscle. Berne et al. (1971) found large increments in tissue IMP and small increases in tissue adenosine during ischemic skeletal muscle contractions in the rat. Bockman et al. (1975a) found muscle adenosine levels elevated after 10 and 25 minutes of contraction in the dog hindlimb but not after fiVe minutes of contraction. Thirty minutes after contraction tissue adenosine content was slightly below control levels. No increase in the venous plasma adenosine levels occurred during the contractions. Bockman et al. (1975b) found venous adenosine levels increased dur— ing contraction in artificially perfused rat hindlimbs. Tominaga et al. (l973d) also found increased levels of venous plasma adenosine and/or AMP during contractions in the dog hindlimb. Levels rose from a control of 259 nmoles/ min./100 gm to 1030 during contractions. An increase also followed ischemic contractions. Watanabe et al. (1973) found that the intracellular changes in ammonia, lactate, inorganic phosphate, potassium, and oxygen which occur during exercise depressed adenosine and AMP aminohydrolases. In this way AMP or adenosine could accumulate during 'I At“? ::t—:::_."~, exercise an found that during cont increased r adenosine c plasma durj adenosine n tration. ’. and/0r adel under cons: Recen“ Sine in th‘ Thomas et ‘ adenosine Berne Et a 0f adenosi 10119le pr arterialh; the blOQd- (1975) fou stimulatic PISSSUre’ adenosine hl’drogen j 43 exercise and produce dilation. Tominaga et al. (1973c) found that recovery of injected l4C—adenosine decreased during contractions in the dog hindlimb, indicating increased uptake of the 14C during exercise. However, adenosine concentration remained constant in the venous plasma during exercise. Therefore, increased release of adenosine must have occurred to maintain a constant concen— tration. Tominaga et al. (1975) found increases in AMP and/or adenosine with contractions and ischemic contractions under constant flow but not with constant pressure. Recently, studies have been done on the role of adeno— sine in the regulation of kidney and brain blood flow. Thomas et al. (1975) found no correlation between tissue adenosine levels and renal artery perfusion pressure. Berne et al. (1974) demonstrated that topical application of adenosine to cat pial arterioles produced dilation roughly proportional to the dose used. However, intra— arterially administered adenosine did not appear to cross the blood-brain barrier. In further studies Rubio et al. (1975) found that brain hypoxia produced by electrical stimulation of the cortex, decreased arterial perfusion pressure, or ventilation with 5% oxygen all increased tissue adenosine levels. It was suggested that adenosine and hydrogen ions may act synergistically to regulate cerebral blood flow. Three served inc: blood cells adenosine c the predomi is via AMP addition, 2 Cells, re51 Rllbio et aj tidase act; and to lOCi to blood v‘ Verts AMP . skeletal m of the blo‘ high Gnoug 0ther 9&83 throu becOme Che reported e the rat Sc When 14C‘A 44 Three possibilities exist for the source of the ob- served increases in adenine compounds: nerve, muscle, and blood cells. Early studies indicated that extracellular adenosine could not have come from skeletal muscle, since the predominant pathway for AMP breakdown in skeletal muscle is via AMP aminohydrolase to IMP (Imai et al., 1964). In addition, adenosine deaminase levels are high within muscle cells, resulting in low intracellular adenosine levels. Rubio et al. (1973) localized skeletal muscle 5'—nucleo— tidase activity by a histochemical technique to endothelium and to localized zones within muscle cells in close proximity to blood vessels in rats and guinea pigs. This enzyme con— Verts AMP to adenosine. Therefore, this study showed that skeletal muscle adenosine is produced only in the vicinity of the blood vessels, where it could reach a concentration high enough to produce vasodilation.* Other studies indicate that adenine nucleotides may Pass through muscle cell membranes and thus could possibly become chemical messengers. Chaudry and Gould (1970) reported evidence for the direct entry of ATP and ADP into the rat soleus muscle. More l4C—ATP was found in the cells when l4C—ATP or l4C~ADP were added to the incubation medium *For a more complete discussion of adenine compound metabol— ism in skeletal muscle, see pp. 56’60, Collingsworth, A. H., M. S. Thesis, 1973. than when 1 et al. (197 (1962) four depolarizat ATP may lea tern of the directly t] Blood nucleotide: found that ing from E Show to c et all, 19 unPublishe Siderable Consi he a sourc published Suggested axon rem cocaine at attributa Cells, Si] 45 than when l4C—adenosine was added to the medium. Chaudry et al. (1975b) further confirmed this finding. Abood et a1. (1962) found efflux of 32P—labelled ATP during muscle cell depolarization. Boyd and Forrester (1968) suggested that ATP may leave muscles by way of the transverse tubular sys- tem of the sarcoplasmic reticulum rather than by passing directly through the membrane. Blood cells may have been the source of the adenine nucleotides observed in some studies. Forrester (1972) found that control levels of ATP were eliminated by switch— ing from EDTA to citrate as anticoagulant. EDTA has been shown to cause adenine nucleotide efflux from cells (Abood et al., 1962; Bockman et al., 1975b; Collingsworth and Liu, unpublished). Damaged platelets could also release con— siderable quantities of ATP and ADP (Holmsen, 1967). Considerable evidence has accumulated that nerves may be a source of extracellular ATP.* Burnstock (1972) has published an excellent review of this area. Hilton (1953) suggested that post—exercise hyperemia was due to a local axon reflex with an unknown chemical transmitter, since cocaine abolished the response. Honig and Frierson (1974) attributed post—exercise hyperemia to intraarterial ganglion cells, since procaine partially blocked the response. “— *For a more complete discussion of this subject see pp. 29-35: Collingsworth, A. H., M. S. Thesis, 1973. I —-:——"—-: Again, a t found ATP stimulatio rabbit. T by erythro vented the Ballard et lator nerv 0f the ade Su (1 thoracic a It was tra transmural accounted tides made Phenoxyben 3H Efflux. Stimulatic siderable hereaSed suggestinS substaHCes In a preparatic reported 1 46 Again, a transmitter was not identified. Holton (1959) found ATP released into the venous effluent upon antidromic stimulation of the great auricular nerve or the skin in the rabbit. The ATP levels observed could not be accounted for by erythrocyte hemolysis. Degeneration of the nerve pre- vented the ATP efflux upon stimulation of the skin. Ballard et al. (1970) demonstrated non—cholinergic vasodi— lator nerves in the canine paw. It was suggested that one of the adenine compounds might be the transmitter substance. Su (1975) found 3H—adenosine taken up by the isolated thoracic aorta, ear artery and portal vein of the rabbit. 3H—ATP within the cells. On It was transformed mainly to transmural stimulation tritiated adenosine and inosine accounted for 80% of the total released 3H. Adenine nucleo— tides made up the rest. Tetrodotoxin abolished the response. Phenoxybenzamine prevented contraction but did not prevent 3H efflux. Kuperman et al. (1964) found that electrical stimulation of the frog sciatic nerve resulted in a con- siderable increase in labelled AMP and ATP efflux. Increased calcium efflux also occurred during stimulation, suggesting a possible binding or interaction of the two Substances. In a study on the rat phrenic nerve—hemidiaphragm Preparation, Silinsky and Hubbard (1973) and Silinsky (1975) reported the release of ATP from motor nerve terminals. No stimulus normal pre} tion, apprc bath after period. S: was presen‘ magnesium-: minimize A‘ ATP levels Control 19' illdUCEd by that the A Since acet the fact t affect the restllt in firmer ev 51 source 47 No stimulus-specific release of purines was found in a normal preparation. However, in a highly curarized prepara— tion, approximately six times as much ADP was found in the bath after stimulation as was found there in the control period. Since no ATP was found, Silinsky suspected ATPase was present, and therefore a perfusate consisting of magnesium—free saline with 0.1 mM calcium added was used to minimize ATPase activity. In these curarized preparations ATP levels in the bath increased to approximately four times control levels upon nerve stimulation and depolarization induced by a hyperosmotic bathing medium. It was suggested that the ATP was released from the motor nerve terminals, since acetylcholine release paralleled ATP release. However, the fact that a hyperosmotic solution might in some way affect the permeability of the muscle membrane and thereby result in the efflux of ATP was not considered. Some further evidence was presented in favor of the nerve termin— al source of the ATP, however. When no calcium was present in the hyperosmotic bathing medium (a condition which could prevent acetylcholine release upon nerve stimulation), no ATP efflux occurred on nerve stimulation. It was suggested that the ATP might function in complexing acetylcholine in nerve storage vesicles. In a study to extend Silinsky's 1973 results, however, Kato et al. (1974) were unable to find ATP released upon stimulation tion avoide Stimulation potassium ; by both ty; tected duri ant type of a5 a possil ”as found :1 from normal but: conta‘n rtisulted i] fibers and HoweVer, m and the so The m. Yet largel and neoant vasodilati at 51L (19 may be Cou leedleman prostaglan Cate an it. prOstaglar 48 stimulation of cat superior cervical ganglion. This prepara- tion avoided any artifacts due to the presence of muscle. Stimulation was produced electrically or with a high potassium perfusate. Although acetylcholine was released by both types of stimulation, ATP release could not be de— tected during either type. However, the fact that a differ— ent type of nerve preparation was used must be considered as a possible source of the negative results. ATP release was found immediately following a change in the perfusate from normal Ringer's to one lacking calcium and magnesium but containing barium and EDTA. This modified perfusate resulted in a transient depolarization of postaganglionic fibers and a contraction of the nicitating membrane. However, no concurrent release of acetylcholine was found, and the source of the ATP was not determined. The mechanism