THEEFFECTS onmoooroxmou - THE REACTIVITY or SMOOTH MUSCLE , ’ Thesis for the Degree of Ph. D. v MECHEGAN STATE UNIVERSITY THOMAS D. BURNS 1973 I‘EJ'T'J [‘9 I, m 1? AR 2" a A\'l.c_:;;£3.q 523‘3 Umvc .. sity ”T 2 l gunman :41ng W Tetrodotoxin extract from the P‘ excitabTe tissues I In the present stuc‘ an: gracilis muscle were employed to s1 insular and visceu the effects of TTX agents (epinephrine SITinants. KC] an: 1n. / ' ' “ppm producm' o'abnh'sh 1) new Zivagal-induced 1' 'esponses to carot‘ ‘nblood flow or d Tami but failed ‘Ttestinal prepare "1 W'95 TTX producn ABSTRACT THE EFFECTS OF TETRODOTOXIN ON THE REACTIVITY 0F SMOOTH MUSCLE BY Thomas Dudley Burns Tetrodotoxin (TTX), also called tarichatoxin, the toxic extract from the poisonous puffer fish and California newt affects excitable tissues by selectively blocking sodium conductance. In the present study in situ canine preparations of the intestine and gracilis muscle as well as isolated arterial (femoral) strips were employed to study the effect of TTX on the responses of vascular and visceral smooth muscle. The study also focused on the effects of TTX on the vascular responses to other vasoactive agents (epinephrine, norepinephrine, acetylcholine, ganglionic stimulants, KCl and changes in osmolality)o TTX in doses (0.05- lO ug/min producing 10'6-10'9 g/ml of blood) sufficient to depress or abolish l) nerve-induced motor responses of gracili muscles, 2) vagal-induced intestinal motility reponses and 3) vascular responses to carotid occlusion produced significant increases in blood flow or decreases in vascular resistance of innervated gracili but failed to effect denervated gracili or innervated intestinal preparations. In cross-perfused innervated gracili muscles TTX produced a maximum vasodilation at doses which afished the re: lassdilatlon C0” 'ener‘v‘atEd gracll n denervated pre ‘xglly infused T ever, occasionall .‘th increases in induced by local :onve'ted KCl-lndl 1"-r‘nstine. TTX al- sisu‘ng little pha 'esponses. Intra- laCl produced vaso abolished the moti chase; thus allowi l" isolated arteri Elmilled as pre-l {10'9'10‘5 g/ml) h Elli storage. TTX 0' am (5 x l0‘5 Fiji" s ~ 90 nephrine, ac. l, W W hypo—05h; l '5' 7.5. and l5.0 “leases in tensw Rich .er Pia-loads. Ea3"? U M). [K“] M) 122 mEQ/l Thomas Dudley Burns abolished the resistance responses to carotid occlusion. TTX induced vasodilation correlated with the level of initial resistance in innervated gracili (r = 0.92) but showed no correlation ( 4 = 0.27) in denervated preparations. In innervated intestinal preparations, locally infused TTX generally produced no vasodilator response. How- ever, occasionally TTX increased resistance which occurred concomitantly with increases in motility. TTX had no effect on the vasodilation produced by locally infused KCl into gracili muscles but frequently converted KCl-induced vasodilation to vasoconstriction in the intestine. TTX altered the motility responses to KCl from a pattern showing little phasic activity to one exhibiting large phasic responses. Intra-arterial or luminal placement of hyperosmotic NaCl produced vasodilation followed by vasoconstriction. TTX abolished the motility responses occurring during the vasoconstrictor phase; thus allowing hyperosmotic NaCl to produce only vasodilation. In isolated arterial strips the responses to all agents studied were augmented as pre-load was increased (l, 2, 4 and 6 gms). TTX (10'9-10'5 g/ml) had no effect on strip before or after 24 hrs of cold storage. TTX depressed the increases in tension to nicotine or DMPP (5 x 10'6 g/ml) but failed to effect the responses to norepinephrine, acetylcholine. epinephrine, tyramine, KCl or hyper and hypo-osmololity. Increasing extracellular KT (fl.5, 4.5. 7.5. and l5.0 mEq/l) above control (4.7 mEq/l) produced increases in tension of vascular strips which were greater at higher pre-loads. When the strips were actively contracted with BaClz (l mM), [KT] < 12.2 mEq/l produced relaxation while [K*] > l2.2 mEq/l produced contractions. Increasing pail osmlality (50 -' gytractions of pass I ietlz, Hyposmolalitll tension before and a‘ for osnolality wereh :‘ociade. These dateh ‘W in neural bloch at‘ion on vascular 5rh “-9an mechanisms; 2 i ll lemoidl of intrinji Eif'acellular K+ andl "all”! responses J m“ and 4) the J lLaltltdtlvely and q"I Thomas Dudley Burns bath osmolality (50 mosm/Kg) above control (300 mOSm/Kg) produced contractions of passively loaded strips but relaxation following BaClz. Hyposmolality (- 30-50 mOSm/Kg) produced increases in tension before and after BaClz. The responses to change in bath K+ or osmolality were not affected by TTX, adrenergic or cholinergic blockade. These data strongly support the contention that: l) TTX in neural blocking doses (10'9-10'6 g/ml) has no direct action on vascular smooth muscle but produces vasodilation through neural mechanisms; 2) TTX may stimulate molility of visceral muscle by removal of intrinsic inhibitory nerves; 3) in the intestine extracellular KT and changes in osmolality may produce neurogenic motility responses which frequently alters the direct vascular action, and 4) the responses of isolated arterial strips may differ quantitatively and qualitatively depending on equilibration pre- load and the degree of active tension. in Parts THE EFFECTS OF TETRODOTOXIN ON THE REACTIVITY OF SMOOTH MUSCLE By if} Thomas D. Burns A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1973 To my family and fm' happims. and 70 cm” a"d Tommy heeven. DEDICATION To my family and friends who have shown me there are many ways to happiness, and To Caroll and Tommy who I hope will receive some rewards before heaven. The author woulc his encouragement. fl for his desire to mail most valuable assista A special apprec Also, a sincere grat‘l 1h. Brody. R.H. Daug Sectt for their value future. The author owes “as. J. Johnston, Hi: assistance in the pr: ACKNOWLEDGMENTS The author would like to thank Dr. J.M. Dabney not only for his encouragement, financial support and guidance but especially for his desire to make learning enjoyable. A special appreciation is extended to Dr. C.C. Chou for his most valuable assistance throughout the course of my training. Also, a sincere gratitude and appreication is extended to Drs. T.M. Brody. R.M. Daugherty.Jr., F.J. Haddy, D.A. Reinke and J.B. Scott for their valuable inspiration and personal concern in my future. The author owes many thanks to Ms. K. Bishop. Mr. C.P. Hsieh. Mrs. J. Johnston, Miss M. Kuhn, and Mrs. Pam Rashid for their assistance in the production of this manuscript. iii Iii-L‘ICIIIEDGEI'IENTS . LIST OF TABLES USTOF FIGURES. . IhTRODUCTION . . . LITERATURE REVIEH Historical Backg The Cardiovascull Effects of Tetro Statement of Pro Nature dfld de 2' crOss. enerv "testlnal arteri 4. Nature place" 5. TABLE OF CONTENTS ACKNOWLEDGEMENTS ....................... LIST OF WABLES~ Historical Background of Tetrodotoxin ........... The Cardiovascular Effects of Tetrodotoxin . . ...... Effects of Tetrodotoxin 0n Nonvascular Smooth Muscle Statement of Problem .................... EXPERIMENTAL METHODS ..................... Preparation of the Animal ................. Experimental Preparations ................. Skeletal Muscle Preparations .............. l. Naturally perfused gracili muscles (innervated and denervated) ................. 2. Cross-perfused gracili muscles (innervated and denervated) with constant flow ......... Intestinal Preparations ............. . . . . . 3. Naturally perfused double segments with intra- arterial (i.a.) infusion of test agents ..... 4. Naturally perfused double segments with luminal placement of test agents ............ 5. Pump-perfused single segment with intra- arterial infusion of test agents ........ iv LIST OF FIGURES. . .... .................... INTRODUCTION ......................... LITERATURE REVIEW PAGE iii viii ix @0300 12 15 18 18 19 19 19 27 27 In Vitro Prepare —6_._ Vascu‘ A. l: 3. Pl C. Fr Experimental Pr: 1. Thee GT in! 2. Effec intes 3- The a on th to 51; Which 4. Effec Vascu Place jejur 5. The ‘ Rhan 12.! Anal evai :ZESULTS . . vascular A . . Cti Gracili "Us“: 1' Eff. and 2‘ Eff 0f 3‘ Eff I In Vitro Preparation ..................... 6. Experimental Protocols .................... 1. RESULTS . . . Vascular strips in vitro .............. A. In Vitro baths ................. B. Preparation of the strips C. Formulation of the artificial solutions The effect of tetrodotoxin on the vasculature of innervated and denervated gracilis muscle . . . . Effects of tetrodotoxin on the responses of intestinal and vascular smooth muscle to KCl . . . . The action of tetrodotoxin and hexamethonium on the responses of vascular and visceral muscle to stimulation by potassium, sodium and drugs which act on ganglia ...... . . ........ Effects of tetrodotoxin on the responses of vascular and visceral muscle during luminal placement of hyperosmotic solutions in the jejunum ....... . ............... The effects of tetrodotoxin, KT, osmolality and pharmacological agents on arterial muscle, in vitro ...................... Analysis of venous samples and statistical evaluation of data ................. Vascular Action 0f Tetrodotoxin (TTX) In Naturally Perfused Gracili Muscles 1. Effect of TTX on venous outflow of innervated and denervated gracili ............... Effect of TTX on the vascular responses to KCl of innervated and denervated gracili . . . . . . . . Effect of TTX on the vascular responses of gracili to neural stimulation . . . V OOOOOOOOOOOOOOOOOOOOOOOO PAGE 34 34 34 37 38 39 39 4O 41 43 46 49 50 50 50 51 54 Vascular Actic Gracili Muscle L Effect vascula gracili Effect action Vascular Actic Dean and Jeju L 2. 3. Effect Effect Effect luminal osmotic ViSCUlal‘ Actio JeJunum . , 1. Action . activit Effect , reactiv "TCOtin ResDonses 0f F l. Acu InN Effect Of Arte agents Effect Of arte Effect BOCIZ Effect: SITTpsr 0" 0f Tet aturdlly Vascular Action of Tetrodotoxin 0n Cross-Perfused Gracili Muscles ..... . . . . . . ........... 1. Effect of TTX on vascular resistance and the vascular responses of innervated and denervated gracili to carotid occlusion ............ Effect of initial resistance on the vascular action of TTX ................... Vascular Action 0f Tetrodotoxin 0n Naturally Perfused Ileum and Jejunum ...................... 1. Effect of TTX on venous outflow of the ileum . . . . 2. Effect of TTX on the vascular responses to KCl . . . . 3. Effect of TTX on the vascular responses during luminal placement of hyperosmotic NaCl, hyper- osmotic KCl and glucose .............. Vascular Action 0f Tetrodotoxin 0n Pump-Perfused Jejunum ........................... 1. Responses 0f Femoral Arterial Strips In Vitro . . ..... 1. Action of Hexamethonium (C5) and TTX on the vaso- activity of isosmotic KCl and hyperosmotic NaCl Effect of TTX on the venous-arteriolar response, reactive dilation, and the vascular responses to nicotine, acetylcholine and epinephrine ...... Effect of preload on the contractile responses of arterial strips to TTX and other vasoactive agents ........................ ' Effect of TTX and cold-storage on the reactivity of arterial strips ................. Effect of K+ on arterial strips before and after BaC12 ....................... Effect of hyper- and hyposmolality on arterial strips before and after BaClz ........... Action 0f Tetrodotoxin 0n Visceral Smooth Muscle In Naturally Or Pump-Perfused Intestine ........... vi PAGE 55 55 66 74 74 75 76 91 91 96 102 103 122 I. Effect ileum 2. Effect to i.a acetYI‘ 3. Effect by NW and 91‘ DISCUSSION The Action Of Vascular SITIOOt Skeletal Intestine Vascular The Action Visceral Smoot SWARY AND CONCLU REFERENCES . . . PAGE l. Effect of TTX on spontaneous motility of time ileum and the motility responses to vagal stimulation ...................... 122 2. Effect of TTX and C5 on the motility responses to i.a. KCl, hyperosmotic NaCl, nicotine, acetylcholine and epinephrine ............ 132 3. Effect of TTX on the intestinal motility produced by luminal placement of hyperosmotic NaCl, KCl and glucose ..................... 134 DISCUSSION ........................... 143 The Action of TTX on the Reactivity of Vascular Smooth Muscle . . . . . . ............. 143 Skeletal Muscle Vascular Responses ........... 143 Intestinal Vascular Responses ............. 152 Vascular Smooth Muscle Responses, in vitro ....... 158 The Action of TTX on the Reactivity of Visceral Smooth Muscle .................... 165 SUMMARY AND CONCLUSIONS ..................... 172 REFERENCES ............................ 177 vii I. Effects of vascular r to luminal Effects of vascular re to luminal Effects of vascular re t0 luminal TABLE LIST OF TABLES Effects of Tetrodotoxin (TTX) on the intestinal vascular responses in naturally perfused jejunum to luminal placement of hyperosmotic NaCl ....... Effects of tetrodotoxin (TTX) on the intestinal vascular responses in naturally perfused jujunum to luminal placement of hyperosmotic KCl ....... Effects of tetrodotoxin (TTX) on the intestinal vascular responses in naturally perfused jejunum to luminal placement of hyperosmotic glucose. . viii PAGE 81 83 86 Figure l. 2. Natural l y pe Gracilis mus Pump-perf use Naturally pe (double-segn Single-segme Ill Vitro mus Effects of T flow of the Effects of 1 naturally be Effect of hi flow of "flu Effect of T”; perfUSEd gra Effect of 71 camera c '"UScle (trac Willi musc varied (Ora; Figure 10. ll. 12. 13. 14. 15. 16. LIST OF FIGURES Naturally perfused gracilis muscle prepartion ...... Gracilis muscle cross-perfusion preparation ....... Pump-perfused gracilis muscle preparation ........ Naturally perfused intestinal prepartion (double-segments) .................... Single-segment intestinal preparation .......... In Vitro muscle strip preparation ............ Effects of Tetrodotoxin (TTX, 5.0 ug/ml) and KCl on blood flow of the naturally perfused gracilis muscle ...... Effects of TTX (0.5 ug/ml) on blood flow of the naturally perfused gracilis muscle ........... Effect of high and low doses of TTX on blood flow of naturally perfused gracili muscles ........ Effect of TTX on vascular resistance of constantly perfused gracili muscles (graph). . . ....... . . . Effect of TTX on vascular resistance and local response to carotid occlusion in cross-perfused gracili muscle (tracings) .................... Effect of changing initial resistance on the vascular resistance responses to TTX (tracings) ......... Vascular resistance responses of cross-perfused gracili muscles to TTX when initial resistance was varied (graph) ...................... Effect of initial resistance on the change in resistance of gracili muscles to local infusion of TTX (graph) ...................... Effects of TTX and KCl on blood flow of naturally perfused ileum (graph) ............ Effects of TTX on the intestinal vascular responses of naturally perfused jejunum to luminal placement of hyperosmotic NaCl, KCl, and glucose (graph) ...... ix Page 21 24 26 29 33 36 53 57 59 63 65 68 7O 72 78 90 F‘;Jte — o (3.) 2T M. 21 U. 3. 2E U. A. Effect 0‘ response: infusron Effect oi motility hyperosnx Effect 01 and venou jejunum ( Effect of responses infusion (graph). Effect of Of fresh responses Nicotine, EffeCt 0f the tensi after phe EffECtS o bQTOre an Effect Of BaC12 (gr Figure Page 17. Effect of C and TTX on vascular resistance responses 0? pump-perfused jejunum to local infusion of KCl and hyperosmotic NaCl (graph ...... 93 18. Effect of TTX on the vascular resistance and motility of pump-perfused jejunum to locally infused hyperosmotic NaCl (tracings) .............. 95 19. Effect of C5 and TTX on the reactive-dilation and venous-arteriolar response of pump-perfused jejunum (graph) .................... 98 20. Effect of C and TTX on the vascular resistance responses 0? pump-perfused jejunum to local infusion of nicotine, acetylcholine, and epinehprine (graph) ......................... 101 21.. Effect of pre-load tension on the in vitro responses of fresh femoral arterial strips (tracings) ....... 105 22. Effect of pre-load tension on the in vitro responses of arterial strips to BaClz, norepinephrine, nicotine, DMPP, TTX, and acetylcholine (graph) ..... 107 23. Effect of TTX, acetylcholine, and epinephrine on the tension of fresh arterial strips before and after phentolamine (tracings) .............. 109 24. Effects of DMPP, nicotine, tyramine and norepinephrine before and after TTX in fresh and cold stored arterial strips (tracings) .................... 112 25. Effect of K+ on arterial strips equilibrated at different pre-loads before and after BaClz (tracings). . 115 26. Effect of KT on arterial strips before and after BaClz (graph) ...................... 117 27. Effect of KT on arterial strips before and after BaClg, TTX, phentolamine, propranolol, atropine and ouabain (tracings) ........... . ........... 119 28. Effect of increasing and decreasing extracellular osmolality on tension of arterial strips before and after BaClz (tracings) ............... 124 29. Effect of pre-load on the tension responses of arterial strips to increasing bath osmolality before and after BaClz (graph) ................. 126 X 3i. Effect of responses vagal stin Effect of motility r ileum (tra Effect of Of Puma-De and epinep Effect of I 01‘ pump-be (graph ). . Effect of ( ’95Ponses I ”Ype'osmot‘ Figure Page 30. Effect of locally infused TTX on the motility responses of naturally perfused ileum to vagal stimulation (tracings) . . . . . . . . . . . . . . 129 31. Effect of TTX on the spontaneous and K+ induced motility responses of naturally perfused ileum (tracings) .................... 131 32. Effect of C and TTX on the motility responses of pump-pergused jejunum to nicotine, acetylcholine and epinephrine (tracings) ............... 136 33. Effect of C5 and TTX on the baseline motility responses of pump-perfused jejunum to KCl, and hyperosmotic NaCl (graph) ........ . ................. 138 34. Effect of C? and TTX on the phasic motility responses 0 pump-perfused jejunum to KC] and hyperosmotic NaCl (graph) ............... 140 xi Tetrodotox' fish (Egg, m has been shown t song the physio 97) and systemic depression of tip (39}. reportedly ante (S4, 95, 97; 35d Paimre (33) H Miller“ Sympat responsible for t iaoflél» (74, 7 hiflotensiOn res“ muscle, A1th0ugh of TTX has been a: The reSPOnseg m be limited to , INTRODUCTION Tetrodotoxin (TTX) the toxic extract from the poisonous puffer fish (fugg, Spheroides rubripes) and California newt (Taricha torgsa) has been shown to affect excitable tissues in animals and man (73). Among the physiological effects of TTX are paralysis of nerves (61, 95, 97) and systemic hypotension (33, 73). Nerve paralysis results from depression of the propagated action potential without depolarization (29), reportedly due to a selective inhibition of the sodium conduct- ance (54. 95. 97) normally occurring during depolarization. Feinstein and Paimre (33) maintain that a blockade of conduction occurring in peripheral sympathetics and nerves innervating the adrenals is responsible for the profound hypotensive action of TTX. However, Kao gt_gl, (74, 77, 88) subsequently reported that TTX-induced hypotension results from a direct relaxant action on vascular smooth muscle. Although the hypotension produced by systemic administration of TTX has been ascribed to numerous factors (33, 61, 74, 76. 77, 78, 82, 87, 88) the mechanism producing vasodilation remains unclear. The responses of nonvascular smooth muscle to TTX are reported to be limited to neural mechanisms (39,“86, 100). Wood (130) cautioned against Gershon's (39) contention that TTX specifically blocked enteric neurons without affecting smooth muscle and reported that TTX altered both the electrical and mechanical activity of isolated cat jejunum but concluded that the stimulation occurs upon removal of an inhibitory neural mechanism. The stimulation of rat jejunum by TTX was reported to be similar to the stimulation resulting from whole body X-irradiation, hexamethonium and physostigmine (71). Kuriyama 1 335.1: (86) “d E :iooal evidence I storach, rabbit : action of TTX. E moth muscle W‘l sany investigatm to separate neurc ITO and lOS). Intrinsic nE of gastrointestir. of intrinsic nerv retains questiona intrinsic nerves afew studies ind directly or indirI end/or visceral SI I12. 113. 114 and ilddenervated lor that no; and KCl WC! part of th sechanisms and pa cologically dener confimed pawn a gt g1, (86) and Gershon (39) presented electrophysiological and func- tional evidence from guinea-pig taenia coli. mouse stomach. guinea-pig stomach, rabbit jejunum. and guinea-pig ileum favoring a non-direct action of TTX. Based on the strong evidence that TTX affects nonvascular smooth muscle primarily gig extrinsic and/or intrinsic neural pathways. many investigators have used this neurotoxin in numerous preparations to separate neurogenic and non-neurogenic mechanisms (6. 39. 71, 73, 100 and 105). Intrinsic nerves have been ascribed important roles in regulation of gastrointestinal secretion and motility (83), however. the role of intrinsic nerves in local regulation of intestinal blood flow remains questionable. Although, little evidence exists relating intrinsic nerves with local regulation of intestinal blood flow, a few studies indicate that local neural mechanisms may be involved directly or indirectly in the responses of intestinal vascular and/or visceral smooth muscle to certain vasoactive agents (23. 26, 112. 113, 114 and 127). Paton and Zar (102) studying innervated and denervated longitudinal muscle of guinea-pig ileum. reported that BaClz and KCl (thought to have primarily direct effects) produce part of their action on visceral smooth muscle by neural mechanisms and part yig,direct effects. Gershon (39). by pharma- cologically denarvating longitudinal strips with tetrodotoxin. confirmed Paton and Zar's findings. Although the data reported by these investigators suggest only visceral smooth muscle affects. avidanca suggesting that intrinsic neural elements may be involved in the intestinal vascular responses to luminal placement of riserosmotic solL In those studies, local anesthetics local vascular re the lumen. Visceral srnc effect on the int interaction betwe has been describe to these authors ls determined by on vascular Smoot 31 any meChan‘ical Wenchm‘ Then heard] Elements 1] cological agent SI vascular as well i The f0TlowinI regarding TTX, Pl" effects of TTX, t hyperosmotic solutions has been reported by Dabney gt.al, (23, 26). In those studies, exposure of the canine intestinal mucosa to the local anesthetics pipericaine and dibucaine significantly altered the local vascular responses to certain electrolyte solutions placed in the lumen. Visceral smooth muscle activity can have a profound passive effect on the intestinal vasculature (24, 27. 