by which adenine compounds dilate is as yet largely unknown. Folkow (1949b) reported that atropine and neoantergan (antihistamine) did not block ATP—induced vasodilation. Denervation also had no effect. Weissel et al. (1973) suggested that the dilator action of adenosine may be coupled in some way to its metabolic effects. Needleman et al. (1974) found that ATP and ADP stimulated Prostaglandin release from many tissues. This would indi- cate an indirect dilator effect of adenine compounds, with Prostaglandins actually accomplishing the dilation. Adenir membrane pe acoupled s ability to potassium. result in 1 determine a dilate. Ether Other than mediators . early impl found incr exerciSe i histamine stricter n histamine. They also were deple Themed the eXerc: Gt al~ (11 increeSed Removal 0 amine f0: 49 Adenine compounds could directly dilate by decreasing membrane permeability to calcium. They could also stimulate a coupled sodium-potassium pump, decrease membrane perme— ability to sodium, or increase membrane permeability to potassium. Any of these effects would hyperpolarize and result in relaxation. Much more work needs to be done to determine the actual mechanism by which adenine compounds dilate. Other Dilator Substances. A variety of substances other than those already discussed have been suggested as mediators of local blood flow regulation. Histamine was early implicated in such a role. Anrep and Barsoum (1935) found increased histamine levels in the venous blood during exercise in dogs. The stronger the contractions, the more histamine appeared. Stimulation of vasodilator or vasocon- strictor nerves alone did not cause an increase in venous histamine. Anrep et a1. (1939) confirmed these results. They also showed that pre—existing muscle histamine stores were depleted during contractions of prolonged duration. Increased histamine was found in the venous effluent from the exercising forearm by Anrep et a1. (1944). Graham et a1. (1964) found that exercise and catecholamines caused increased histamine forming capacity in mouse muscle. Removal of the adrenal medulla prevented the rise in hist- amine forming capacity caused by exercise. Schayer (1965) reported tl smooth mus adjusted to contractio Zveifach, Howev venous his cnenius. histamine tiou in tr noII-Specii 1965). T} 1016 histe Fox 1 Potent va: (1964) f0. exercise could not large inc arterial increaSe no inCrea humans. that Cart "'asodilat RUScle Cc 50 reported that histamine is continually synthesized within smooth muscle cells and that its rate of synthesis can be adjusted to meet various conditions. Antihistamines caused contraction of microvessels in the rat (Altura and Zweifach, 1965). However, Emmelin et a1. (1941) found no increase in venous histamine during tetanization of the canine gastro— cnemius. Altura and Zweifach (1967) found that intrinsic _histamine formation was not regularly associated with dila- tion in the rat. Also, histamine was shown to be a rather nonvspecific dilator of the vasculature (Altura and Zweifach, 1965). Therefore, it is still controversial as to what role histamine might play in exercise hyperemia. Fox et al. (1961) demonstrated that bradykinin is a potent vasodilator in the human forearm. Allwood and Lewis (1964) found no increase in venous bradykinin following exercise in the human forearm. However, the substance could not be excluded as a mediator of the hyperemia, since large increases in forearm blood flow occurred upon intra— arterial bradykinin infusion without a detectable venous increase in bradykinin. Carretero et a1. (1965) also found ~n0 increase in venous bradykinin following exercise in humans. More conclusively, Webster et al. (1967) showed that carboxypeptidase B, an inhibitor of bradykinin—induced vasodilation, had no effect on the vasodilation produced by muscle contraction in the canine gracilis muscle. ......I! Barcr ganic phos exercise v blood flow until a £0 flow very to dilate produced b and Chir, the cat re increaSes at al- (19 the artifi or the exe found no i cise in th after Stat homage j ‘ flow. eMia is Cc StUdies. Hadd) heed arts page (15 51 Barcroft et al. (1970) found evidence regarding inor— ganic phosphate in humans. During sustained or rhythmic exercise venous plasma phosphate increased by 20%, while blood flow increased ten times. Infusion of phosphate until a four-fold increase had been produced changed blood flow very little. However, in the cat phosphate was shown to dilate adequately enough to account for the hyperemia produced by a short tetany (Hilton and Vrbova, 1970; Hilton and Chir, 1971). Hilton and Hudlicka (1971) found that in the cat release of inorganic phosphate closely paralleled increases in blood flow associated with exercise. Dobson et al. (1971) found no increase in phosphate release from the artificially perfused, exercising frog sartorius muscle or the exercising canine hindlimb. Tominaga et al. (1973a) found no initial increase in phosphate release during exer— cise in the dog hindlimb. There was a gradual increase after stabilization of the vascular resistance, but the increase in phosphate did not correlate with the increase in flow. Therefore, the role of phosphate in active hyper— emia is controversial and appears to depend on the species studies. Haddy (1960) reported that local magnesium excess pro— duced arteriolar dilation in the dog forelimb. Whang and Wagner (1966), following forearm exercise in humans, found an increase in venous magnesium, presumably due to a after two Hedw PGEl was and hindp on vasoco norepinep] both pre- Hovever, 1 prostagla: Acet and venou studies w Frolich seven in 1109 fore fumarate, 52 decrease in the amount of water present. Scott et a1. (1970) reported that the magnesium increase was only 5% greater than the control value and had disappeared by the fifth minute of gracilis exercise with natural flow. Radawski et al. (1972b) found no arterio—venous magnesium difference after two hours of exercise in the canine gracilis muscle. Hedwall et a1. (1971) showed that the prostaglandin PGEl was a potent vasodilator in the dog gracilis muscle and hindpaw. The substance also had some inhibitory effect on vasoconstrictor agents such as nerve stimulation and norepinephrine. Daugherty (1971) found that PGEl dilated both pre— and post-capillary vessels in the dog forelimb. However, no studies were found regarding a specific role of prostaglandins in active hyperemia. Acetate has been shown to produce arteriolar dilation and venous constriction (Overbeck et al., 1961) but no studies were available on its role in active hyperemia. Frolich (1965) specifically tested the vascular effects of seven intermediary products of oxidative metabolism in the dog forelimb. The metabolites were acetate, citrate, fumarate, malate, d-ketoglutarate, oxaloacetate, and succin— ate. Submaximal doses of each substance (2.47 nmole/min.) produced prompt vasodilation which was localized to the arterioles. Venous lactate and pyruvate increased during and after leg exercise in humans (Carlson and Pernow, 1961). degree of versial Inte been done ing activ among the might int (1966) re tension a tension d forearm w blood flo arm that to that resulted the isch greater 53 Lactate remained elevated for a 20 minute period of exercise in dog calf muscles but declined to control levels within a 60 minute period of exercise (Morganroth et al., 1974). Thus, although these substances produce dilation, their degree of change during alterations in blood flow is contro— versial and in some cases unknown. Interaction of Substances. Relatively few studies have been done to determine the combined effects of factors dur- ing active hyperemia. Billings and Maegraith (1937) were among the first to suggest that an accumulation of substances might interact to produce vasodilation. Kontos et al. (1966) reported that the decrease in venous blood oxygen tension and the increase in venous blood carbon dioxide tension during five minutes of mild exercise in the human forearm were too small to entirely account for the increased blood flow. Kontos et al. (1970) found in the human fore- . arm that combined hypoxia and hypercapnia equal in magnitude to that observed during ischemia of five minutes duration resulted in vasodilation averaging 64% of that produced by the ischemia. The combined effect was not substantially greater than the additive effects of hypoxia alone and hypercapnia alone. Skinner (1967) found that perfusion of the isolated canine gracilis muscle with blood low in oxygen and high in potasssium produced greater dilation than perfusion With blood low in both strictor response more by perfusion potassium than by stances. Skinner canine gracilis mu vere never associa oxygen levels rema deficient blood, the greater and mo resistance. In f and Skinner and Co creased oxygen and dilator than oxyge: during sympathetic deficient, hyperka dilation both befo than perfusion wit Stowe et al. and pH caused a gr resistance than ei Produced by reduc' heaVy exercise (2 moderate decrease fieerease in resis 54 blood low in both oxygen and potassium. Likewise, the con— strictor response to sympathetic stimulation was reduced more by perfusion with blood low in oxygen and high in potassium than by perfusion with blood low in both sub— stances. Skinner and Powell (1967) demonstrated in the canine gracilis muscle that increases in venous potassium were never associated with maximal dilation as long as oxygen levels remained normal. When perfusing with oxygen deficient blood, the higher the potassium concentration, the greater and more rapid was the decrease in vascular resistance. In further studies Skinner and Costin (1970) and Skinner and Costin (1971) found that blood with de— creased oxygen and increased osmolality was a more potent dilator than oxygen deficient blood alone, both before and during sympathetic stimulation. Perfusion with oxygen deficient, hyperkalemic, hyperosmotic blood produced greater dilation both before and during sympathetic stimulation than perfusion with oxygen deficient and hyperkalemic blood. Stowe et al. (1974) found that reduction of both PO and pH caused a greater drop in canine gracilis muscle resistance than either alone. A 40% drop in resistance was produced by reducing both P02 and pH to levels found during heavy exercise (23 and 7.10). Mild exercise produced only moderate decreases in P0 and pH (34 and 7.28) but a 38% 2 decrease in resistance. Stowe (1974) reported that the enhanced vasodila state exercise ca and Po to pre—ex 2 remained decrease rection of the ef necessary. Honig (1968) produce greater i of the separate e cellular levels 0 lack, a mechanis contractile acti of phosphate and muscle almost com hydrolysis . 