112 and 114). This interaction between visceral and vascular smooth muscle reactivity has been described and emphasized by Haddy gt_al, (49). According to these authors the net vascular response of any vasoactive agent is determined by three interrelated effects: 1) the direct action on vascular smooth muscle, 2) the effect on tissue metabolism, and 3) any mechanical effect resulting from the activity of nonvascular parenchyma. Therefore, any attempt to study the role of intrinsic neural elements in local blood flow regulation by use of a pharma- cological agent such as TTX requires that the action of TTX on vascular as well as visceral smooth muscle must first be defined. The following review briefly presents a historical background regarding TTX. previous investigations directed at the cardiovascular effects of TTX. the action of TTX on nonvascular smooth muscle. LITERATURE REVIEW Historical Background of Tetrodotoxin, TTX - Descriptions of the toxic puffer fish (tetrodons. the first recognized source of tetrodo- toxin) can be found in ancient Chinese literature over 2000 years ago (73). Although tetrodon eggs were used as a therapeutic agent for gesmries, 3 deti Septic relevance lt-Ie Great Herbal so: the toxic pul Tito century (70) descriptions of t l-r‘eot (.73). G. producing numbnes heiress (34). The first so ieyin 1884 (104; WlenIng was com; Interested in all 'Vlllve toxicity RT-als, and began and coined the ten ills, given a molec and Clinicaliy unt' Shh . - ‘% T‘Ubl‘l' e Interim centuries. a detailed description of the tetrodon fish and its thera- peutic relevance didn't appear until the pharmacopea Pen-t'so Kang Mu (the Great Herbal) was published around 1600 A.D. Tetrodon poisoning and the toxic puffer fish first became known to Europeans in the 18th century (70) when many cases of poisoning as well as clear descriptions of tetrodon fish were reported by early visitors to the Orient (73). G. Foster, Cook's naturalist, described the illness as producing numbness of the limbs, flushing and generalized muscular weakness (34). The first scientific study on tetrodon poisoning was published by Remy in 1884 (104). However, the first extensive study of tetrodon poisoning was completed by Takahashi in 1890 (cited by Kao. 73). Interested in all the species of toxic tetrodon, Takahashi studied the relative toxicity of various organs, their physiological effects in mammals, and began work to chemically extract the toxic agent. Tahara. working in Japan, completed extraction of the toxin in 1894 and coined the term "tetrodotoxin" (121). The extract from Spheroides eggs. given a molecular formula of C15H13N015, and was used experimentally and clinically until 1950 when crystallization of the toxic agent from Spheroides rubripes, called “spheroidin”, was reported by Yokoo (135). Interest in tarichatoxin, the poison from California newt (Iagighg_tgrg§g), developed following transplantation studies by V. Twitty in 1927 (126). Twitty. an experimental embryologist at Stanford, reported that transplantation of eye vesicles from local salamander embryos into embryos of eastern salamanders (Amblystoma) produced prolonged paralysis of the host (126). Subsequent apartments showed ‘- se‘ectively WW“ gar: resulted in ex' preaaration availabl nutty tarichatoxirl preparation 10 timel t'ew'ously reporter.I term. indicated thaI M. "spheroicl to the toxic princl Quanura (125) was tarichatoxln (14) Structure of tetro the puffer fish pcl c‘E'hydiaquinazol ir (ml. wt. = 319.23. SW95 0f Puffer lily ‘ ..\ a1 - to". dlStrlf helm I fife of 30 m' Crystalline ' Fest potent non-p e‘fective than CO' Merful neural b Ishihara (61) in I using isolated Sr that TTX in dosemv cvm> Peaceme on» cmaoegu acme mew: AHV mcowuuwwcw opsmpmxm .xcmpgm mw_wueem on» o» Eemcpmczou Agmmme mcpuumppou a one? sopmaao mg» umpumgwu Au>v mmpaccmo maoco> .Aucm= asp m:_uu:u ma umpm>em=mu we: «pawns mzo .cowuegeamca m_um=E mwpwuecm ummamgwa xppegspm: ms» mo Azmw> —mgucm>v mcwzmcu uwumsosum _ mezmwm 21 vas cannulated containing dext' Blood from via the femoral nscle was peril on a top-loadin. has continuousl. twain llith const; animal (donor) 5 itacili muscles were sePat‘ated . its one muscl e. The gracil' described excep was drawn from (finger.type Du "miles of the pmduced a Peri Venous cannulae containing dex ailing]: S fe'hor 22 was cannulated (PE 240) and its flow diverted into a glass reservoir containing dextran9. Blood from the reservoir was continuously returned to the animal via the femoral vein by means of a pumpio. Blood flow through the muscle was periodically determined by weighing timed venous collections on a top-loading semi-analytical balance“. Abdominal aortic pressure was continuously recorded via a femoral arterial cannula. 2. Cross:perfusedfigracili muscles (innervated and denervated) with constant flow (Figures 2 and 3) - In this preparation one animal (donor) served as the blood source for perfusion of the gracili muscles of another animal (recipient). Neurogenic responses were separated from direct vascular responses by surgically denervat- ing one muscle. The gracili muscles of the recipient were prepared as previously described except the muscles were perfused at a constant rate. Blood was drawn from a femoral arterial cannula in the donor and pumped (finger-type pump)10 into the arterial supply of the isolated gracili muscles of the recipient. The pump flows were set at a rate that produced a perfusion pressure approximately equal to aortic pressure. Venous cannulas (PE 240) directed the blood into a glass beaker containing dextran9. The blood was continuously returned to the donor animal's femoral vein. 9Cutter Laboratories, Berkeley, California IOStgma Motor Inc., model T-GSH, Middleport, N.Y. tlnettier Co., Model P 1200, Princeton, N.J. 23 C. Q sz_n:0wm QOZOQ .mmwgmpee pesoEmw esp soc» mpeswce neon cw vmgouwcos Aamvocammmga owemumam use cowmaFuuo com nmuepom_ mew: Aw_ou AaHV gang :o'maecp c< .Aaav massn copmamgma mg» on Emmcpmczou umeouwcos «so: Aug ecu —av mwezmmmea .eocov ecu mo A>uv cwm> pagosmm one on “aav assa e »a umcgzpmg we: we ;u_=z scam ewo>ewmmg a cue? 3o_uu=o on» uwuuocpu mmpacceu mzoco> .Aucmwawumcv Peepce cmguoce mo Acmum>gmcou mco .vmue>cm==w mcov mmpumas wpwuegm vmumpomp one we z—nazm Fewcopcm on» one? mung ucoamcou e um umasaq use Agocovv —msm:m mco seem execu mm: coopn Am mm \E <0 mozoo 25 .Am was A>uv cwu> Fosasmm on» ou:_ mums msu: Amy mcowuumncw uweoumzm .Aumv mpaccmu cgzumg a gmsogsu Locou mg» o» vacgauwg mu: coop; «ch .Amv gwo>gmmog a one? zopmuso ms» umuuogpu Au>v mapaccmu maocm> .u< on» you: mucwon sop» vagouwcos mgmz “Nam ucm pang mmgammmgn cowmamsoa .o_um=e comm mcpapnaam agougu any cw umumpa Augm= as» mcwupsu an umum>gmcmu ma: mpumas mco .cowumgmamga mpumae mwpwumgm camamsmn mesa ms» mo Azmp> Fogucm>v mcwzmgv ovumsmgum m usamwm 26 >n_ 8:00 9. Am” om aEza :05:th The per need1es (21 the arterial test agents muscles. S; ammals frOI femoral ve1 injections, bOth amma] SUmulatior Int ' M 3= Nature \ 27 The perfusion pressures were monitored from small hypodermic needles (2l gauge) inserted through the rubber tubing connected to the arterial cannulas (15 gauge, blunt-end hypodermic needles)]2. All test agents were infused]3 or injected behind the pumps perfusing the muscles. Systemic pressures were continuously monitored in both animals from femoral arterieso A cannula was inserted into the femoral vein of the recipient for subsequent systemic intravenous injectionso Prior to heparinization the carotid arteries and vagi in both animals were isolated for subsequent occlusion or electrical stimulation‘4o Intestinal Preparations 3° Naturally-perfused double-segments with intra-arterial (i.a[l infusion of test agents (Figure 4) - A loop of jejunum (20 cm, aboral to the ligament of Treitz) or ileum was exteriorized through an abdominal incision and divided into two adjacent segments (10 cm), then placed on a supporting platform constructed with towels and gauzeo The carotid arteries, cervical vagal trunks, the single vein draining each intestinal segment and small side-branch arteries (upstream to segmental supply) were isolated at least 15 minutes prior to heparinizationo Venous (PE 240, ID = 1067 mm; DD = 2.42 mm) and arterial (PE 50, ID = 0058 mm; OD = 0096 mm) polyethylene 12Arista Surgical Co., New York, NoYl 13Harvard Apparatus Co., Model 600-910/920, Dover, Mass. 14Grass Instrument, Square-Wave Stimulator, model 55, Quincey, Mass. 28 Figure 4 V IBF Schematic drawing of the naturally perfused intestinal pre- paration (double-segment) showing venous (VC) and arterial (AC) 3 cannulas. Test agents were infused (I) intra-arterially and the venous outflow returned from the reservoir (R) to the femoral vein (FV) by a pump (P). Motility, recorded as intra-balloon pressure (IBP), was monitored by balloons (B) with intra- T0 F luminal cannulas (ILC). Systemic pressure (SP) recorded from the femoral artery (FA). The preparation was maintained at 37°C. 29 reservc purged] WE 32: tollec load-in R (condo secure added an inc D'Ever bl ca- h‘ag j iO'ta drugs 1Efu5 One lume the 30 cannulas8 were inserted into the vessels and secured by ligatures (4-0). The venous cannulas directed the outflow into a collecting reservoir initially containing dextran (6% in saline)9. The blood was pumped10 back into the dog continuously gig_a femoral vein cannula (PE 320, ID = 2.69 um; 00 = 3.50 mm)8. Timed venous outflows were collected in clean glass beakers and measured by weighing on a top- loading semi-analytical balance“. Rubber tubesl5 (ID = 2.5 mm; on = 5.3 m) to which balloons (condoms) had been tied were inserted into the intestinal lumen and secured by tying both ends of the segments. Fluid (10 ml, H20) was added to the balloons and intraballoon pressure (IBP) which served as an indication of intestinal motility was monitored and recorded. To prevent collateral circulation the attached mesentery was separated by cauterization. A cannula8 (PE 280, ID = 2.5 mm; OD = 3.25 mm) was inserted through the femoral artery and positioned in the lower aorta for monitoring systemic pressure. Test solutions, agents. and drugs were delivered through the arterial cannulas by a constant rate infusion pump‘3. 4. fiaturally perfused double-segments with luminal placement of test agents (Figure 4) - This preparation was similar to the one previously described. However, after washing the intestinal lumen with saline. rubber tubes15 (ID = 2.5 mm; OD - 5.3 mm) were inserted directly into the lumen for introducing and withdrawing the test agents or saline. The tubes were connected to pressure lSDavol Inc., Levin Type No. 16. Providence. R.I. t'ansduc Inthose i.a. car Maod f' hganen Paced towels caroti( drainil “emain an art 1‘Satu ifiter; artEF. a sna‘ tubln the s c0hta throL into F1Ul: (IBP 31 transducers4 for direct monitoring of intraluminal pressure (ILP). In those preparations designed to employ intra-arterial (i.a.) infusion, i.a. cannulas (PE 50) were also inserted. As previously described, blood flow was measured by timed collections which were then weighed‘]. 