2. Reactive Hyper No major th vasodilation foll Conheim in 1872. build up during t Freeburg and Hyma Vasodilation fol and a second £10 a metabolic caus 55 enhanced vasodilator activity of venous blood during steady state exercise can be completely abolished by returning pH and PO to pre—exercise levels. These two parameters remainid decreased throughout sustained exercise. No cor- rection of the effluent for potassium or osmolality was necessary. Honig (1968) found that phosphate and AMP together produce greater inhibition of ATPase activity than the sum of the separate effects of the two substances. Since intra— cellular levels of these metabolites may rise with oxygen lack, a mechanism may exist for chemical feedback on the contractile activity of smooth muscle cells. Concentrations Of phosphate and AMP equivalent to those found in ischemic muscle almost completely inhibited contractionecoupled ATP hydrolysis. 2. Reactive Hyperemia of Skeletal Muscle Two major theories have been proposed to explain the vasodilation following arterial occlusion first observed by Conheim in 1872. The metabolic theory states that chemicals build up during the occlusion and produce the dilation. Freeburg and Hyman (1960) found a 30—60 second delay in vasodilation following arterial occlusion in the human leg and a second flow maximum 30 to 45 seconds later, suggesting a metabolic cause for the dilation. A stable metabolic dilator could re The finding that proportionate in this theory (Pat additional findi amount of blood does not support debt was conside that the decrea produces the di factors are inv Early stud' the mediator of first to suggest found increased blood following duration. Rest] greatly enhance: method histamine following the or levels returned emia. Billings venous blood fr (1941) , however levels in man 0 56 dilator could recirculate, producing the second maximum. The finding that prolongation of the occlusion caused a proportionate increase in excess blood flow also supports this theory (Patterson and Whelan, 1955). However, their additional finding that there is great variability in the amount of blood flow repayment under the same conditions does not support this theory as well. In most cases the debt was considerably overpaid. The myogenic theory holds that the decreased blood pressure during the occlusion produces the dilation. A third possibility is that both factors are involved in mediating reactive hyperemia. Early studies focused on the vasodilator histamine as the mediator of reactive hyperemia. Lewis (1927) was the first to suggest this possibility. Anrep et al. (1944) found increased plasma levels of histamine in human venous blood following arterial occlusion of 10 or 20 minutes duration. Restriction of venous outflow by compression greatly enhanced the amount of the increase. With this method histamine levels were elevated for two minutes following the occlusion. With a free circulation histamine levels returned to normal long before the end of the hyper- emia. Billings and Maegraith (1937) found histamine in the venous blood from ischemic rabbit tissues. Kwiatkowski (1941), however, found no increase in venous histamine levels in man or rabbits following release of arterial occlusion- In a‘ not alter the hY. confirmed theSe 3 These results We to insufficient . samples at a tim' Folkow et a also found no ev‘ reactive hyperem magnitude whethe: tive to histamint blood vessels cor arterial occlusi< the reactive hyp: neoantergan, ant emia. Duff et a Prolonged arteri. indeed reduced bj that histamine m. fOlloving prolonr all the evidence candidate for mer Horton (196 reactivg hyperem‘ although no pla S] 57 occlusion. In addition, a cysteine block of histamine did not alter the hyperemic response. Emmelin et al. (1941) confirmed these negative findings in both humans and dogs. These results were attributed by others (Anrep et al., 1944) to insufficient control of venous flow and collection of samples at a time when histamine levels had already declined. Folkow et a1. (1948), using more refined techniques, also found no evidence that histamine was a mediator of reactive hyperemia. Reactive hyperemia was of the same magnitude whether an animal's blood vessels were very sensis tive to histamine or relatively insensitive. Rendering the blood vessels completely insensitive to histamine before arterial occlusion also had no effect on the magnitude of the reactive hyperemia. Administration of benadryl or neoantergan, antihistamine agents, did not alter the hyper— emia. Duff et al. (1955) found that hyperemia following prolonged arterial occlusion (longer than ten minutes) was indeed reduced by antihistamines. It was therefore suggested that histamine may partially mediate reactive hyperemia following prolonged arrest of the circulation. Considering all the evidence, histamine does not appear to be a good candidate for mediating reactive hyperemia. Horton (1964) examined venous blood collected during reactive hyperemia in dog hindlimbs for increases in kinins. Although no plasma kinins were detected in this blood, serotonin increa However, this su hyperemia, since blood flow. Its clotting functic similar results. evidence for kin The kinin blocke hlPeremic reSpon Oxygen is a tive hYPeremia. in tissue Oxygen that reduced art after Circulat or the subSQquent r [1956) measured hYPeremia and fc stage of the hYE considerably bej thus cOIISidered the entire hYper reactive hYperen results. KontOC in deep forearm to . hYpoxla avere 58 serotonin increased seven-fold above control levels. However, this substance was not implicated as a mediator of hyperemia, since its intraarterial infusion did not alter blood flow. Its increase was instead attributed to an anti— clotting function. Allwood and Lewis (1964) obtained similar results. webster et al. (1967) also found no evidence for kinin participation in reactive hyperemia. The kinin blocker carboxypeptidase B had no effect on the hyperemic response to occlusion. Oxygen is another substance which could mediate reac— tive hyperemia. Arterial occlusion would lead to a decrease in tissue oxygen levels. Dornhorst and Whelan (1953) found that reduced arterial oxygen saturation both before and after circulatory arrest had no effect on the duration of the subsequent reactive hyperemia. In addition, McNeill (1956) measured venous oxygen saturation during reactive hyperemia and found it to be decreased only during the early stage of the hyperemia. Control oxygen levels were reached considerably before the end of the vasodilation. It was thus considered unlikely that tissue hypoxia existed during the entire hyperemic period and that oxygen deficit mediated reactive hyperemia. Kontos et al. (1965) confirmed these results. Kontos et al. (1970) found that for equal decreases in deep forearm venous blood P , the vasodilator response 02 to hypoxia averaged only 26% of that produced by ischemia. Also, the hyperel breathing 100% 0: P02 at a level a] room air breathi: In support 1 emia, Crawford e canine hindlimb degree of hypoxi t1956) occluded three to ten min limbs were perfu from the reactiv blood was reoxyg OxYsenaticm reco the OXYgen lack via release of a ments_ It was s not a major Caus (1973a) also she Period of reac ti SiOn. Baetjer (19 creases in Venou the arterial inf Radawski (1971) 59 Also, the hyperemia following ischemia was not modified by breathing 100% oxygen, thereby maintaining deep venous blood P02 at a level above that seen with free circulation during room air breathing. In support of oxygen as a mediator of reactive hyper— emia, Crawford et al. (1959) showed that blood flow in the canine hindlimb increased nearly proportional with the degree of hypoxia. More conclusively, Fairchild et al. (1966) occluded arterial flow to the canine hindlimb for three to ten minutes. 0n release of the occlusion, the limbs were perfused with deoxygenated blood. No recovery from the reactive hyperemia occurred until the perfusion blood was reoxygenated ten minutes later. Following re— oxygenation recovery occurred within a few minutes. Whether the oxygen lack directly caused the dilation or only did so via release of a mediator was not answered in these experi— ‘ ments. It was suggested that washout of metabolites was not a major cause of reactive hyperemia. Tominaga et al. (1973a) also showed that venous P02 correlated with the period of reactive hyperemia following a two minute occlu— sion. Baetjer (1935), working with cat hindlimbs, found in— creases in venous blood potassium following reduction of the arterial inflow by 20% or more. However, Scott and Radawski (1971) measured control and hyperemic levels of ..Va! osmolality, magr. from the canine arterial occlusi occurred in the Tominaga et al. Billings an report that tiss blood Supply. 