5. Pgmp-perfused single segment with intra-arterial infusion of test agents (Figure 5) - A loop of jejunum (20 cm. aboral to the ligament of Treitz). exteriorized through an abdominal incision, was placed on a supporting platform constructed with saline dampened towels and gauze. Fifteen minutes prior to heparinization the carotid arteries, cervical vagi, single artery supplying and vein draining the segments were isolated such that the paravascular nerves remained intact. A blunt hypodermic needle12 (15 gauge), serving as an arterial cannula, was inserted into the artery and secured with ligatures (00). The segment was perfused with a constant volume pump10 interposed between the arterial cannula (l5 gauge)‘2 and a femoral arterial cannula (PE 280). Perfusion pressure was monitored from a small hypodermic needle12 (21 gauge) inserted into the rubber tubing just upstream to the cannula. A polyethylene cannula (PE 240) inserted into the vein draining the segment directed the venous blood into a collecting reservoir containing 200 ml dextran9. The blood was returned to the animal through a femoral venous cannula (PE 320). A rubber tube15 to which a condom had been tied was inserted into the intestinal lumen and secured by tying both ends of the segment. Fluid (10 ml, H20) was added to the balloon and intraballoon pressure (IBP) served as an indicator by cauterization to further prevent 32 \ \ .uenm an cmcwepcwms we: cowuegmnmea use .Auonv npzcceo Pageanp -mgucw no car: Amy cooFPma m za AamHv meammoga coop—maimeucm mm uwgopwcos we: zap—wuoz Auv :_m> Pagosme on» on up emcezoog Away gaza e cows: Ease “my spo>emmme a one? zoquao on» umuumgwu «>V cpm> ago one? umoepa opaccmo < .Apav gang on» an awesome: AHV nopomncw museum ammo wee Eemgum -czov mu_m a sage umeouwcoe we: Aamv mgammwea cowmsmgma .ucmsmmm woumpomw «no we Awm one Am u zv mmzpm> new: .AXH» + Fug .m> pox .m.wv emguo seem umcmmme umemqeou mew; mcompapom pom“ as» ugmwe we“ no apes: mm—omae umue>emcwu op nmeeaeoo mew: umum>emccw .uemy we» so cameo on» cm .:_s\~s c_ mmmwumnm we» co topmowucw we moon; mcwmmmLUCw xpmsemmmemoea um mmpomaa _pwooem Amsm oop\cmE\Pe mn.e~ u zopo coopn cemev umue>emcmu use AmEm oop\cws\~s Nm.m_ u zoFe voopn ceoev uwum>gmccm ecu oucm ummsch zpmsomcmppaewm we: A4 .:wxouou05pmp + pox omuoEmomw use ma ups\m: m .cwxououoeumu ”Fox omaosmomw no .m:w_mm peacocv somezflom pomp zoom .mammwp ea mam oop\cws\msm cw mmuchueo on» so umumowccw mm :zonm we zopeuao maocm> cm mucosa .A_ mean?“ .Pe ameav mpomae mwpmomem ummaeema appegsue: Ammcefi :mxoeov cmpm>emcmv new Ammcwp nepomv cmpm>emcce on» mo zap» voogn co Apuxv mn_eo~;o Eaemmmwoa cum cexooouoeumg we muommmm N oeamwu . 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E C 3.2. g l 2.50.43. x: O s _ 2. o (“Gal/WI“) mum snow I" N on Inl lncreas pressur Howevel deuerv fusior shows prudu lu < that 60 Intra-arterial infusion of a TTX solution (0.5 ug/ml) at increasing rates (0.1-2.0 ml/min) progressively decreased perfusion pressure of both innervated and denervated muscles (Figure 11). However, after subtracting the effect of the saline diluent the denervated muscles showed no change in resistance during TTX in- fusion at any dose from 0.05 to 1.0 ug/min (Figure 10). Figure 10 shows that progressively increasing the dose of TTX every 3 minutes produced a decrease in resistance that differed significantly (p < 0.05) from the pre-infusion value at 0.5 ug/min. Notice that the average control resistance of the innervated muscles was higher than that of the denervated muscles and the resistance values were not different following a TTX dose of 0.5 ug/min. Figure 11 shows that at an infusion rate of 2.0 ml/min (1.0 u9/min), when vascular resistance of the innervated and denervated muscles were the same (Figure 10) the response of perfusion pressure to carotid occlusion was totally abolished and the perfusion pressures (innervated = 55 mmHg; denervated = 65 mmHg) were similar in both muscles. In these cross-perfused muscles carotid occlusion of the recipient animal produced an increase (10-40 mmHg) in perfusion pressure of the neurally intact muscles. Bilateral occlusion of the recipient animal's common carotid arteries increased systemic pressure (10 to 60 mmHg) in all of the animals (N = 8) studied, while bilateral occlusion of the donor animal for 3 to 6 minutes failed to alter perfusion pressure of either muscle even though the circuit lag-time was always less than 60 seconds (Figure 11). Cutting the nerve trunk innervating one gracilis produced, 61 simultaneously; 1) a decrease in perfusion pressure of the denervated muscle, 2) an increase in perfusion pressure of the neurally intact muscle and 3) an increase in systemic pressure of the recipient animal. Following denervation, perfusion pressure gradually returned to near control levels in 15-30 minutes and denervation was sub- is: stantiated by carotid occlusion. TTX (0.5 ug/ml ) infused at pro- ‘i‘ gressively increasing rates (every 3 minutes) yielded a dose range of 0.05-1.0 ug/min. TTX progressively decreased the change in local h resistance to carotid occlusion (Figure 11) with a significant £ inhibition occurring at a dose of 0.1 ug/min. Although a decrease in vascular resistance occurred before the carotid reflex response was completely abolished, when it was abolished the effect of TTX was the same in both innervated and denervated muscles. The mag- nitude of the responses to carotid occlusion returned to control levels 30 to 45 minutes after cessation of TTX infusion. Notice that during the infusion period TTX had no systemic hypotensive effect on either the recipient or donor animal even though the venous outflow containing TTX was returned to the donor animal. The total TTX dose returned to the donor animal was generally less than 10.0 ug with only 5.70 ug infused during the normal test period of 15 minutes. In about 50% of the animals the donors systemic pressure gradually decreased to less than 75% of the control values 45-60 minutes after the first test sequence. In all animals a significant hypotensive (< 75 mmHg) effect appeared 62 .INzoflwhd‘sru-uzz. 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E v Q N O i l- -5- o“.- ‘F- 31.25% i . , . .¥i§¥ £5. a an; m... 66 in the donor animals after the second or third TTX infusion sequence 60 to 90 minutes following the initial administration of TTX. 2) gjfgct of initjgl resistance on the vascular action of TTX - Increasing initial resistance by neural mechanisms potentiated the vasodilator action of TTX in the neurally intact gracilis, while in the denervated muscles increasing resistance by direct means failed to alter the effect of TTX (Figures 12, l3, l4). In the cross-perfused gracili (prep #2, Figure 2) TTX (0.l and 0.25 ug/min) infused at a volume (0.1 ml/min) insufficient to show a dilutional effect produced a decrease in perfusion pressure of only the innervated muscles. This vasodilator action of TTX was dramatically decreased or abolished when resistance of the inner- vated muscle was reflexly decreased (Figure 12, l3). Intravenous infusion of norepinephrine (NE, 10 ug/min) into the recipient animal increased systemic pressure 15 to 50 mmHg. simultaneously decreasing perfusion pressure of the innervated muscle l0-40 mmHg; thus, decreasing or abolishing the vasodilator effect of TTX (0.1 and 0.25 ug/min). Hemorrhaging the recipient animal until 25 to 50% of the calculated blood volume (8% of body wt = 100%) was removed immediately increased resistance of the innervated gracilis, con- commitantly augmenting the response to TTX (Figures l2, l3). However,TTX failed to produce a resistance change in the denervated muscles where initial resistance was simultaneously increased by local i.a. infusion of NE (0.l ug/min). Notice in Figure l3 that in the controls which were taken before initial resistance was 67 .A=Pe\mn F.ov mz mo copmamcw .m.w Paco, an ummmmgucw Apmaomcmu_:epm «Pumas umum>cucou mg» mo mucmumwmmc new Fmspcm “cowgpumg as» mcwmmgegosm: x5 uammwcucw x—xopmms mm: mpumss umum>cmccp an» we aucmumwmoe empaumm> pm_uF=H .A:_e\m: op .mzv mewgzamcwamgoc ea Pmewcm aca?a_ume as» ope? commzwcw mzocw>mgucw an «Fangs umpm>cwccp as» xpco a? ommaocumv »_xmpwos cos» we: mocaumwmmc gm_=umm> Fawppcp .uemp me“ :o czogm use mmmcoammc Focucou .mzocgm mzu an umpmowncm mm memugmum mm_um=5 span ope? Acwe NV vmmzmcw a—maomcmp—sspm mm: Acwe\mn m~.ov xph .Aaamv pmswcm ucmpqmuuc wsu mo mesmmoga uwsmpmAm use .mmpomas Auaav umum>gwccw can Anamv umum>cmcmu oz» mo mezmmmca copmaweoa .Aaamv mgammmcq owsmpmAm mpmswcm Locou as» Low czosm mew pms_=m opacwm a sage mmcwumgk .Am ucm N mgammm .~* nmgav umem> mm: mucmumwmme Pawpwcw can: mucogmwmwc empaumm> co Axhhv :Pxouovocumu eo uumewu ~_ ac=m_e 68 ..>._.:_E\n: o: ”2 , will TL" . «L _ 69 Figure 13 Mean vascular resistance responses (N - 6) of cross-perfused innervated (solid lines) and denervated (broken lines) gracili muscles (prep #2, Figures 2 and 3) to local i.a. infusion of TTX (0.l and 0.25 ug/min; on abscissa) when initial resistance was varied. Control responses are those obtained before either raising or lowering initial resistance. Initial resistance was lowered in the innervated muscle by i.v. infusion of norepinephrine (lO ug/min) into the recipient animal; and initial resistance increased in both muscles by hemorrhaging the recipient animal and simultaneously infusing norepinephrine (0.l ug/min) into the denervated muscle. Resistance (mmHg [ml/min] 100 gm) 7O -----------.. — IMERVA‘I‘ED (Nib) 'IIIA DENERVATED (Nib) Durhg nonpi. (La. , 0.1 Jag/min) (mum) During nor . ) (i.v., lOyg/mlnwedplom 0.25 0.1 TTX (pg/min) Ii 71 .mmpumzs Au~.o u c v umpm>gmcmv vcm Amm.o u gv vmpu>gmccp may anon com =m>wm one macmpuwymu -ou cowumpmccou .mmcm cmumgm mg» an couocmv mes mmcwp cowmmmcmmg on» we mucmwcm> .mpmswcm m seem :mxaa mm=Fu> oucmpmpmmc mp com czozm mew mcoppmzco =o_mmmcmoc swag» saw: mmcwp copmmmgmmm .Am can ~ «gnaw; .me macaw mmpumas Ppwumem Ammcwp :mxogn use mwpugwu cmaov umum>gw=mu ucm Amway, ace meow uppomv vmpm>cmccm one? A:_E\ma m~.o .xhhv :Fxououosuop eo cowmzmcp .m.m pcuop op Amumcmugo .mav museumwmmc cw mucosa mg» no Ammmwumnm .Hmv magnummmmg papaya? mo pummmm eP ucamwu 72 .96: 6133; flu! .86....3.z.§ I .c....\oan«.o. .2... 73 changed by any neurogenic or direct means, resistance of the inner- vated muscles was greater than that of the denervated gracili. In the innervated muscles the dose response curves during TTX infusion got progressively steeper as initial resistance was increased. when neural activity was essentially eliminated by i.v. infusion of norepinephrine into the recipient animal TTX failed to produce a r— . change in resistance. a Figure l4 shows l8 accumUlated initial resistances from 6 animals with the corresponding vasodilator response (decrease in i resistance) to TTX (0.25 ug/min). The change in resistance of the innervated muscles to TTX positively correlated (r a 0.92) with the level of initial resistance. However, in the denervated muscles initial resistance appeared to have no relationship (r = 0.27) to the vasodilator action of TTX. Regression analysis of these data showed the responses from neurally intact gracili to have a slope of -0.37 with a y-intercept of 2.37 while those from the denervated preparations show a slope of -0.02 and a y-intercept of 0.19. A period (I or 2 minutes) of ischemia produced by turning the perfusion pump off resulted in a lower perfusion pressure when the pump was turned on, thus a decrease in resistance (reactive- dilation). However, when venous pressure was increased 12 to 18 mmHg by elevating the venous cannula (20 cm) vascular resistance was elevated (venous-arteriolar response). TTX in neural blocking doses (0.5 - l0.0 ug/min for 5 to l5 minutes) had no effect on the venous- arteriolar response or reactive dilation of innervated or denervated gracili muscles. LIn foureperfused denervated gracili muscles when u 74 perfusion pressure (80 :tlo, 102 1 20 mmHg) thus vascular resistance (8.0, 9.5, and l0.7 mmHg/min/ml/lOO gms) was varied while keeping flow constant (X = 15 ml/min/lOO gms) turning the pump off for one minute produced a subsequent fall in perfusion pressure to the same absolute level (40-55 mmHg = 2.8-3.7 mmHg/min/ml/lOO gms). In the same animals elevating venous pressure (X = 14 mmHg) produced r- similar increases in perfusion pressure (X = 28 mmHg) even when initial resistances differed (is = 5.0, 6.5, and 9.5 mmHg/min/ml/ lOO gms) thus producing mean increases in vascular resistance of 1.0, 1.0 and 0.7 mmHg/min/ml/lOO gms. TTX in a dose (0.25 uglmin, ii” i.a.) sufficient to block vascular resistance changes in 5 minutes had no effect on reactive dilation or the venous arteriolar response. Vascular Action 0f TTX On Naturally Perfused Ileum And Jejunum 1) Effect of TTX on venous outflow of the ileum - TTX (5 uglml) given locally into the naturally perfused neurally intact ileum (prep #3, Figure 4) produced an increase in venous outflow which progressively became greater as the infusion rate was increased from 0.1 to 2.0 ml/min. However, at no point in the dose range of 0.5 to 10.0 ug/min did the change in venous outflow during TTX infusion differ statistically from that of the saline controls even though TTX frequently stimulated motility of the intestinal segment (Figure 15). Electrical stimulation of the decentralized vagi and carotid occlusion produced an increase in motility and venous outflow respectively from both intestinal segments. Following the infusion 75 of TTX into one of the segments (2 wt. = 25.9 g; 2 blood flow = 16.4 ml/min) vagal stimulation failed to affect motility of that segment and carotid occlusion produced a greater increase in blood flow as compared to the pre-infusion control. Thus, indicating that at some point in the TTX dose response curve local blockade of the neural elements occurred. In two animals (not included in Figure 15) where the neural blocking action of TTX was studied at each dose, 2.5 uglmin (0.5 ml/min) infused for 5 minutes was sufficient to signifi- cantly (p < 0.05) depress but not abolish the vagally induced motility response. 2) Effect of TTX on vascular responses to KCl - Figure 15 shows that infusion of isosmotic KCl into the intestinal segments, not previously receiving TTX, produced an increase and then a de- crease in venous outflow. However, segments which had previously received TTX and were then given KCl with TTX (5 ug/ml) showed only a decrease in venous outflow. The increase in venous outflow reached a maximum at 0.5 ml/min. Infusion of KCl at rates of l.0 and 2.0 ml/min always produced an increase in intestinal motility which accompanied the decrease in venous outflow. KCl in combin- ation with TTX often increased motor activity at a lower infusion rate (0.5 mllmin). Furthermore, in this series of experiments the vasodilator response to KCl alone was abolished when TTX was present. In all of the animals studied, i.a. infusion of KCl increased systemic arterial pressure. This increase was not abolished by local infusion of TTX. However, when the blood containing TTX was allowed to return to the animal systemic pressure began to gradually fil- 76 decrease 30 to 45 minutes after completion of the first TTX test sequence and the systemic pressure response to local infusion of KCl was then depressed or abolished. The total dose of TTX given before a decrease in systemic pressure was observed was always greater than 20 pg and in 5 of the 7 dogs was greater than 40 pg. 3) Effect of TTX on the vascular responses to luminalgplace- ment of hyperosmotic NaCll_hyperosmotic KCl, and glucose - Hyper- osmotic NaCl (1500 mOSm/Kg), hyperosmotic KCl (1500 mOSm/Kg), and glucose (2.5, 5.4, 20 and 50%) were placed in the lumen of natur- ally perfused jejunum (prep #4, Figure 4) to study the local vascular responses, motility and fluid movement before and after neural blockade by TTX or dibucaine. The vascular responses to intraluminally placed and i.a. glucose have been reported (Chou gt al,, 22). .3391 - Luminal placement of hyperosmotic NaCl (l500 mOSm/Kg) increased venous outflow in the first 3 minutes. This increase in venous outflow was significantly greater (p < 0.0l) than that during luminal placement of isosmotic pOlyethylene glycol solution and was not affected by an i.a. TTX dose (0.5 - 2.0 ug/min) suffi- cient to block the vagally induced motility responses (Table 1). Systemic arterial pressure gradually decreased during the course of the experiments showing a significant hypotensive effect of TTX about 30 minutes after TTX was returned to the animal (Table l)° Table 1 shows that the calculated vascular resistance of the segments decreased during the first 3 minutes and increased slightly over the next 8 minutes but remained significantly (p < 0.0l) lower than if" 77 .Amo.o v av mcommgmaeoo nmcwmg Lee mwmxpmcm.m m_ucmn:pm x3 wouerucP mm emcee sumo soee pcoemmwmu appcmuwmwcmwm mew: :ms\~a m.o um Xe» V m:_p nepom oz» can mmapm> Aisusuv m:_P :mxoea 03» one .cwe\pe 0 mo mum; cowmzwcp pm + egg was Fox xpco use "mum; cowmzmcm come no emguo comm ow umemasou mew: mmzpm> A cm>wm mzo_m Poegcou new; .czozm mew weoeeo nemucmpm ewes» wee AN u zv mmapm> :mwz .cws\ps cw mmmwumnm one co nmyeowucw me move; cowmswcm newmmmeUCW um Ao-uu-ov A_E\m: my xhh + A_\Emos oomv sue we. .Ao----o "_\Emoe oomv Fog .A........ ”Fe\m my xee .Aolullule m_\Emos oomv Fonz mo cowmaecw meempemuegucw mcweau Ampmcwneo on» co cm>mm mm .ozmmwu we mew ooP\:we\Emv czozm mum mmmcoamme zopmpao maocm> .Aq wezmwu .me aoeav Sump? ummaecma >~_m segue: on“ mo zope noofln co Apuxv mcweopnu aswmmepoa new Awav :exopouoeuwu eo mpumeem m_ aezmwa 78 RIZV .:_E\_EV 30¢ comma... o.— n6 «.0 —.o o 55.9.3.0 5.50883 9:50 . ._E\maextlo /. 2:505 38 Uozlo ON 8 S 8 (wBom/ugw/wfi) moumo snouaA 8 8 79 the control resistances. However, during TTX infusion hyperosmotic NaCl produced a decrease in resistance which continued to fall over the entire test period (Table 1, Figure 16). This slight increase in resistance over the last 8 minutes to hyperosmotic NaCl was accompanied by an increase in motility which was abolished by TTX. Hyperosmotic NaCl placed in the lumen significantly altered ,. venous blood osmolality. Na+ and KT concentrations and the volume of fluid recovered from the lumen. Osmolality of the venous effluent was significantly ( p < 0.01) increased in 3 minutes, increasing 2 more after 11 minutes, and was unchanged by TTX (Table 1, Figure 16). Na+ concentration of the venous blood significantly increased before and after TTX. However, potassium concentration of the venous blood increased during luminal placement of hyperosmotic NaCl only before TTX infusion (Table 1). Notice though, that the K? values before and after TTX were significantly different only during the control. Recovery of the fluid in the lumen at the end of the test period showed a significant increase which was attenuated by local TTX infusion (Table l)° §§1_- Luminal placement of hyperosmotic K01 (1500 m05m/Kg) increased venous outflow before TTX but decreased flow after TTX (Table 2, Figure 16). During luminal placement of KCl before TTX, vascular resistance of the segments significantly decreased in 3 minutes then increased over the next 8 minutes remaining below the control values. However as compared to the controls, after TTX resistance was unchanged during the first 3 minutes but pro- foundly increased over the remainder of the test period. KCl increased motor activity of the segment before and after TTX. 80 .poepcou as» ea cowuqu—ou tum ecu Ease umcwmuno omega op umemaeou mew: mmzpm> Ppe peace emzop as» :H .mxmwemumm ma umuocmu mew Am .m> m ..o._v _oeacou muewsaocaae swung soup ucmsmeemu xppeumumwumum mmapm> .czocm mew meageo usmncmum ecu mwapa> came one .Acws ~FV vowsoa ammo we“ we Aces my cowuomP—ou wen co .ucm .ump one megawvcw mcszpou umgmnsac as» .Acps\m: o.~-m.ov xh» mo cowmzmcw —mwcmpsaumeucp A umcm>ouos use .ouceumvmmg eepzume> uwumpaupmu .wszmmoga Fewempse owempmxm .Amcowpmeucmucoo +x ace .+mz .zuw—mpoEmo .3opmv mmmcoammg maocm> one .Am¥\smos oompv Fonz umposmogmmxa use Awma .Fogpcouv Fouzpm mcmpxgumxpoa umaosmomm we APE opv acmemumpa poemsap op Eacahmn vmmzwgoa appeezumc cw mumcoqmmg em_:umm> Pmcwpmmacw we“ co Axehv cwxopouospmu mo mpumemm . P mpnmh 81 he: I.“ . C! .H I. flee ween Heem Emma mumsH mam “me < as: . . 33: when m. “mm 32 . < 42:28 HO V a n to 89 ._ a . El .m.m . m ..whfio.mH Hugo.m mues.m muHHNmH m.HHomH sHMNM HHmHm m Asmwav .I . .l. . . I I4 , AJHNZV a s m H e N m .8H “38 m 358 _ m . m _ H m < H _ m H as :35 335 333 m§._o> 628 +¥ .uzou + HHe ugece gezep we» :H .mxmweeume an eeeecee use Am .m> m ..e.wv Hecucee eaeweeegeee green eegw neeeewwwe apHeewu -mwueum meeHe> omen» .exegm one mgegee eeeeeeem ecu meeHe> sees as» .Acws HHV eeweee amen one we Hews mv eewueeHHee eem ge .ecm .em. exp epeewecw meEeHeu veganssc mew .Hces\mn e.~ - m.ev xww we cowmswew Heweeeee-eee=e H eege>eeeg eee eeeeumwmee gepzeme> eeueHeuHee .egemmeee Hewgeuge ewsepmam .Ameewueepeeucee +¥ ece +ez .auwHeHeEme .zeva memeeemeg meece> ecw .Amx\amos oomHv Hug ewpeEmegeeaz ece Hume .Heeeceov Heesz eeeszpezHee eweesmemw we APE oHv ecoEeeeHe HeewE=H e» Eugenen eemewgee xHHeszue: cw memeeemee LeHeeme> Hecwumeecw ecu ce Axwwv ewxeueeegueu we puewwm . N eHeew — H . in“! in: .. flu H e. “Ne o.HNN.m an? H.NNHmH ENE {HHHHM 33% H dc. HO.V n. u to . I 89 . l. . H. 3.2 N. N? m. 