3 002 levels in ve emia brought abo sions in the can rapidlY and then foll‘mng blood Venous pH change PC02' HYPOcapni the hyperemic re ProduCed by acet hYPeremic reSpOn from 7.38 to 7.2 hindlimb. Konto in the human for creased ten time 0.03 units and P round also that la d no effeCt 0n 60 osmolality, magnesium, and potassium in the venous blood from the canine gracilis muscle. Following a five minute arterial occlusion, no changes in any of these substances occurred in the venous outflow. Rudko and Haddy (1965) and Tominaga et al. (1973a) obtained similar results. Billings and Maegraith (1937) were among the first to report that tissues become acid upon occlusion of their blood supply. Kontos et al. (1965) measured venous pH and CO2 levels in venous blood during rest and reactive hyper— emia brought about by 30 second or larger arterial occlu— sions in the canine hindlimb. Venous PCO2 increased rapidly and then declined gradually to control levels, following blood flow with a delay of several seconds. Venous pH changes were inversely proportional to those of PCOZ. Hypocapnia produced by hyperventilation decreased the hyperemic response to a 30 second occlusion; hypercapnia ' produced by acetazolamide administration increased the hyperemic response. Scott et al. (1970) found that pH fell from 7.38 to 7.28 during reactive hyperemia in the canine hindlimb. Kontos and Patterson (1964) found similar results in the human forearm. Flow during reactive hyperemia in- creased ten times the COntrol value; venous pH fell by only 0.03 units and PCOZ rose 10 mm Hg. In this study they found also that intravenous infusion of sodium bicarbonate had no effect on reactive hyperemia, indicating mediation of the hyperemia et al. (1970) ob after circulator dioxide breathin increases after administration v. forearm venous l: capnia averaged a1. (1971) conf reduced the vas in humans. How lation between following a two whether or not i ischemic tissue: is still contrO‘ The adeninn of reactive hypi levels in the vi arterial occlus duration of the Change the leve Venous blood fr 61 of the hyperemia via carbon dioxide rather than pH. Kontos et al. (1970) observed greater increases in blood flow after circulatory arrest in the human forearm during carbon dioxide breathing then during room air breathing. Flow increases after ischemia were maintained until carbon dioxide administration was stopped. For equal increases in deep forearm venous blood PCO , the vasodilator response to hyper— capnia averaged 60% of that following ischemia. Kontos et al. (1971) confirmed this latter result using acid infusion in the canine gastrocnemius muscle. Kontos (1971) found that the alkaline buffer amine, tromethamine, markedly reduced the vasodilator response to ischemia in the dog and in humans. However, Tominaga et al. (1973a) found no corre— lation between venous pH or P and vascular resistance CO2 following a two minute arterial occlusion. Therefore, whether or not the accumulation of carbon dioxide in ischemic tissues may act as a mediator of reactive hyperamia is still controversial. The adenine compounds have been suggested as mediators Of reactive hyperemia. Gordon (1962) found that AMP levels in the venous plasma from the rabbit kidney following arterial occlusion rose to 30 ug/ml. However, varying the duration of the occlusion from three to 30 minutes did not change the level of AMP found. No AMP was detected in venous blood from the rabbit hindlimb following a 30 minute arterial occlus: (1964) likewise slight increase: [MP levels incre as early as four femoral arteries of the coronary adenosine level. et al., 1971) canine skeletal and adenosine. trol values, fr (1971) confirms muscle adenosin to 1.5 nmole/gm (1972) found no minute occlusion Kontos et a mole on the reS] period of ischeI action of adeno: tion had no eff: arrest. Tominaga er Study venous bl< Sion in the can: 62 arterial occlusion. Berne et al. (1963) and Imai et al. (1964) likewise found no increase in adenosine and only slight increases in AMP in ischemic rabbit skeletal muscle. IMP levels increased. ATP levels decreased in rabbit muscle as early as four hours following ligation of the iliac and femoral arteries (Grigor'eva et al., 1965). Interruption of the coronary circulation increased cardiac tissue adenosine levels. In further studies of this type (Berne et al., 1971) the venous blood from contracting, ischemic canine skeletal muscle was analyzed for adenine nucleotides and adenosine. Only the adenosine levels rose above con— trol values, from 0.03 nmoles/ml to 0.12 nmoles/ml. Dobson (1971) confirmed these results and also found that the muscle adenosine content rose from a control level of 0.7 to 1.5 nmole/gm during ischemic contraction. Forrester (1972) found no ATP in the venous plasma following a four minute occlusion of the human forearm. Kontos et al. (1968a) studied the effect of dipyrida— mole on the response of the canine hindlimb to a 30 second period of ischemia. This drug potentiates the vasodilator action of adenosine, AMP, and ATP. However, its administra— tion had no effect on the hyperemic response to circulatory arrest. Tominaga et al. (1973a) used a bioassay preparation to Study venous blood following a two minute arterial occlu— sion in the canine hindlimb. The venous effluent had renal vasoconstrictive adenosine might renal vasculatur similar results. adenosine and/o a three minute 0 parison of const Tominaga et al. creased in the v occlusion only constant pressur muscle to effect metabolites, so deaminases. Th adenine compound products. The myogeni for reactive hyp canine hindlimbs Greased intravas to be the cause, injected into at blood into the a arterial occlusi brachial artery 63 vasoconstrictive activity, suggesting that AMP and/or adenosine might be present as these compounds constrict the renal vasculature. Scott et al. (1965) had obtained similar results. Tominaga et a1. (l973d) found that venous adenosine and/or AMP rose above control levels following a three minute occlusion in the canine hindlimb. In a com— parison of constant pressure and constant flow preparations, Tominaga et al. (1975) found that adenosine and/or AMP in- creased in the venous blood following a three minute occlusion only with constant flow. They theorized that with constant pressure sufficient blood was supplied to the muscle to effectively wash out mediators and accumulated metabolites, some of which.were suppressing the activity of deaminases. Thus, with increased enzyme activity, the adenine compounds would be converted to nonvasoactive products. The myogenic theory has frequently been used to account for reactive hyperemia. Folkow (1949a), in denervated canine hindlimbs, found vasodilation in response to de— creased intravascular pressure. Chemicals did not appear to be the cause, as the blood was not vasoactive when injected into other vessels. Patterson (1956) brought extra blood into the arm by suction and trapped it there during arterial occlusion. This maintained the pressure in the brachial artery distal to the occluding cuff at 45 mm Hg, compared with a}: subsequent hyper ing for the ext: was not complete flow was changed confirmed these tion to the myog mediate reactive Wood et a1 ments and obtai blood reduced was not abolish creased the rea fected by maint Kontos et a Venous congestic vasodilator res; seconds) but on] arterial occlusi volume in respor 1y well with the correlation was (1975) found the With the duratic sure and constar 64 compared with about 10 mm Hg in the non—packed arm. The subsequent hyperemia was considerably less, even after allow- ing for the extra trapped blood. However, the hyperemia was not completely abolished by this procedure, and the peak flow was changed very little. Freeburg and Hyman (1960) confirmed these results. Therefore, other factors in addi- tion to the myogenic mechanism were considered likely to mediate reactive hyperemia. WOod et a1. (1955) performed a similar series of experi- ments and obtained similar results. Packing the arm with blood reduced the resultant hyperemia by 20 to 50%, but it was not abolished. Mild exercise during the occlusion in- creased the reactive hyperemia considerably and was unaf— fected by maintenance of intravascular pressure. Kontos et al. (1965) further studied this problem. Venous congestion induced by venous occlusion abolished the vasodilator response to short arterial occlusion (five seconds) but only decreased it in response to a longer arterial occlusion (30 seconds). The reactive hyperemia volume in response to a short occlusion correlated moderate— ly well with the decrease in intravascular pressure. This correlation was poor for a longer occlusion. Owen et al. (1975) found that the recovery time from ischemia progressed with the duration of the ischemia, with both constant pres— sure and constant flow. Therefore, myogenic factor The reactive hy may be adequate However, if the seconds, the op role in causing metabolic media adenine compounv 65 Therefore, in considering the data, both metabolic and myogenic factors are probably involved in reactive hyperemia. The reactive hyperemia following a short arterial occlusion may be adequately explained by the myogenic hypothesis. However, if the occlusion lasts much longer than five seconds, the opinion is that metabolic factors also play a role in causing the hyperemic response. The most likely metabolic mediators appear to be oxygen, carbon dioxide, and adenine compounds. 1. Surgical Tech Mongrel dog fifteen kilogram tory Animal Resc sodium pentobart A sustaining dos administered int was observed. 1y isolated. T The femoral art the upper and l< femoral artery x and cannulated ; acoustant flow 0f 95% oxygen, Via a model T8 Placed on the f artery. Thus , M *In some experi lated rather t METHODS 1. Surgical Technigues Mongrel dogs of both.sexes weighing between seven and fifteen kilograms were obtained from the Center for Labora— tory Animal Resources and anesthetized initially with 25 mg sodium pentobarbital per kilogram body weight intravenously. A sustaining dosage of six mg per kilogram body weight was administered intraperitoneally when a strong corneal reflex was observed. One gracilis muscle was exposed and surgical— ly isolated. The gracilis nerve was isolated and sectioned. The femoral artery was isolated and all branches between the upper and lower gracilis artery were tied off.* The femoral artery was then tied above the upper gracilis artery and cannulated just distal to the tie with PE 260 tubing, and a constant flow Ringer's perfusate bubbled with a mixture of 95% oxygen, 5% carbon dioxide was immediately commenced via a model T8 Sigmamotor pump. A second occlusive tie was Placed on the femoral artery just below the lower gracilis artery. Thus, all perfusate either entered the upper or *In some experiments the upper gracilis artery was cannu— lated rather than the femoral artery. 66 lower gracilis with PE 90 tub' were tied off. 6V, 6 cps, 1.6 model SS stimul cells from the perfused for an experimental pr Arterial b artery cannula ducer and a Gra before the star pressure as clo from 8 to 11 ml Shown in Figure 2. Ion Exchange A gradient tus similar to (1952) and Hurl adenine compoun were used in th first modificat 15 feet from th fOrnate solutio the reservoir b 67 lower gracilis artery. The gracilis vein was cannulated with PE 90 tubing. The origin and insertion of the muscle were tied off. The gracilis nerve was stimulated at either 6V, 6 cps, 1.6 msec or 22V, 10 cps, 1.6 msec with a Grass model SS stimulator for one minute to wash out residual cells from the muscle vasculature. The muscle was then perfused for an additional 20 minutes before starting an experimental procedure. Arterial blood pressure was measured from a femoral artery cannula with a Statham model P23AC pressure trans— ducer and a Grass model 5D polygraph. Flow was adjusted before the start of the experiment to obtain a perfusion pressure as close as possible to 90 mm Hg. Flows ranged from 8 to 11 ml/min. The preparation described above is Shown in Figure l. 2. Ion Exchange Chromatography A gradient elution ion exchange chromatographic appara— tus similar to that described previously by Busch et al. (1952) and Hurlbert et al. (1954) was used to separate the adenine compounds. Three modifications of these procedures were used in the studies described in this thesis. In the first modification a 500 ml reservoir bottle approximately 15 feet from the floor was filled with a l M ammonium formate solution. Tubing which could be clamped connected the reservoir bottle to a mixing chamber containing 500 ml 68 ((lrIQ C...- MF .. ..... -V \ m35uomno oco mucomonmon pcflom 30mm .N ondmflm be caonm we poms mdponmmmc mse .ocwamm mo HE N CH sfidaoo oflu Op poops mp3 mpcsomaoo one mcwswwpcoo GOHpDHom a mo in 33195 .ofimoaoom one .msa KHZ .mea Hem cuoupmm wamoumoumfionno omnofloxo GOA sOHpSHo ucoflpmno .m onsmflm 73 74 m onsmflm mumZDz w4a2I¢<¢GOFIQummno mco mucomonmou ucaom comm .ocflamm mo HE m cw cabaoo map on poops moz mpcsomfioo map mcwcwopcoo GOHpDHom m mo HE mansioco .ocemoaoom one .mzm .mzmo .mzH .mom .mea mom cuoppmm mQQMHmOmeOHQO omnmfioxm cOH soapsao pcoflpmnw .v musmflm [NJ v ousmflm mmmEDZ MJQ2IQ<¢OOFEJU QKnomno oco upcomoumou oaonflo comm .moamamm mEH pcom topmou mcHouHo some “moamfiom mzm ucomoumou moHouHo powoao .ouDGHE mom mucsoo ocflfimouop Ob humus mzd one coconnu sou oHoB msoeponucoocoo caocx Ce mSH Ho mzd msacamucoo moamsdm .o>nco Unccccum m2< .m onsmflm DON 82 com m madman 00o _ .=>_\oz oov oon m>mDU Qm__ Sigma Chemica Sulfate. The fuged for two added to appr 98 17. Epinephrine.——Adrenalin chloride solution was obtained from Parke, Davis and Company. A low dose infusion was prepared by adding one mg of epinephrine to 100 ml of distilled water. Eight ml of this solution were then added to 32 ml of distilled water. Two ml of this solution were added to 50 ml of perfusate. The resulting 80 ng per ml solution was infused at 0.76 ml per minute. A high dose infusion was prepared by adding one mg of epinephrine to 100 ml of distilled water. Eight ml of this solution were then added to 32 ml of perfusate. The resulting 2 pg per ml solution was infused at 0.76 ml per minute intraarterially. 18. Ouabain.-—Ouabain was obtained from Nutritional Biochemical Corporation. Five mg of ouabain were added to 50 ml of perfusate. Following a one minute sonification, the solution was infused at 0.76 ml per minute intraarteri— ally. 19. ATP Infusion.——ATP obtained from Nutritional Bio— chemical Corporation was added to perfusate in the amount of 10 mg per 100 ml. 20. ADP Infusion.——Sixty mg of Ba—ADP obtained from Sigma Chemical Company were added to 8 ml of 0.2 M sodium sulfate. The resulting solution was stirred and then centri— fuged for two minutes. The supernatant was decanted and added to approximately 800 ml of perfusate. 21 . y chemical Co. of 10 mg pe: 14( 22. 10 uCi) was 23. gag elution ion in 88% formi supernatant mately 20 ti the AMP assa ethanol and supernatant . three times finally washn 18. Statisti Data fr voltage nerv completely r unequal sampi epinephrine 1 nerve stimula complete blOr analysis of ‘ 99 21. AMP Infusion.e—AMP obtained from Nutritional Bio— chemical Corporation was added to perfusate in the amount of 10 mg per 100 ml. 22. l4C—ATP Infusion.——One—half ml of l4C—ATP (14 pg; 10 uCi) was added to 30 ml of perfusate. 23. Resin Regeneration.——The resin used for gradient elution ion exchange chromatography was washed three times in 88% formic acid by soaking overnight and decanting the supernatant each time. The resin was then washed approxi— mately 20 times with distilled water. The resin used for the AMP assay was washed three times in a 1:1 mixture of 30% ethanol and l N HCl by soaking overnight and decanting the supernatant each time. This resin was then further washed three times in 88% formic acid as described above and was finally washed approximately 20 times with distilled water. 18. Statistical Methods Data from nerve stimulation plus phentolamine and low Voltage nerve stimulation experiments was analyzed by a completely randomized one—way analysis of variance for unequal sample size. Data from low dose epinephrine, nor— epinephrine plus phentolamine, and curare plus low voltage nerve stimulation experiments was analyzed by a randomized complete block model analysis of variance. Prior to the analysis of variance the sample mean variances were determined t from the rem done was ana paired repli: as statistic; performed on sponding peri was determine 100 determined to be homogenous by using the F max test. Data from the remaining experiments in which AMP assays were done was analyzed by the Student's t—test modified for paired replicates. Initial, non—experimental values served as statistical controls. A linear regression analysis was performed on all venous AMP concentrations and their corre- sponding perfusion pressures. The correlation coefficient was determined. 1‘ Afi—PP 33:2: 9 . To dete: through the g were Perform were added t< gr"leilis arte fir‘afll/‘lucii Shows the res Virtually no tory leVels With infIISior ATP can be de amOllnts. Alt between Preps Small Variabi results as We These results to quantify 1 within the gr or reaCtiVe h RESULTS 1. ATP Survival on Passage through the Gracilis Muscle Vasculature To determine whether or not ATP can survive passage through the gracilis muscle vasculature, six experiments were performed in which increasing concentrations of ATP were added to the Ringer's perfusate and infused into the gracilis artery. The venous effluent was analyzed by the firefly—luciferase method for ATP concentration. Figure 6 shows the results of these experiments. It can be seen that virtually no ATP appears in the venous effluent when dila— tory levels (0.5 ug/ml) of ATP are infused intraarterially. With infusion concentrations of ATP of 10 ug/ml and higher, ATP can be detected in the venous effluent in increasing amounts. Although flow rate varied from 8 to 11 ml/min. between preparations, it is considered unlikely that this small variability could have produced such consistent results as were observed in this series of experiments. These results indicate that it would be almost impossible to quantify low levels of ATP which might be released from within the gracilis muscle into the perfusate during active or reactive hyperemia. Approximately one—third of the 101 "lw/ D Mul-HVWDE mulu~0~>maw th< m u 2 .on0m 00H o co coupon ma coaumuucoocoo mam amanouum .mMmmm med wsoco> oco mucommumou Honamm comm can .Hmaflcm oco scum cunt can mucomonmou Honfihm mo omhp comm .cofiumnucoocoo med How momma ommquHosH Imamouwm may ha UonHmcc mMB pcosHmmo mcoco> one .mcowpmuucoocoo mcflwum> ca oaomsfi mflaflomnm om“ oucH NHHMHHopnmmupcfl comsmcfl was mad .ousumasomm> odomsfi mHHHomnm map smsounu oatmmmm co Hm>fl>u5m mam .m ousmflm 102 103 w oudmwm :4}: Tim— 43552 000— 00— IL _ O.— I O. I 8’. 1w/9I1 [div] SflONBA I m 0. UJUWDZ m_4_U_>~5m n:.< effluent sam} described thl for ATP by tl (greater thar sample. N T0 deter through the g of Perfusate ‘ muscle. Figu The Venous ef eXChange Chro: Effluent O.D.‘ °°lleCtion, ProdUCt of AT] Peak is Presex Figure 8 “ATP throug ATP enabled br sion of Very 1 formed. The thOUgh inflow 104 effluent samples which were analyzed by the AMP assay, as described throughout this Results section, were also tested for ATP by the firefly assay. No significant quantity (greater than 10 ng/ml) of ATP was ever detected in any sample. 2. Breakdown Products of ATP on Passage through the Gracilis Muscle Vascula- 21$ To determine breakdown products of ATP on passage through the gracilis muscle vasculature 100 pg ATP per ml of perfusate were infused intraarterially into the gracilis muscle. Figure 7 shows the results of five such experiments. The venous effluent was collected and analyzed by ion exchange chromatography for ATP breakdown products. Effluent O.D.260 was determined to be constant before sample collection. It can be seen that AMP is the major breakdown . product of ATP in the gracilis muscle vasculature. A small peak is present in the nucleoside area. Figure 8 shows the results of infusing 500 ng/ml of l4C—ATP through the gracilis muscle. The use of labelled ATP enabled breakdown products to be determindd with infu— sion of very low levels of ATP. Two experiments were per- formed. The major breakdown product again is AMP, even though inflow ATP concentration here is very small. .m.rx\ .unu mWhnufiZUnuwrl va>>nunuv&dnm~mm~ m u z .c0flpm>uomno oco mpcomoumon uQHom comm .Hopoaouocmouuoomm om cmfixoom o co 15 com um coon mm? onEMm uouooaaoo coflpocnm zoom .v oMDmHm :H QSOQm ouspwoonm Oflcmmmm IoumEOpco ogcmcoxo coH on“ ha couhamcm cam cowooaaoo mm? ucosammm mDoco> oaB .Hfi\w: ooa cw oaomdfi mHHHomHm osu occfl maamflmocnmmnucfl comsmcw mm? med .oHDDMHSomm> oaomcfi mflaflomum cap monounu ovummmm co mem mo mPUDUOHm csopxmonm 105 .b ousmflm h ounmflm mmmZDZ MIKE/am ow. om. o: om. oo_ om oo 9. cu _, _ _ H . _ _ _ _ 6 ...X..3.~.:..X..82.233-223................:....a. 2.3.. c - Me. In — u m . m ..u 08 _ .. 1 in. do ,5 age as: azfi .22 w .... S m 14 S n_._.< LO mbUDn—Omd Z>>Oox_\/W:me nitcxu m u z .coapc>uomao oco upcomoumou pcflom comm .Houofioc losmoupoomm mo cmfixoom c so 15 com cc cmou was camficm Houooaaoo coflcocum comm .m oucmem mo QOHDHOQ HoSOH on“ CH QBOSm cnscoooum oesmcumoucfiouco cmccaoxo 20H cap en comwawcm can cocooaaoo was “cocammo mdocc> one .HE\m1 ooa mo coaumnucoocoo m cm oHomSE mHHHomum one once maacflnouuccupce comdmce was mse .oucpmasowc> maomcfi mHHHoch may cmsounp cmcmmcm co Hm>fl>ucm mad .OH oucmflm 114 115 0H cheese mmmEDZ m4d§: n:>_< SBOISOB'IOON J<>_>m3m a E 4 virtually 4 muscle int; 7.6%, as s] and AMP re.‘ venous eff: U1 L1?! l—h l—h m 0 rr Three effect of j graCilis m Table 5. I I 116 virtually all of the AMP infused was able to pass out of the muscle intact. Mean adenine uptake by the muscle was only 7.6%, as shown in Table 4 (page 113). Therefore, ATP, ADP, and AMP released within the muscle would all appear in the venous effluent as AMP. 5. Effect of Flow Rate on AMP during Passage through the Gracilis Muscle Vasculature Three experiments were performed to determine the effect of flow rate on AMP breakdown and uptake by the gracilis muscle. The results are presented in Table 5. Table 5. Effect of flow rate on AMP during passage through the gracilis muscle vasculature. PER CENT UPTAKE* . Slow Flow Fast Flow 14 3 14 9 12 4 Mean 13.3 5.3 PER CENT O.D.26o IN NUCLEOSIDE AREA Slow Flow Fast Flow 13 6 7 9 12.5 5 Mean 10.8 6.7 *Based on O.D.260 At a flow : was 5.3 peJ mately 3 m.