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I . amn— m .ma m e .mo : e.mH m s.Hm H.Nm meow “No wN “Hm ”N H.» mm HeHuzeHsoewzoe m N H m N H m N H Hze oo~szz\xe\e= zzv He: zzv Haze ooa\sz\zev ewzmwzeu 3,2533. $833. zed .2223 87 did not change with the 5.4 percent glucose solution. Fifty percent glucose caused a greater increment in flow, venous osmolality, glucose concentration, and lumen volume than did 20% gluCose. Although 20% glucose and 34% PEG solutions have the éame osmolality, and both solutions raised venous osmolality and lumen volume to the same extent, the 20% glucosegsolution raised venous outflow, yet 34% PEG solution did not. Similarly. the osmolality of 50% glucose and 85% PEG are the same, and both solutions raised E venous osmolality and lumen volume to the same extent; but the rise in flow caused by 50% glucose was significantly greater than 5 that caused by 85% PEGO Fifty percent glucose significantly increased venous outflow and motility before the Jejunal mucosa was exposed to dibucaine, but after dibucaine administration, 50% glucose did not signifi- cantly alter flow or motility. For example, when 50% glucose was placed in one segment there was an increase in flow and motility but the other segment which contained isosmotic PEG showed no change in flow or motility° However, after exposure to dibucaine there was no response to 50% glucose, yet the other segment responded to 50% glucose with increases in flow and motility. Thus, the effects of 50% glucose and dibucaine appear to be local since they affected only the segment exposed. The possibility that dibucaine might have paralyzed the vascular smooth muscle making it unresponsive was examined by intra-arterial infusions of vaso- dilators (isoproterenol, isosmotic KCl. or hyperosmotic NaCl solution) before and after luminal placement of dibucaine. The increased flow 88 caused by intra-arterial infusion of the vasodilators was not altered by luminal placement of dibucaine. The increases in venous osmolar- ity. glucose concentration, and lumen volume caused by luminal placement of 50% glucose were the same before and after dibucaine. Dibucaine per se did not alter blood flow or lumen volume but did increase motility. This increase in motility, however, waned and disappeared shortly after the removal of dibucaine from the lumen and its replacement with isosmotic PEG. ‘ Hyperosmotic glucose (50%, 3000 mOSm/Kg) solution placed in the lumen increases venous outflow and decreases vascular resist- ance before (22) and after i.a. infusion of TTX (Table 3, Figure 16). In this series of experiments the glucose solution was introduced into segments previously receiving NaCl or KCl and TTX. Glucose decreased resistance. This vasodilation progressively increased throughout the test period (Table 3). Venous osmolality, glucose concentration, and volume recovered from the lumen all increased after TTX° Before neural blockade by TTX, 50% glucose increases motility of the isolated segment; however, after TTX motility was unchanged during luminal placement of hyperosmotic glucose. Intra-arterial infusion of 2.5, 5.4, and l6.4 percent glucose solutions all increased glucose concentration in the venous plasma but only l6.4 percent glucose at or above infusion rates of 0.5 ml per minute significantly increased blood flow as compared to normal saline. Venous plasma osmolality was increased by the 16.4 percent glucose solution and lowered by 2.5 percent glucose. 89 .xee ueocan mmogp soc» Hmo.o v av Heacaeewu premu upmwcmpm cowmaecp xp» newcau mo=Hm> omega ouocmu Hey mcoum .ummp mgu co umpm_p wave: omega cw mmzpm> ucmmmcamc agave ecu co cm>em mean cowpmcaHHmu .Hmpou uwHom mm umpocmuv accruapom “mop asp acwosuocucw cmumm mmuzcws HH new .5 .m saga use ocmN me?» an mmpucpu gene we umuocmu “Houxpm mcmecpmapoa owuosmomwv =o_p=Hom Hoeucoo ugu mo “guacamHa Hmcwszp mcwcau czogm mew amazp «:9 seem umcm>ouoc A>v wszpo> on» use Hwy amouzpm voopn m=o=m> .A+xv coppmcucmucou anmmmpoa coo—n mzocm> .A+mzv cowuueucmucou anvom voopa unocm> .on xuwpmposmo woo—n m=o=m> .Amv wucmpmwmmc cmpzumm> umumpaopmu .A=Ps\m: o.~ . m.ov Nee mo co_m=e=H HeHcmpee-ecp=_ HmmcpH augmeev newcae use HmacHH uHHOmV acoemn Ho u zv mmouaHm mom use He u zv Hug opposmocoq»; .AeH u zv Humz uwaosmocan»; we “HE oHV “cosmomHa Hmcm53H on He mesmwm .ee aocav Escznmn uwmsmcma HHHmeapm: mo mmmcoamoc ceHaomm> Hm=HHmmucH 0H mesmHe 90 NaCl KCl Glucose (l500 mOsm/Kg) (1500 mOsm/ Kg) OOOOmOsm/Kg) ‘ , , R 95‘ é\\ t / I Q \\ (mmthm/min) MT! O\:———O‘”. coo- - -. O H 4 (mam/x.) Na+ (Inlqll) (mom/100ml) ’ M“ gear}; 7.? ,5 V (ml) Time (min) 9l Vascular ActiOn 0f TTX 0n Pump-Perfused Jejunum 1) Action of 05 and TTX on the vasoactivity of isosmotic KCl and hyperosmotic NaCl (H:NaCl) - Local i.a. infusion of isosmotic KCl produced a biphasic vascular response in the pump-perfused jejunum (prep #5, Figure 5), dilation at low infusion rates and constriction at high infusion rates. Hexamethonium (05) or TTX stimulated motility of the segment and concomitantly increased initial resist- ance, but failed to alter the biphasic response of resistance (Figure l7). As with isosmotic KCl local infusion of hyperosmotic NaCl produced a biphasic change in vascular resistance. The increase in vascular resistance occurred concomitant with an increase in motility. However, during infusion of TTX, hyperosmotic NaCl pro- duced only dilation which progressively increased as the infusion rate was increased (Figure 17 and l8). Furthermore no increase in motility was seen during infusion of H-NaCl when TTX was present. Figure 17 shows the calculated vascular resistance during i.a. infusion of 300 milliosmolar KCl and 1500 milliosmolar NaCl into jejunal segments having a mean weight of 3l grams with a mean blood flow of l6 ml/min (54 ml/min/lOO 9). KCl at infusion rates of 0.5 ml/min or less, (increasing blood [K+] 4.7 mEq/l) significantly decreased resistance while infusion rates of l or 2 ml/min (increasing [K+] 9.4 and 18.8 mEq/l) increased resistance. In all cases infusion of C5 (0.5 mg/min = 2.5 x 10‘5 g/ml of blood) or TTX (1.0 ug/min = 5 x 10’3 g/ml of blood, l.5 x l0-10 M) increased initial resistance of the segment and simultaneously increased motility. However, the biphasic response to KCl was unchanged by 05 or TTX. 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H... v— N.N— «.0 120 Figure 26 shows that before actively contracting the strips, increasing K? from 4.7 to 19.2 mEq/l produced only an increase in tension which was greatest in the strips at higher preload levels. After BaClz, increasing K+ in the bath over the range of 4.7 to 9.2 mEq/l produced only relaxation; however, increasing K? to levels above 10 mEq/l produced an increase in tension not different from that seen before BaClz. Figure 27 shows that this dilator action of K3 in the presence of active tension was not blocked by TTX (1.0 ug/ml), phentolamine (50 ug/ml), propranolol (5-10 ug/ml) or atropine (1-5 ug/ml); but appeared to be greatly attenuated by ouabain (10'5 N). These data show that in the presence of active tension in— creasing extra-cellular K+ in vitro produces responses which closely duplicate those seen in vivo. 4) Effect of hyper- and hyposmolality on arterial strips before and after BaCl; - Increasing osmolality decreased the tension of arterial strips which had been actively contracted by BaClz. However, in the absence of active tension, hyperosmolality frequently increased tension of strips equilibrated at high pre- loads (4 or 6 gm) (Figure 28 and 29). Hyposmolality produced an increase in tension before and after contracting the strips with BaClz. The responses of vascular strips were quantitatively and qualitatively different depending upon the manner in which osmolality was altered. Figure 28 shows that before BaClz, increasing bath osmolality to 345 mOSm/Kg by the addition of a concentrated Krebs—Ringer 121 solution, thus increasing bath concentration of all ions, produced a significant increase in tension. Notice that the increment of increase for any given change in osmolality was greater in the strip receiving a concentrated Krebs-Ringer solution than the change in tension produced by a hyperosmotic solution in which osmolality was increased by d-mannitol. After actively contracting the strips with BaClz, increasing osmolality to as much as 390 m05m/Kg pro- duced only a relaxation. This decrease in tension was greater when osmolality was increased by d-mannitol as compared to the concen- trated Krebs-Ringer solution (Figure 28). Although the increase in tension induced by hyperosmolality was potentiated at higher preloads, a small decrease in tension was frequently seen when osmolality was increased 10 to 40 mOSm/Kg in strips equilibrated at 4 or 6 gm (Figure 29). Hyperosmolality generally failed to increase tension until osmolality exceeded 350 mOSm/Kg. The relaxation produced by hyperosmolality following BaClz increased in magnitude as preload was increased and appeared to be related to the level of active tension, this relaxation was not sensitive to phentolamine (10-50 ug/ml), propranolol (1-5 ug/ml), atropine (1-5 ug/ml), or TTX (0.1-1.0 ug/ml) and closely duplicated the vascular responses to hyperosmolality which have been observed 11m (in). Decreasing osmolality by the addition of distilled water gener- ally failed to change the tension of strips equilibrated at high or low preloads; however, distilled water occasionally decreased tension in strips preloaded with 6 gm. Following BaClz the addition of . 1.7 122 distilled water produced an initial decrease in tension followed by a gradual increase, thus showing little net change in tension when bath osmolality was sequentially decreased to 255 mOSm/Kg. However, when osmolality was lowered by decreasing only the Na+ concentration, an increase in tension developed before and after actively contract- ing the strips with BaClz (Figure 28). This increase in tension induced by hyposmolality was unaffected by phentolamine (10-50 ug/ml), propranolol (1-5 ug/ml), atropine (l-5 ug/ml) or TTX (0.l-1.0 ug/ml). Action of TTX on Visceral Smooth Muscle in Naturally_0r Pump-Perfused Intestine 1) Effect of TTX on spontaneous motility of the ileum and the motilityduringvagal stimulation - Local intra-arterial infusion of TTX produced either a decrease or increase in spontaneous activity of the naturally perfused ileum (Figure 31). In intestinal segments exhibiting spontaneous motor activity, infusion of TTX in low doses of less than 1 ug/min occasionally depressed the ongoing motility. The decrease in motility occurred within one minute after starting TTX, and generally remained depressed during the infusion period. Spontaneous activity of the segment failed to return to pre-infusion levels within 20 minutes after stopping TTX. In quiesent segments showing little or no spontaneous motor activity TTX in doses of 1.0 ug/min or more generally stimulated motility (Figures 18, 31, 32). The increase in motility induced by TTX wained with time and generally returned to pre-infusion 123 .mEesm N wsumusous wsows usw ow uses sonesesHeu .3 xn uuwosuu me uusmez use uussesu usu: mswee usw HH< .sosmsuw usesm u uwsuuusous ossoesw soeu so wsuH usw ow mesmeu use .Huez so uuwuHouu sonsHom usuossximousx usonmooxs Hos use 0N: uuHHmeHu Hos .Hostsesiu so uumeusoss we: HwHHeHoEmo sossz sH Hox\smos oooHV sosweHom suoswmimousx onoEmosuoxs Hmv uuueususs usuz msos HHe sossz sH Hm¥\smoe oooHv sonsHom sumssmimousx osonmosuQAs HHm me\smosv muoHe> oswosmo usw ow oX\Emoe com so HHoswsoo usw Eoss uumeusouu so uumeususH usu: msweo usw so stHeHosmo .3osse usw we uuweoHusH me su>Hm Hz; o.HV NHoem he uumeusoss we: sosmsuw suwse use usosue Ho use ov AwHHeHosmo ossmeusuuu use Hm use uuosw msswosuu exmwsuwme usw swsz .szosm use msossu useusewm ssusw use As u 2V muuHe> seuz .Howsssesuu en uumeususs we: AwwHeHoEmo sows: sw soswsHom sumssm -meusx e swes usw ow ossuue an eumsomse usw so szosm me uumeususw me: awHHeHoEmo .NHuem suwse sosusuw u>kue msHo ueoHuso assusoouussoo usw use usosus sosmsuw ueoHuss usesu>e usw su>so ms u>s=o soeu so wsmss usw ow .NHuem zs o.H swsz mosswu usw mswwueswsou aHu>wwue HmussH uusmeuv suwse use AmusHH uHHomv usosuo .Ho ussmss .us susov osws> sH mssswm HeHsuwse smuss so sosmsuw usw so stHeHoEmo mssmeususw use mueoHuse wsususssu uussw so wuussm eN essess A TENSION (cams) 126' H B m“ (BACL2 1.0 hm .n. AFTER ' = P<0£5 (6.0) (1.9) (1.1) (1.1 + 1.8) (N 7) W 300 320 340 ‘ 360 380 400 OSNOLALITY (MOSH/KG) 127 values in 10-20 minutes. The magnitude of increase in motility varied from slightly detectable increases to large rhythmical contractions producing increases of lo to 30 mmHg in luminal pressure. The local neural blocking action of TTX was studied by evaluat- ing its efficacy in abolishing the motility responses to electrical stimulation