‘ The effect consistent. formation c was little two flow re total 0.1),2 flow a mear Gates that greater Wit adenine rir 6. $3,: \ In one infused int mall/zed by shown in th that MP is intact. Th the Presenc rather than not bin d to Some cells . 117 At a flow rate of approximately 10 ml/minute, mean uptake was 5.3 per cent. When flow rate was decreased to approxi~ mately 3 ml/minute, mean uptake increased to 13.3 per cent. The effect of flow rate on nucleoside formation was not as consistent. In two of the three experiments nucleoside formation did increase with low flow. In the third there was little difference in nucleoside formation between the two flow rates. At fast flow a mean of 6.7 per cent of the total O.D.26o was in the nucleoside area, whereas at slow flow a mean of 10.8 per cent was in this area. This indi— cates that nucleoside formation or accumulation may be greater with slower flow rates, thus accounting for greater adenine ring uptake at low flow. 6. Survival of IMP on Passage through the Gracilis Muscle Vasculature In one experiment 100 ug/ml of IMP were intraarterially infused into the gracilis muscle. The venous effluent was analyzed by the ion exchange chromatographic procedure shown in the lower portion of Figure 3. Figure 11 shows that IMP is able to pass through the muscle vasculature intact. The small peak in the nucleoside area may be due to the presence of hemoglobin and/or dextran in these samples rather than nucleosides, since these two substances would not bind to the resin and would therefore elute at once. Some cells were noted in these samples and dextran was Figure 11. “h. 118 IMP survival on passage through the gracilis muscle vasculature. IMP was infused intra— arterially into the gracilis muscle at a concentration of 100 ug/ml. The venous effluent was collected and analyzed by the ion exchange chromatographic procedure shown in the lower portion of Figure 3. Each fraction collector sample was read at 260 my on a Beck— man DB Spectrophotometer. Each point repre— sents one observation. N = 1 OD 260 119 IM P SURVIVAL the gracilis used intra- cle at a U) :venoush. ‘5’ AMP IMP ADP 'zed by t e_ m <7; ire shown in ,6 _1 8 Each fraction _; I mu on a Beck- 3 point repre- .1 2 O. D. .4 260 02 d . :. U.- . r... ..'°I.‘"'.. ... n o“ ......no-Ooo... l T I l 1 20 40 so so too SAMPLE NUMBER Figure 11 ‘1 Present i] IMP was f¢ the above appeared j Cate that 7. m Figur of the per figure the OCClusion, the nine e3 increase wa 120 present in the perfusate in this experiment. No uptake of IMP was found. Thus, if extracellular IMP had formed in the above studies with adenine nucleotides, it would have appeared in the venous effluent. These studies also indi— cate that venous AMP cannot come from extracellular IMP. 7. Effect of Occlusion on AMP Outflow Figure 12 shows the results of a two minute occlusion of the perfusate on effluent AMP. It appears from this figure that AMP efflux did increase immediately following occlusion. However, an increase occurred in only three of the nine experiments of this type; in the remaining six no increase was observed. Statistical analysis of the data showed no significant difference between the second control and the first post—occlusion sample. Perfusion pressure decreased in six of the nine experiments, indicating vaso— dilation. In only two of the six experiments with vaso— dilation did effluent AMP levels rise. In contrast, muscular exercise produced by stimulation of the gracilis nerve at 6V, 6 cps, 1.6 msec did produce a statistically significant increase in AMP efflux. However, the increase occurred in only six of the nine experiments and was quite small in most cases. The reason for such a weak response may be that the stimulation parameters 6V, 6 cps, 1.6 msec are borderline, producing weak sympathetic stimulation in some preparations and little or none in 121 UC\O”0 mun?— QE< WDOZB) 20 2053¢)00 r .unu .mucofienomxo one: we ccofi onu munomonmou Dnflom nomm .ecmmm And onu en coweacco mos copooaaoo oamfiom unocammo noom .cowpmHsfiepm one mo cco one nowwm mopccflfi cop cocooaaoo mos Honccoo Hmcflm n .cowncasswum menu mnAHcc couooaaoo omos moamfimm one .oome m.H .mmo w .>w um o>non mflaflomum onu mo cOHchBEHum onscHE oounu m an cosoHHOM .couooaaoo mos Homecoo m gonna mopccwfi noouMHm .comooaaoo onos moamfiom osu .coficmoh was 30am Houmo eaoucacoEEH .moucnefi 03p 90m coQQOpm mos oaomsfi mHHHooum on» on 30am .moamfinm Houpcoo 03p mo cowuooaaoo mcwsoHHom .msn muono> no coeumaseflum o>noc cam cosmsaooo mo uoommm 00~ Lkvwhflw .NH enemas 122 NH oucmflm mmeDZ_§ In m¢ O? 9 on on nu on . 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Mos mo memo OBe come m H m oonp moanoauoHSEHpm oucnflfi o>Hm m en pokoaamwcwmomwwwwmm . . no onu Houmm mo . . one Houccoo c coamcmnfl on» mo c . o oo 00 one A posoHHom p c HH .coapooaaoo cocoon c an mHoumwpoEE. mud onu pd . 0mm> mo coHcooHpnfl u .m onEcm ucosammo no cowpmHflp on com um pomcmna mm? .o omcfi mflaflooum one once ousnfiE\ .. oconwuoumoma .maouucoo 03c mo cowpooaaoo mcHBOHHom om2< .va oucmam H msoco> no moapmadfiaum o>uoc can HonoHououmomH we no mmm . 128 mv oe on we ousmfim wmeazi on mm o_~ m__ o. ... _ _ jozmmmeomdog o IIIIIIIII x IIIII OIIII ; |:2\oz \ ./ // Ina x ,7]. as; Ion .. maozm> ,0. u? 232 13 mI z: is mmnmmmma lllllllllll .I I. L: zoaamfia \\\0I l/IoIIo 241mg Ioo. E24 mDOzm> ZO zo_._.<43_2_em m>mmz 02d. nOZmEMeOmeQ .10 eomuum II ...) .1085 0:0. MHGGMQHQTH #CHOm £00m ->ommo I| neuromuscul gracilis mu of experime series (N = 6 cps, 1.6 above contr thereby ind no muscular Sure also i Vasoconstri Source of t leaSed due final Stimu resIllts: i Pregame. In the Stimulation thetic nerv It Can be S motor nel‘ve in AMP outf not rise, also produc, skeletal mu latiOn OCCu 129 neuromuscular blocker gallamine was infused into the gracilis muscle. Figure 15 shows the results of two series of experiments in which this drug was used. In the first series (N = 7) the gracilis nerve was stimulated at 6V, 6 cps, 1.6 msec. Venous AMP did significantly increase above control levels during stimulation in this series, thereby indicating nerves as the source of the AMP since no muscular contraction occurred. However, perfusion pres- sure also increased during the stimulation, indicating vasoconstriction. Therefore, a second possibility for the source of the AMP existed in that it could have been re— leased due to some effect of the vasoconstriction. The final stimulation without gallamine block produced the same results: increases in both effluent AMP and perfusion pressure. In the second series of this type (N = 10) nerve stimulation was at 5V, 2 cps, 0.5 msec. Presumably sympae thetic nerves would not be stimulated with these parameters. It can be seen from Figure 15 that in this case, although motor nerves were being stimulated, no significant increase in AMP outflow occurred. Likewise, perfusion pressure did not rise. The final stimulation without gallamine block also produced no increase in AMP efflux, even though skeletal muscle contraction was vigorous and marked vasodi« lation occurred, as seen from the decline in perfusion 130 o\/ ./I \\- IIIIIIIIIIIII olllolIo LGEOOUQO nu In \ {min/o o o zonmbkmmnd ZQWE 005 .mpnofifinomxo cop mo nmofi onu oncomoumou oucscm como noom .mucofifluomxo co>om mo ccofi onu oncomoumou oHoHHo pwaom nomm .eommo mzn onu en conHmco mos poucoaaoo onEMm unoSHmmo noom .noauca Infiepm Honey menu mneusp cocooaaoo mos onEBm ono .nowpoacaflpm o>uon ochAE 03p o en cosoaaom .popooaaoo mos Houucoo c coemsmnfl onp mo poo onp Hopmo mononefi coe .oouooaaoo oHoB moamaom 03p oEHu noens mcflusp .mouscefi o>em Mom copoHDEHnm mos o>uoc meafloohm onu none .pouooaaoo mos Houucoo nnusom c gonna moycnflfi epnose .COHmsmcH onu mo buopm onu Mopmo pose cocooaaoo mp3 Honucoo cuflnu 4 .ocHEoHHom mo opccfle\m1 oem mo coamcmce en cosoaaom .copooaaoo onos maoupcoo ose .moemom nuon How Hooepcope mos Hooononm Hopnoafluomxm .Ho>oa mo. onp no ochOAMHcmHm ucomonmon moaomflo como .oomE m.o .mdo m .>m pm mos cOHu IoHcEHum o>noc noene 2H moHHom onp pcomoumon woucsgm como .oomE m.H .mmo m .>m no mo? cowuoasaepm oPHoc noens CH moeuom on» unomonmon moaoneo pwaom .oucmwm menu CH ssonm ohm occofienomxo mo moeuom ose .coemcmcfl onflacaaom mcHHDp mad muono> co nOHpoHcEHpm o>uoc Mo poommm .ma oucmflm 131 me enemas mmFDZ_2 mac 0? 00 on 0N 3N n. 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SGHE m>am1 usmze . . mm3 Houpmoo npusow m “mama mmp . .ou .m omSE “zoo cyan“ ¢ H mm m msp Hmumm pmsfl ©m¢omaaoo mmB a WwHwomwm m3“ ounfl mpDGHE\m1 om um U®m5mcfl mmB mumuso ~maouucoo . mcaysw mzd m 00 mCHBOHHom cowmsmcfl muwuso . ozu mo qoauo HH~mmo N .>m pm cowumadfiaum m>umc mo nommwm m50fi®> go mea wtfi .FH mgsmflm 136 5H mnsmflm 852:2 IoH 0.0 0.» onV o_n ow m. _ mm zo wommzmiomm\w.>& 20.2325 m>mmz ...o Swim .wHCGEHHGQXG HSOH HO Emma OCH 42\oz QE< mDOZm> Z_ m: i mmDmmme 2053“.me Z_ the initia cise. It ate the la series of minute per lating the Figure 18 During the Significan tension, e the Stimul Possible h in three 0 TheSe resu likely to emia. 13. Effect W Cs 137 the initial vasodilation which accompanied muscular exer— cise. It was still possible, however, that AMP could medi- ate the later stages of active hyperemia. Therefore, a series of six experiments was performed in which a twenty minute period of muscular exercise was produced by stimu- lating the gracilis nerve at 5V, 2 cps, and 0.5 msec. Figure 18 shows the results of this group of experiments. During the entire twenty minutes of muscular exercise, no significant increase in AMP outflow occurred. Muscle tension, excellent at first, gradually fell toward zero as the stimulation continued, indicating muscle fatigue and possible hypoxia. Perfusion pressure definitely decreased in three of the six experiments, indicating vasodilation. These results suggest that adenine nucleotides are not likely to play a role in the later stages of active hyper- emia. 13. Effect of Nerve Stimulation at 22V, 10 gps, 1.6 msec on Venous AMP dur— ing Phentolamine Infusion To further investigate whether the source of the ob- served AMP was nerve, the gracilis nerve was stimulated at high voltage after the alpha adrenergic receptors had been blocked with phentolamine. The results are shown in Figure 19. Perfusion pressure declined from a mean control value of 82 mm Hg to 50 mm Hg during the stimulation, indicating vasodilation. Venous AMP levels did not change hummer m.0 0ww\N t>nu Q§< w: 0. 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During norepinephrine infusion perfusion pressure increased from a mean control value of 74 mm Hg to a maximum of 172 mm Hg. Venous AMP also significantly increased above con- trol levels. The AMP level of the first sample collected after the start of the infusion was not significantly dif- ferent from controls, although some increase was observed. This small increase can be explained by the fact that per— fusion pressure had also risen only a small amount above control levels at this time. It can be seen that the third sample collected during the infusion shows a decline in AMP outflow. This is due to the fact that mean values have been plotted, and in one of the six experiments the AMP level was extremely high in the second sample collected. In the remaining five experiments AMP levels were fairly comparable between the second and third infusion samples, indicating that the AMP outflow can be maintained for at ..l..|lll._ Figure 20. 143 Effect of norepinephrine on venous AMP. Following collection of two controls, nor— epinephrine was infused into the gracilis muscle at 760 ng/minute for eighteen min- utes. Three samples were collected at periodic intervals during the infusion. One final control was collected ten minutes after the infusion was stopped. Each venous sample collected was analyzed by the AMP assay. Each point represents the mean of six experiments. MEAN PERFUS'Oi PRESSUR MM H3 MEAN VENOUS AMP NG/ML 'enous AMP: :ontrols, nor- the gracilfl zighteen min- .lected at 3 infusion. ted ten minutes ad. Each venous j by the AMP 5 the meanof 144 EFFECT OF NOREPINEPHRINE ON VENOUS AMP N = 6 ° = P (.05 200— ’ MEAN '” PERFUSON ,/””‘— \\\‘ PRESSURE w°‘ ,»”’ \\\\ MM H9 ...—O ..L. 200— MEAN I60— VENOUS xf“ AMP 120— // \ \ \ N I \\ G/ML 80— // 0‘ \ / \ \ 40— ,-l \ \ \ " {' NQREMNEPHMNE 1, I l 5 IO IS are 2‘5 30 MINUTES Figure 20 least fi were dec infusion 15. Eiifi AM§_ Phe nephrine outflow results nephrine Vasocons With the 16. EEEE M Fig ute Of e PEIfusio mm Hg. mean Con 131 and 1‘1 with It can b immediat that a s outflow. 145 least fifteen minutes. Both AMP and perfusion pressure were declining toward control levels ten minutes after the infusion had been stopped. 15. Effect of Norepinephrine on Venous AMP during Phentolamine Infusion Phentolamine was infused prior to and during norepi— nephrine infusion in four experiments to determine if AMP outflow would be blocked along with vasoconstriction. The results are shown in Figure 21. It can be seen that norepi- nephrine produces no increased outflow of AMP when its vasoconstrictor action is blocked. This is in agreement with the results of phentolamine block of nerve stimulation. 16. Effect of Epinephrine at High Dose on Venous AMP Figure 22 shows the results of infusion of 1.5 ug/min— ute of epinephrine (N = 5). This dosage produced a rise in perfusion pressure from a control mean of 65 mm Hg to 141 mm Hg. Venous AMP likewise significantly increased, from a mean control level of 57 ng/ml to mean infusion levels of 131 and 150 ng/ml. Phentolamine was then infused concurrent— ly with the epinephrine and a sample collected at once. It can be seen that both venous AMP and perfusion pressure immediately fell to control levels. These results show that a second vasoconstrictor agent is associated with AMP outflow. 146 7REWDhZ. m2:ZOJOFZMIQ m§EQDQ Aiz< wDOZm> ZO mgéfllagfflmtoz hO Powhhm .mucofianomxo HDOM mo sooE was mesomonmon psflom zoom .wommo mz< opp we powwamso mMS oopooaaoo onEom uMWMWWWMpMMMM maom 039 .mostHE o>Ho3p Mom . .Uopooaaoo onoB moa mm3 .oussaE\ms ooh .osauflmo: opp mpHB washounsocoo oomSMsH do mcassamop opp pm tamouos cone .Uoflnom mflap mo pco one o . . . .wouooaaoo ouoB maoupcoo Hocowuflppo mww wonWWMHMoMowcwmw may 0 . . . opssflfi\m1 om um oaomsfi mflafioon msHBO Hom .coam5msa I m .m on :00 03¢ mo soauooaaoo . a . . osMWWMMMQoSQHmstDU m2¢ msoso> so ocflummosflmosos mo poomwm .HN mesons 147 Hm humans 5 $5222 [Wm mm o_~ m__ 0%. .m F mzijoezmra _ Eziramzammoi 22\oz lmN .-ununxiiiitllil--. n:>_< '8 maozm> [2 24.22 fi if E. [on . uuuuuuu .n n. ..... 1-1-... mmammmma loo. ZO_mDnEma euz 2&2 ZO_mDuZ_ wZ:>_O|_O._.ZMIQ OZEDQ n:>_< mDOZm> ZO wZEIQmZEmmOZ “.0 Homuum Figure 22. 148 Effect of epinephrine at high dose on venous AMP. Following collection of two controls, epinephrine was infused intraarterially into the gracilis muscle at 1.5 pg/minute for ten minutes. Two samples were collected during this time. Phentolamine, 50 ug/minute, was then infused concurrently with the epineph- rine for ten minutes. Two additional samples were collected. Each effluent sample col- lected was analyzed by the AMP assay. Each point represents the mean of five experiments. PERF PRES MM VE h dose on venous f two controls, aarterially into g/minute for ten ollected during ug/minute,Wfi th the epineph' dditional sarules nt sample col- MP assay. Ban five experiments. 149 EFFECT OF EPINEPHRI NE L5 ON VENOUS AMP [ ”(S/MW] 200— N=5 MEAN o :P< '05 PERFUSION ’ I PRESSURE Ioo- MM H9 .___.// \\\._ ..- ISO“ ’ ,‘O MEAN /°’ ’ ’ \ IZO— / VENOUS ,’ \ AMP 80-— // \ NG/ML \‘ 40— O- E E E T E. IPHENTOLAMINE [—1 EPINEPHRINE l 5 no IS 20 25 MINUTES Figure 22 17. E m Ep: which w; These rt remainet AMP als< between 18. Egg T0 Would a. infused results during a maxim AMP Sig three 1 crease PIESSur 50meWha Slire. perque tain a SiVe de 80me ty 150 17. Effect of Epinephrine at Low Dose on Venous AMP Epinephrine was infused at 61 ng/minute, a dosage which was designed to be too low to produce vasoconstriction. These results are shown in Figure 23. Perfusion pressure remained at control levels throughout the infusion. Venous AMP also did not change, again suggesting the association between AMP outflow and vasoconstriction. 18. Effect of ADH on Venous AMP To determine if a non—neurogenic vasoconstrictor agent would also be associated with AMP outflow, vasopressin was infused into the gracilis muscle in five experiments. The results are shown in Figure 24. Perfusion pressure rose during the infusion from mean control levels of 85 mm Hg to a maximum mean infusion level of 232 mm Hg. Mean venous AMP significantly increased above control levels during all three infusion samples. In the first two samples, the in— crease in AMP outflow paralleled the rise in perfusion pressure. However, in the third sample venous AMP declined somewhat, despite the continued increase in perfusion pres— sure. Perhaps this is due to the use of an artificially perfused preparation, where it might be difficult to main— tain a supply of high—energy phosphate compounds if exces- sive demands were made on them. It could also indicate some type of a washout effect of adenine compounds from 151 u§< WDOZM> ZO m2:2\02.®..~ MZEIQMZEM uO Eomuhm .mpsoEHHomxo snow W0 . % oN H sooE opp mpoomonmoH unflom noom moons mzd wmwmmmum mos was no? oopooaaoo oamfiom psoSwao Soom mo3 Honusoo 1:0Am5msa opp nopmo mopssHE sou pouooaaoo o opooHHoo .oso ..cOAmnmcH mflap msflndp mHo>uopsH u SMHE\mG Hm Hocflm mo mfiom.oon£9 .mopDCHE soopmam How op a. omsmoa onww oHMmDE maaaooum opp OpGfl haamwmwwwmmwwcmcwzoaaow . ..m on 200 03p mo . . mos omwmemmwmw> ow owoo 30H mo ocflnsmooflmo mo poommm ~ zo fizz/$261 szIaozim no Soto nz\oz did mDOZm> Z coo: Houucoo vaflonmso msflonodo NCHMQMDO Hsfloamso Houucoo Houucoo .mzm wsoco> so :HonSO mo uoommm .@ oHQoB made fc ting ce apparer from th samples able. served therefo to rele An in AMP decreasv the vaSI It was ' the eff; rate W01 ent. Hc Substant crease j time did Therefoz aCCOUnt 157 made for each sample. Figure 25 shows the results of plot— ting cells against venous AMP. It can be seen that no apparent relationship exists between amount of cells removed from the sample and venous AMP. For example, in some samples originally containing many cells, no AMP was measur— able. In other samples where few, if any, cells were ob— served a considerable amount of AMP was measured. It is therefore unlikely that the AMP observed can be attributed to release from blood cells in the samples. Another possibility considered was that the increases in AMP observed during vasoconstriction were due to the decreased venous effluent flow rate which often accompanied the vasoconstriction even though arterial flow was constant.* It was theorized that if a continual release of AMP into the effluent was occurring, then any decrease in the flow rate would increase the concentration of AMP in the effluv ent. However, no such relationship is evident in Figure 26. Substantial increases in venous AMP occurred with no in— crease in collection time. When increases in collection time did occur, the effect on venous AMP was not consistent. Therefore, it is unlikely that a decrease in flow rate can account for the increases in venous AMP observed during vasoconstriction. *The muscles often became very edematous at this time, and fluid leakage from sources such as cut vessels often occurred. Figure 25. 158 Effect of cells on venous AMP. Each sample used in the AMP assay was first centrifuged at 10,000 x G for five minutes at 0° C. A qualitative estimate of the amount of cells (or cell debris) in each sample was made immediately following centrifugation and the estimate recorded. Each point represents one sample. A = no cells; B = very few cells; C = few cells; D = moderate number of cells; E = many cells. Mp. Each samPle irst centrifuged tes at 0°C- he amount of ach samPlewas centrifugation Each POint no cells; 3‘ ls; D = moderate ells. VENOUS A M P NG/ML 159 350 - 0 . O 300 - 250 - : O . C 200v‘-I ~ g . v . . O ‘ z ‘ 3 O . 0 O O . : t i 5 i , O 50- 3 3 ‘ i s ' I ‘ f . 2 * 2 1 ' I I I i A s c 0 EV CELLS Figure 25 160 T CCT.