of the decentralized vagi (6v, 6 cps, and 16 msec). TTX in doses as low as 0.1 ug/min was effective in depressing the motil- ity response to vagal stimulation. However, TTX doses of less than 2 ug/min generally required prolonged infusion periods of more than 15 minutes to produce an effective block. If the TTX dose was increased to 2.5 or 5.0 ug/ml complete block to vagal stimulation was obtained in less than 10 minutes and could be maintained by a TTX dose of 1/10 to 1/4 that of the initial blocking dose. Figure 30 shows that TTX gradually depressed the motility response to vagal stimulation, and completely abolished the response at a TTX dose of 2.5 ug/min. Placing TTX (10 ug/ml) in the intestinal lumen attenu- ated the motility response to vagal stimulation only after a 15 minute exposure period. Increasing the period of exposure to 30 minutes failed to abolish the response. Hhen TTX decreased spontaneous motor activity, vagal stimula- tion still produced an increase in motility; but when TTX stimulated motility, vagal stimulation generally had little effect. When the motility responses to vagal stimulation were effectively depressed or abolished the changes in resistance to carotid occlu- sion were also abolished but the increase in systemic pressure to local infusion of KCl was only attenuated. Fri: 4* mt." ' 128 .sonsssw .e.w so uuowsus usw mussumussus mssoesw sueu 3oHue uses HewsoNHsos sssw usw swsz sws oN . m so uuossus wsumussus mmssuesw usw ss useusm .wsuH usw so szosm use How mumsoomus Hoswsou Hms ousov Hm use woo usw so Hmseo HewsoNHsos gosswv sowweHasswm HeosswuuHu ow axes ss mumsosmus Hsswuusmus mHesusss use uses HewsoNHsos xossw usw so uuweosuss use HsHE\HsV sowmesss so muwes use uuossus uss .wsmss usw so uses soneseHHeo usw an uuweususs me @125 ss szosm use Heva mumsoeeus usemmuss HessssHeswsH .Hox\smos com "HuxiHv uuHsoHosu Easemewos onoEmomH use HHs\mn my ssxow -ouoswuw so sonsssH H.e.sv Hessuwseieswss ow mumsoomus awsstoe HanuHHv HesswmuwsH He assess In:1 If e . . o o f H H...— F' .— v as... . I -. s. . . s. . . __._.H . . -._ - . -..-,. . . 1 _.- . .H .H -. ___. _._. -..-.... .. ——.—-.. -_. _, 0 —o u . ._..._ __ . s .H How—.— _ s- ._ _- . . 7&7 s. .7 . .o.-.-..-._..--- -7 s.. . '- I 2. .5 In. Infusion (ml/min) .V'l Tf -A IN “no-I nut 3— 1: 132 Notice in Figure 30 that systemic pressure gradually decreased when TTX was allowed to enter the systemic circulation. This hypo- tensive action was generally seen if 30 to 50 ug of TTX had been re- turned to the animal. As systemic hypotension developed the cardiac response (bradycardia) to vagal stimulation progressively decreased. 2) Effect of TTX and C5 on the motility responses to i.a. KCl, hyperosmotic NaCl, nicotine, acetylcholine, and epinephrine - Intra- arterial infusion of isosmotic KCl, hyperosmotic NaCl, nicotine and acetylcholine all stimulated intestinal motility; while epin- ephrine exhibited an inhibitory action (Figures 31, 32, 33 and 34). (“*- Hexamethonium (C5) (0.5 ug/min) and TTX (1.0 ug/min) also stimulated motility; however, C5 converted the response to nicotine from stimulation to inhibition and TTX depressed or abolished the response to nicotine and hyperosmotic NaCl (Figure 32). Neither C5 nor TTX in the doses studies changed the response to acetyl- choline or epinephrine. EEl_- Local infusion of isosmotic KCl stimulated motility of both natural (2 weight = 19.8 gm, R flow = 10.5 mllmin) and pump- perfused (x weight = 31 gm, 2 flow 8 16 mllmin) intestine (Figures 31, 33, and 34). Figure 31 shows that when isosmotic KCl was infused locally into a neurally intact, naturally perfused ileal segment intra- luminal pressure (ILP) increased. Although numerous rhythmical contractions appeared to occur at an infusion rate of 0.5 ml/min the responses were generally too small to be recorded. Increasing the rate of infusion to 1.0 mllmin produced a generalized 133 constrictor response exhibiting little evidence of rhythmical phasic contractions. Occurring concomitantly with the increase in ILP was a decrease in venous outflow. KCl at 2 mllmin produced a rapid increase in ILP along with a complete blanching of the segment. Isosmotic KCl in the presence of TTX (5 ug/ml) produced a stimulatory response exhibiting large rhythmical contractions. During infusion of TTX, the stimulatory effect of KCl occasionally appeared at lower infusion rates (0.2 and 0.5 mllmin) but was most evident at 1 and 2 mllmin. The effects of KCl on motility of the constantly perfused jejunum were similar to those of the naturally perfused intestine (Figures 33 and 34). KCl alone produced an increase in motility exhibiting large changes in baseline pressure with little evidence of phasic rhythmical responses. C5 had little effect on the motility respon- ses to KCl but TTX attenuated the increases in baseline pressure (Figure 33) and augmented the phasic pressure responses (Figure 34). Hyperosmotic NaCl - Local infusion of a hyperosmotic (1500 mOSm/Kg) NaCl solution produced both an increase in baseline pressure and phasic contractions (Figures 33 and 34). The stimulatory effect of NaCl (1500 mOSm/Kg) was often evident at an infusion rate of 0.2 ml/min but was not significantly altered until infusion rate was increased to 0.5 m1/min. CG (o.5 ug/ml) did not change the increase in pressure produced by NaCl (1500 mOSm/Kg) and only slightly decreased the phasic responses at the highest infusion rate of 2 ml/min. However, TTX (1.0 ug/min) completely abolished both the increases in baseline pressure and the phasic responses. 134 Nicotine, acetylcholine, and epinephrine - Figure 32 shows representative tracings of the intestinal motility responses to 1 ml intra-arterial injections of nicotine (50 ug), acetylcholine (3 ug) and epinephrine (3 HQ) before and during infusion of Hexamethonium (C5) (0.5 mg/min) or tetrodotoxin (TTX) (1.0 ug/min). Infusion of C5 or TTX stimulated motility which decreased in magnitude with time. Bolus injections of nicotine (50 09) into the relatively quiesant gut stimulated motility before 05 but during C6 infusion nicotine inhibited motility. TTX abolished both the stimulation and inhibition produced by nicotine. Injections of acetylcholine (3 ug) stimulated and epinephrine (3 ug) inhibited motility before and during 05 or TTX. 3) Effect of TTX on the intestinal motility produced by luminal placement of hyperosmotic NaCl, KCl and glucose - Luminal placement of hyperosmotic NaCl (1500 mOSm/Kg), Kcl (1500 mOSm/Kg), glucose (1000 to 3000 mOSm/Kg) and polyethylene glycol (1000 to 3000 mOSm/Kg) all increased baseline luminal pressure and elicited rhythmical contractions of the jejunum. TTX abolished the respon- ses to NaCl, glucose and polyethylene glycol but only attenuated the responses to KCl. Hyperosmotic NaCl - Placing 10 ml of a hyperosmotic NaCl solution (1500 mOSm/Kg) in the lumen of naturally perfused jejunum (x wt - 19.8 gm, i flow - 10.5 ml/min) stimulated motility. An increase in both baseline pressure and phasic pressure changes frequently appeared within the first 3 minute collection period. Generally the increase in intra-luminal pressure exceeded 30 mmHg [4TH L‘n—I 135 .mzosse usw so uuwosuu use mwsume wmuw use mossu usw so sosweswmssssu< .wsuH usw so szosm use Hxss mssssu use .mo mssssu .mssuiusov msoswwusou wmuw HHe sos Hus mumsoouus Hoswsoo .ozes ss ussmmuso sooHHeseswsH uonomoe usw uweosuss wsmss usw so uses sonesssHeo .Hm usomss .ms ousov Eosowun uumassuo wasewmsou usw owss Hsss\m: o.H .xssv stowouoswuw so Hsss\me m.o “may Esssoswusexus so sosmesss .e.s HeuoH assszu use usosun Hm: m .Hemv ussssoussou use Ho: m .soswewsumussum Ne assess 136 REESE! Elli]: U u m h S < a z c U h l as... sefiiz): 3:. 34.132.315.511? H. H,.,.. 5 H... .2.; s2u3§93HHHHH 1350.5. 5 _ H H _.H._ H .H ______ , . , xww use... i _ H i ..... . . . , w . a . .2... iii... 3...... \JII/i 2.3.. see 3...}... no. .. .. , cu net-en H _ .3...“ I o j..3fi11(ii/.p....,...§. J.HHHS$§{3...H..;....2€<1 .H,H...JH..H.H.....HHH.._.HH.., .>11 gegéasgsgfii g 5009 050...: Ha... ES. .1. H___.H.H_..__..._.., _oswcou .. ... H . 3 H H midi-2..- OZ‘ -2_§Ua>=u¢ .3: avg-hag! .0 02039-3: 0.50. .<.. >h330£ amemgmoga um HcPe\mn o.F . 12.2 mEq/l) (Figures 25, 26). Increasing extracellular osmolality (> 50 mOSm/Kg) before increasing strip tension with BaClz (1.0 mM) produced small increases in strip tension: however after BaClz hyperosmolality (+10-90 mOSm/Kg) pro- duced only relaxation (Figure 28, 29) while hyposmolality (-15-45 mOSm/Kg) 159 produced increases before and after BaClz (Figure 28). Cold storage (24 hrs at 5°C) attenuated the responses to all vasoactive agents tested. The responsiveness of isolated vascular muscle has been report- ed by Bohr (12) to be greatly attenuated immediately following removal of the tissue from the animal. Burnstock gt_gl, (18) reported that this decreased sensitivity following dissection re- sults from intracellular loss of K+ and gain of Na+ ions. The increase in sensitivity of isolated strips which reaches maximum in 3 to 5 hours (2, 37) appears to be related to cellular gain of K+ (12, 18, 41). The sensitivity, cellular gain of K+ and loss of Na+ has been reported to be accelerated by increasing extracellular K? concentration or equilibrating the strips under tension (41, 133). Therefore, in the present study arterial strips were equilibrated for at least 1 1/2 hours at preloads equal to and greater than those used by Furchgott (36) and A1tura (2) for similar sized strips. An attempt to simulate the conditions of various degrees of initial resistance in vivo was accomplished by equilibrating isolated strips from the same femoral arterial segment under four different pre- loads (1, 2, 4, 6 gm). Various vasoactive agents were then studied before and after contracting the strips with BaClz which has been reported to act directly on smooth muscle by decreasing K+ conduct- ance (57, 123). Figure 21 shows that the constrictor response to norepinephrine (l x 10'6 g/ml) were progressively higher at higher pre-loads, thus suggesting that l) intracellular KT accumulation was higher at higher 160 pre-loads thereby increasing the sensitivity of the strips and/or 2) equilibrating the strips under greater degrees of stretch increased the area or sensitivity of the cell membrane receptor sites. Figure 21 also shows that when the capacity to relax was increased as at higher pre-loads or following BaClz, the relaxation produced by a given dose of acetylcholine was augmented. Acetylcholine also exhibited a constrictor action shown by other investigators (2) how- ever this occurred only at the lowest pre-load, possibly due to a low or inability to relax. Drugs acting directly on smooth muscle or via neural stimulation produced progressively greater responses as pre-load was increased. Yet, TTX over the range of 10"6 g/ml failed to produce any signifi- cant change in tension (Figure 22) and cholinergic as well as alpha and beta-adrenergic responses were demonstrated in the presence of TTX (10"6 g/ml). Neither TTX (10'7-10“6 g/ml) nor 24 hours of cold storage, known to render neural elements nonfunctional (21, 84), blocked the responses to norepinephrine (10"6 g/ml), tyramine (5 x 10"5 g/ml), or high concentrations of nicotine or dimethylphen- ylpiperazinium (5 x 10"5 g/ml) but both TTX and cold storage blocked the responses to low levels (5 