“ .Honpsoo ope Op touomfioo mo oamfiom HopsoEHuomxo oco mpcomonmou usflom comm .Honusoo o>opo mzm msoco> 2H omoonosfl ucoo mom mcflccommonnoo one pmsfiomo coupon can popoasoaoo comp moB Honusoo Op conomfioo mo COHUOAMpchOOmo> moansc oEflu coep looHHoo GH omoouosfl usoo nod oSB .ponnsooo QOHUOHHumcoo toch soap? ca oucofiflnomxo Ham How ooCHEHouoU mos oamaom psodammo mo oEDHo> psoumcoo o pooaaoo Op confldqon oEHw mo psdoso ope .m2< msoso> so ouon 30am commonooo mo uoommm .mm enemas 161 em enemas m2; 20.55.60 $352.. A. on. 00. OO. mt on mN _ _ A F 0_ w o o o M H 00 o H0 omIOOn . .looo. n:>_< .. m302m> . o 100.... mmw ~COHDMHDEHDM 0>H0G H m “BOHM GOHMDHOOOIDWOQ H m «HOHHQOO H U "NOW «NH OHDWHM ~muH5m®mv OTMNM .H OHQQH. .mzfi wDOQ®> QHOmSE MHHHOMHm CO COHHMHDEHUW ODHQQ USN EOHMSHOOO M0 u 205 lIxIxa1IIIl1llslznxnlxlstnilllllsxxu m m m 0H «a me we me a m m a. m OH m S .m.m ow as as me as we we we AH am am am mm mm em we came mm on as mm om ow mm mm o o o o 0 ms mm o a mm on om om ova one one mas o o o o o o m o m ma OH OH ow mm om mm mm an em me mm mm ma NH me A om om mm om mm ma om om am as me me mm mm me me e om me me om om ow om om om mm as we om om ma mm m om ow ow mm mm mm ow mm om mm ow em mm mm mm me 4 ms om om men can can mNH owe m mm mm mm me mm me mm m on om om mm mm mm mm mm o as ma as om mm om N m cm ms om me me me me me we we mm O we ma om ma H o am am o «m am 0 o 0 mm Hm 0 mm Hm o o # moo mm BE .onnmmoum coflmsmnom HE\mc .mzm mdoco> . . .ooma w.H .mdo m >m compmasaaum o>nwc n m “soam coflmSHUOOIDmom n m “Houpsoo u o "wmm ANH mnsmnm .mnasmmmv .msm mnoqo> maomsfi mHHHOMHm no GOHDMHDEHum o>noc was GOHmDHooo mo Domwmm .H magma Table 10 11 Mean 206 Table 2. Effect of nerve stimulation (6V, 6 cps, 1.6 msec) on gracilis muscle venous AMP. (Results, Figure 13) Key: C = control; S = nerve stimulation Venous AMP, ng/ml Perfusion Pressure, mm Hg Dog # C s c c c c l 26 44 66 — 95 150 - — 2 14 31 — 23 125 175 — 125 3 23 70 — 18 80 100 - 75 4 8 26 23 0 75 100 100 100 5 13 38 l3 17 100 135 110 95 6 l4 7 3 7 100 100 95 100 7 28 38 18 8 80 100 80 60 8 8 18 14 16 150 150 150 150 9 18 48 10 12 75 60 60 60 10 61 95 54 57 85 85 95 100 ll 46 115 51 35 95 135 100 95 Mean 24 48 28 19 96 117 99 96 S.E. 5 10 8 5 7 10 9 9 .mQU m .>w ~GOH¥MH5EH¥M ®>H®C N OHOWDE mH m “HOQGHQDOHQOMH AwH QHDmHh HHOQHO CO HOSOHQDOHQOmH M0 GOHmSN Gfl Hafihmfl H H “HOHUQOO ” U n>anh .mUHDMOMV .AE¢ m502®> Hmwhuflfl MO HUUMMM .M QHQMB 207 OH OH OH OH HH HH OH OH O OO OH OH HH HO OO OO .m.O OO HO OO OO OO OO OO OO OH OO OO OH OH OH OO OH OOO: OOH OHH OOH OO OO OO OOH OOH O OO O O O O O O O OH OH OH OH OH OH OO OO O n O O O O O OH O OO OOH OOH OO OO OO OOH OOH O O O O O HH O O O OOH OOH OOH OOH OOH OOH OOH OOH O OO HO OH OO OO OOH OOO O OO OO OO OO OO OO OO OO O O O O O O O O H OO OOH OOH OOH OO OO OO OO O OH OO O O OH OO O O OO OO OO OO OH OH OO OO OO OH OO O O O O O O OO OO OO OO OO OO OHH OHH OO OOO OHO OOH OO OOH OO OO H o Om HO 0 OH HH o o o OO HO 0 OH HH o o H mom mm as rousmmwum coawsmuwm HE\mc .m2< mzocm> .mmo w .>O .GOHDMHDEHHm w>uon waomsfi mHHHomnm so HocwHoHOHQOmH mo Goamdmcfl Hafiumuuwmupcfi mo pommmm n m “HoawnoDOHQOmH H “HOHuQOO n U Ava onsmflm .muHSmomv .oomE O.H "wom .m24 msoco> .m wHQwB "NON OOHEMHHMW H U OGOHUMHDEHHM H m «HOHuQOU H U AmH OHSOHW OWUHDMOMV .GOHMSMQH OCHEMHHOU OmQU N ~>mv COHfiMHDEflUW ®>H®C N0 #OOMMW .V GNOME mflHHSU m2< m50fi0> CO AOOME m.o 208 mH mH mH mH mH mH OH OH O m mm NH O m m w No mO mO OO mO mO mO mO mN Nm mm ON mH 0N NN HN OHN mHN mHN mHN mHN OHN OON OON NH NH o o o o o 0 mm 0O 0O 0O mO 0O 0O 0O mH ON 0 o o o o 0 mm ow mm om om mm ow ow o o o o o o v m mw mm om om mv mv mv mv 0H 0 o o o o o 0 mm mm 00H 00H 00H mHH OHH OHH mm mm No mw mv om mv 0O mH 0N mm mm mm mm mm mm NH o o o 0 MN 0 o om mm mm mm ow mv mv mv mm mm mO MH m mm ow NN mN mm mm om om om om om NN Nm mm mm Hv mN mm 0N om mO 0O 0O om mm mm mm mm mm ow mNH OH mN 0H om mO OHH ONH mNH CMH mHH ONH ONH Hm 0O mm mm mv om No mm OO 0 OO Hm w+o 0+0 0 o OO 0 OO Hm m+o w+o o o mm 85 .mnsmmonm cOHmsmuom HE\mc .mz< msoco> ocHEmHme u w HnOHuwHSEHHw u m “Honucoo u o "mom mcHHsc m24 msoco> so HommE m. AmH qumHm .mUHDmomv o .mmo N .GOHmDmcH MCHEOHHMG .>OO quHmHssHpm o>Hmc Ho Hoommm .v OHQMB ¢CTENFPKt I. r\ ..‘r .. HmH mnsmflm .muHSmmmO Ommo m ~>mv GOHUMHSEHUM ®>H .QOHWDMQfl OGHEMHHNC mflHH5© m2¢ wDOQ®> GO OOOmE m.H OE M0 HOOMMM .m OHQMB 209 mH 0H mH VH MH NH NH NH mN wH ON 0H m m HH mH mm Hm wO mm mm ow Hm Hm mm ow wO mm NN mN mN mm omH om mHH om om om om om mmH om 0O mm o o o 0 mm mO OMH mNH mHH om om om wN m ON m o o o 0 mm mv mO 0O om ow mO mO o o m o o o o 0 0O om mm om mO mO mO mO om Om MO mm av OH mv om 0N mN om om mm mN mN mN I I Hm mm Nv NN Nm mH OH OH ON ON OH 0H 0H 0H mO mN Om mv mN mv Ow Om mm mO 0O om mv om om om OmH ONH ONN mO mm wv mm Hm mm 0 mm Hm w+0 0+0 0 0 mm 0 mm Hm 6+0 6+0 0 0 mm EE .oHSmmon GOHmDmHom HE\mc .mzd msoc0> O moo quEmHHmm H w “COprHDEHpm n m “Honugoo n U “mom AmH wnsmHm .muHmemv .QOHmDMcH msHEdHHcm mGHHsU ASH msoso> so Aoomfi O.H .mmo w .>OV QOHumHsEHHw o>uoc mo poowwm .O OHQOO KUF air—twh U+Frnuqax 1CC.—.U.:rhvc._. «Lamknnuc UCflhfigw % mgocmkw. OHOMDE MHHHOMHm. 30 A009: w.H OMQO OH ~>0Nv QOHHMHDEanm 0>H®OH MO “COMMON .m OHQMVB 210 OH MH MH m m m m Hm Om «N m OH 0 o .m.m mm mMN mMH om mm Hm Hm Om HmH mm m OH O o con mm OON 00H me no om ow mm mm mv NN o o o H 00H omN omH om OO 0O 0O mO mHH OO MH o o o m mm omN omH mv mm mv mv mm mvN mm o ow o o N om omN omH om om om om omH MHN mmH o o o o H U Nm Hm 00 00 U U 0 mm Hm 00 00 o o # moo mm Ea .ohsmmwum COHmsmmmm HE\mc .m24 msocm> wumnso mDHm QOHHOHDEHpm o>uwc n m Honchso mSHm Honucoo u 00 “Houucoo n o “OoM AOH wMDmHm .muHSmwmv .GOHmDmcH menno mGHHDU m2< msocm> mHomDE mHHHomum co AoowS O.H .mmo 0H .>oNV GOHHMHDEHum o>noc mo poommm .m anme @HUmDE WHOOCQ¥C CC ACQUE K. O .UCL O OVU\ ((Wlfir. rrrEWIfil‘ 211 O O oH O O O OH OH OH OH O OH O MH OH ON ON mm vm om OM OM OM ON HN OH HH HH MH OH OH OH OH OH OH OH OH OH mN O O m OH O m ow OH mm om Ow OV om Om mv mm O o O O O mN mN om mm om Om om om O O O O O O O Ow ow OH OH OH OH mN mN vm Nv OH mm OH mm om mm H U U Nm Hm DU 00 O O O 0 NM Hm 00 DO U 0 # mom mm EE .mndwmem cOHmsmnom HE\OG .m24 msocm> mHmHDU msHm COHudeEHpm o>noc u m HonHDo mfiHm Houucoo u 00 “Houucoo H U "moM AOH oHDOHm .mpHDmomO .QOHmSmcH wnmuso OQHHSO mzm msoco> oHomSE mHHHomnm co Aommfi O.H .mmo N .>mO GOHumHDSHum m>uoc mo poommm .O mHQwB @HONSE MHHHOMHO G0 Aommfi m.o ‘ r ~m®0 N Aww OHDmHh .MWHDWONO .m2¢ m50£®> .>mv GOHHMHDEHUM 0>HOQ MO HUONMN .m QHQMB 212 OH OH OH HH OH OH O O O OH O O O OH O HH .m.w OO OO OO OH OO NO NO NO OH OH HH OH NH ON ON HN gmmz I I I ON I Om OO OO I I I O I om Om ON O I I I OO I OO OO OO I I I mm I OO OO OO O OH OH OH OH OH OH OH OH ON Om ON ON OH OH ON O H OO OO OO OO OO OO OO OO mm ON ON Hm om OH Om OO O I I I I I I I I O O O O O O O O N Om Om Om mm mm OH OO OO O O O O O O O O H U O HO mm Nm HO O U U O HO mm Nm HO 0 O H moo mm E8 .oudmmoum COHOSMHom HEKOC .mEH mdocm> GOHHOHSEHpm o>umc u “Houpcoo n 0 "wow oHomsfi mHHHomHO so Howmfi 0.0 HOH OHOOHO .manmmmO .mmo N .>OO GOHHMHDEHum o>uoc mo uomwwm .m24 msocw> .O OHQMB Table Mean 213 Table 9. Effect of nerve stimulation (5V, 2 cps, 0.5 msec) on gracilis muscle tension. (Results, Figure 18) Key: C = control; S = nerve stimulation Average Muscle Tension, mm Deflection Dog # c c 31 52 s3 54 c c 1 o o 17 17 11 8 o o 2 _ _ - _ _ _ _ _ 3 o o 21 35 33 10 o 0 4 o o 18 14 6 2 o o 5 o o 15 — 6 — — - 6 o o 8 — 4 — — - Mean 0 0 l6 17 12 7 0 0 OOH @HDUHh .muHDmGNC .CCFLSuIH (IuiIrI OmQU 0.... ~>NNO EOIHHMHHHEIOUW O>H®.C W0 UOWMMWH .OH OHQME MEAN m50§®> cHO Hommfi m...” 214 O OH O m O O OH HH OH O O .m.m OO OO NO OO OO OO OH NO OO NO OO OH cum: OH OH I OO OO OO HO HO I OO OO OO O mm mm OO OO OO OO OO OO OO OO OO OO O OH OH OO OO mm mm mm OH OH om OH mm H OH OH OOH OO OO OO OO OHH OOH OOH OO OO O OO OO OO OO OO OO mm mm ON OH ON om N OO OO OOH OOH OO OO OO OO MO I mO Om H Om Hm m+o m+o o 0 OO Hm m+o m+o o o H Ooo mm ES .ousmmmum COHmDMHom HE\OC .mzd msoco> ocHEmHOHcmflm u m “SOHHMHDEHum n m “Houpcoo u o "Omm HOH wusmHm .OHHsmmmO .QOHmszH wGHEwHOHQOSQ OGHHDO ASH muonm> co Homma O.H Hmmo OH .>NNO cOprHDEHum m>noc mo poommm .OH OHQOB F CO OQHHHHOHO 215 OH OH NH O O O HH OH HO ON O O .m.m OOH OOH OOH HOH HO OO OH OOH ,NOH OH OH OH cwmz OO OOH OOH OO OO OO OO OOH OO OO NH O O OOH OON OOH OOH OO OO OH HOH ONH O O O O ONH OOH OOH OOH OO OO HO OHH OOH OO OH OO H OHH OOH OOH OOH OO OO OO OO OH O O O O OO ONH OOH OO OO OO O OO OH O O O N OHH OHH OOH OHH OO OO OO OHH ONO OOH HH OH H 0 Oz Oz Hz o o 0 Oz Oz Hz 0 o H moo mm ES .mndmmonm COHmdmuwm HE\OS .m2< msocm> OQHHSQOGHQOHOS M Z OHOHHQOO H O "me HON wnsmHm .mpHsmmmO .m2< wsocw> co GCHHSQmCHQoHo: mo powwwm .HH mewB .GOHmDMQH OGHEMHOHGOQQ mGHHDU m EN m502®> CO OQHHQQOGHQOHOC WC IHQGUHHD 216 OH HH HH OH OH OH HH OH HH OH NH HH .m.m OO HO HO OO OO OO OO OH OH OO NO OO cam: OOH OOH OOH OOH OOH OOH OO OO OH OH OO OO H OO OO OO OO OO OO NH O OH O O O O OH OH OH OH OH OH ON HO OH OO ON OO N OO OO OO OO OO OO OO OO OO OO OH OH H m+2 m+2 m+o m+o o o m+2 m+2 m+o m+o o o H Ooo mm 88 .oHSOOme QOHmsmumm HE\OG .m2< msosm> HHO OHOOHO .manmme .SOHmSMGH GQHEOHOHGQSQ OGHHSU HEH msoco> co QQHusmmchmnoc mo pomwmm .NH mHQmB mGHnsmwcHQmHoc n z HQCHEOHOHgmcm u m “HOHpGoo I o .OQM P”"*‘ CO OQHHSQOHHQO wmtfi. IIIIII AIFF CHE\m1 (JOZUGQH IQE< MSOC®> OHOMSE MHHHOMHO O.HO COHmDMEH HMOHOHHMGHHGH MO HOOHNW .MH @HQMH 217 O O HN HH O O O O ON OO O O .m.m OH OH HHH OO OO HO OO OO OOH HOH OO NO cums OO OO OOH OO OO OO OO OO OOH OHH HO OO O OO OO OOH ONH OO OO NO OO OOH OOH OO OO H OO OO OOH OOH OO OO OO OO OOH OO OO OO O OO OO OOH ONH OO OO OO OO ONN OON OO OO N OH OO OO OO OO OO OO HH OO OH OO OH H m+m m+m m m o o m+m m+m m m o o H Ooo Om EE .oHSmmem GOHmDmem HE\OC .m2¢ msoqw> OQHEMHOHCOSQ n m OwcHusmocHaw H m “HOHHQOO H U "ham ANN mHDOHm .muHDmmmO .mSH msocm> wHomsfi mHHHomum co onHHHmwchw mo A.QHE\O: O.HO GOHmSHCH HMHHGHHMOHHCH mo pommwm .mH OHQMB II.III.\ I((. F U \ ‘(WUrrufrnr QC. TLIC¢C WCQ HMHHOHHGMHUCH NO “COMMON. .HH mHnms 218 MH OH OH OH ON O O O O O O OO OO OO OO OO OO NH OO OH NH OO OO OO OO OO OO OO OO OH OO OH OO OHH ONH OOH OHH OOH OH OH OO OO OO OH OO OH OO OO OO OO OH HN ON OO OO OO OO OO OO OO OO OH OO OO OO OO 0 OH Om o o o Om OH HO 0 o mm SE .musmmwum QOHOSMHmm HE\OG .m2< msocw> oaHuamwchw u m «Honpcoo n 0 OOO wHHHOMHO no A.sHE\Os HOV GOHm HON oHDmHm .mHHsmme DMnH wcHufimwchm HMHHo .m2¢ mDocm> mHomsE HHMMHHCH mo powwmm rag—Hm- .\ (IIIIII .I. . 219 OO OO OH OH HH OO OO OO O O .m.O OOH HOO OOO OOH OO OO HO HO HOH HO OH HH OOO: OOH OHO OOO OOH OO OO OH OO OOO OO O O O OOH OOO OOH OOH OO OO OO OO OOO OHO OH OO H OOH OOO OOO OOH OOH OOH OO OOH OOH OOH OO OH O OOH OHO OOO OO OO OO OO OOH OO O O OO O OOH OOO OOO OOO OHH OHH O O O OO O O H 0 OH OH HH 0 o 0 OH OH HO o o H moo mm 58 .wHDmmem COHmsmem HE\O: .mEH mdocm> AHN mHSmHm .muHsmmmO mad u < “Hoaucoo n o "mom .mZH msocw> so mo< mo Homwmm .OH anma III IIIHIII 1293 03046 6308 III MICHIGAN STATE UNIVERSITY LIBRARIES 3 1‘ ......ng . .i .51. «5.: .