x 10"6 g/ml) of the gang- lionic stimulants (Figure 24). These data indicate that: l) Equilibration of vascular strips under increased pre-loads increases the sensitivity of the strips to both vasoconstrictor and vasodilator agents, 2) TTX in concentrations over the range of 10‘8-10'5 g/ml has no effect on vascular strips either fresh or following cold storage; 3) Responses to tyramine as previously 161 shown by Bell (5) to be independent of Na: conducted action potentials, are unaffected by TTX or cold storage; and 4) The ganglionic stimulants, nicotine and dimethylphenylpiperizinium, in low concentrations produce release of adrenergic mediators yja_Na+ related action potentials but in higher concentrations appear to possess a direct action. Increasing extracellular KT concentrations in additive incre- ments (+ 1.5, 4.5, 7.5 and 15.0 mEq/l) above control (4.7 mEq/l) generally produced only contraction of isolated arterial strips before actively constricting them with BaClz (1 mM) (Figure 25, 26). However, after increasing the tension with BaClz the strips demon- strated biphasic responses with relaxation at low K+ concentrations (< 12.2 mEq/l) and constriction at high concentrations (12.2 and 19.7 mEq/l). The magnitude of both contractions (increases in tension) and relaxations (decreases in tension) were augmented at higher pre-loads; however, the magnitude of the relaxation appeared to be related to the level of active tension present following BaClz (Figure 26). It is well known that Ba++ depolar» izes nerves (98) and muscle cells (30, 44) and alters membrane phenomena by specifically decreasing KT conductance of excitable and nonexcitable tissue (57, 123). This divalent ion also in- creases cardiac pacemaker activity (45, 103, 123) which can be altered by different experimental conditions including reduction of intracellular K? (19, 91). Therefore the excitatory effect of Ba++ demonstrated in the present study which results from its affect on K+ conductance apparently is counteracted by the ATPase stimulating effect of elevated extracellular concentrations 162 of K+ (20). Neither in the present study on vascular strips nor in that reported by Hermsmeyer and Sperelakis (57) on frog ventricular . muscle did TTX alter responses produced by Ba++ or KT. ‘Figure 27 shows that the relaxing affect of K+ at low concentraf tions (< 12.2 mEq/l) appears not be be sensitive to TTX, phentolamine, propranalol or atropine, however, ouabain significantly inhibited the relaxation phase and augmented the constrictor phase. Figure 27 clearly shows that ouabain (10'5 M) failed to abolish the response. Although, the control responses before subjecting the strips to the blocking drugs are not shown, the strips clearly exhibit relaxations which were not significantly different from their controls except following ouabain. The presence of K+ in the control solution may account for ouabain's failure to aboliSh the relaxation, as it is known to decrease the efficacy of ouabain binding to cell membranes (43). These data appear to indicate that: 1) Similar to in vivo responses (48, 51, 52) increasing extracellular K1 concentrations over the range of 4-12 mEq/l produced relaxation of isolated vascular strips only if the strips were first actively contracted; 2) The magnitude of relaxation was related to the level of active tension present; 3) The K+ - induced relaxation phase was not significantly altered by TTX, cholinergic, or alpha and beta- adrenergic blockade; and 4) Ba++ and K1 - induced responses were significantly augmented in strips equilibrated at high pre-loads. Increasing extracellular osmolality always produced relaxa- tion of isolated arterial strips which were actively contracted with BaClz (Figures 28, 29). Decreasing osmolality generally 163 produced increases in contractile tension before and after contracting arterial strips. Both contraction and relaxation were greater in strips equilibrated at the higher pre-loads. Increasing blood osmolality has been reported to produce vasodilation in many tissues and has been implicated in many local regulatory phenomena (38, 53, 39, 110, 111). In vitro studies have clearly shown that isolated guinea-pig taenia coli (124), frog sartorius muscle (46) and rat portal vein (64) respond to hyperosmolality by hyperpolarization, decreased mechanical activity and reduced cell volume. Jonsson (67, 68, 69) has postu- lated that altering extracellular osmolality may produce responses by altering intra and extracellular ion concentrations as well as permeability of the cell membrane. In the present study hyperosmol- ality was studied over the range of 300 (control) to 390 i 10 mOSm/Kg by adding concentrated Krebs-Ringer solution (3 x normal), thus increasing all extracellular ions, or by increasing osmolality with d—mannitol which is known to not cross cell membranes. Figure 28 shows that before the strips were contracted with BaClz, increasing extracellular osmolality and ion concentrations produced increases in tension. These increases, which became significant when osmolality was increased more than 20 mOSm/Kg, were greater at higher pre-loads and substantially higher than those seen when d-mannitol was used. When osmolality was increased by using d-mannitol significant increases did not occur until osmo- lality was increased more than 50 mOSm/Kg. However, both solutions produced relaxation of the strips stimulated by BaClz. A greater relaxation was produced by the d-mannitol solution. These data 164 seem to indicate that increasing extracellular ion concentrations and osmolality activates two opposing responses with the predominating response dependent upon the capacity to relax or contract. Figure 29 clearly shows that when the capacity to relax is elevated, as indi- cated by the degree of active tension, the response to any given change in osmolality is augmented. These data may suggest that, in. 3112, blood vessels may show greater vasodilator responses when initial vascular resistance is increased. These results are compatable with both in vitro (38, 89, 110) and in vivo (63, 69, 124) studies reported by other investigators L. m. and are explainable by altered ratios of intracellular to extracel- :5 lular ions concentrations. When extracellular ions are increased H20 tends to move from the cell thus increasing intracellular ion concentrations, predominantly KT, thereby favoring hyper-polariza- tion by increasing [KT]I/[K+]o ratio. However, occurring concomitantly would be movement of Na+ into the cell due to the large extracellu- lar pool thus favoring depolarization. Increasing the pre-load tension may lead to increased permeability thus aiding Na+ move- ment, augmenting depolarization and producing contractions as seen in the present study. Following BaClz when the cells are pre- sumably depolarized the intracellular concentration of Na+ is probably elevated thus decreasing the [Na+]0/[Na+]I gradient thereby decreasing the effects of sodium movement and augmenting the hyper- polarizing effects of elevated intracellular K1 on the membrane. Decreasing osmolality would have somewhat different effects. Decreasing extracellular ion concentrations and osmolality may ,_ 165 decrease intracellular concentrations by loss of ions to extracellular fluid as well as by increasing intracellular volume (68, 73). Decrease in [K*]I/[K+]o ratio would favor depolarization. However diluting other extracellular ions appears to favor hyperpolarization as shown by the strips subjected to distilled H20 (Figure 28) which demonstrat- ed no significant increase in tension. Yet when only extracellular NaCl was decreased the strips responded with an increased tension even after actively contracting the strips. This seems to indicate that hypo-osmolality favors depolarization by decreasing the intra- cellular K* concentration. This occurs when H20 moves into the cell thus increasing cellular volume (68, 73). Increasing cell volume may also alter Na+ movement by increasing membrane permeability. All responses to change in extracellular osmolality were augmented at higher pre-loads, thus suggesting that permeability changes occur- ring in strips under stretch facilitate both ion and H20 movement. These changes in strip tension during either hyper or hyposmotic conditions appear to result from direct membrane phenomena not sensitive to TTX, adrenergic or cholinergic blockade. The Action of TTX on the Reactivity of Visceral Smooth Muscle Local intra-arterial infusion of TTX occasionally depressed spontaneous motility (Figure 31) but generally exhibited a stimu- latory effect (Figure 18, 31 and 32). KCl increased motility which following TTX was converted from contractile responses showing little phasic activity to responses showing an active phasic pattern (Figure 31, 33 and 34). However, the increases in motility '7b‘ “S. 166 to hyperosmotic NaCl, hyperosmotic glucose, nicotine and dimethyl- phenylpeperizinium were abolished by TTX (Figures 18, 32, 33 and 34). Depression of spontaneous motility by TTX occurred at low con- centrations (0.1 ug/min, Figure 31) insufficient to block vagal in- duced increases in motility. Figure 30 shows that TTX infusions of 0.5 ug/min (0.1 ml/min) depressed vagally-induced motility changes and only after the infusion was increased to 2.5 ug/min were the responses completely abolished. In this series, using ileal segments TTX infusion was started 15 sec before vagal stimulation. In sub- sequent series TTX, in concentrations as low as 0.5 and 1.0 ug/min, was effective in inhibiting motility changes induced by vagal stimulation or ganglionic stimulation by nicotine (5-50 ug) and DMPP (5-50 ug). TTX concentrations of less than 2 ug/min generally required prolonged infusion periods of 5-15 minutes before they were effective in abolishing vagally induced contractions. During periods when TTX showed depression of spontaneous activity vagal stimulation still produced significant increases in motility but when TTX stimulated motility, vagally induced contractions were blocked. Therefore, these data may indicate that when excitatory neural activity is present that small doses of TTX may affect it by blocking more susceptible excitatory fibers, however, when in- trinsic inhibitory mechanisms are removed by TTX motility is stimulated. This stimulation wains with time but never returns to control values. Therefore it appears as though the removal of local inhibitory neural mechanisms is overcome either by adapta- tion of the excitatory neural components or alteration in visceral 167 smooth muscle activity. The later seems more logical in that excita- tory as well as inhibitory nerves are susceptible to TTX blockade (16, 17, 85, 105). The ability of TTX to stimulate motility has been demonstrated in rat (71), and cat jejunum (130); yet Gershon (39), Rigimaru and Susuki (105) and Taira gt_al, (122) fail to mention any stimulatory affect on their isolated smooth muscle preparations. However Kagnoff and E. Kivy-Rosenberg (71) studying the affect of whole body X-irradiation reported that isolated rat jejunal strips of irradiated animals produced similar motility patterns as non- irradiated strips subjected to TTX (0.l-8.3 x 10"6 g/ml). They i‘ showed that the motility pattern produced by TTX was of regular, rhythmical contraction with primarily augmentation in magnitude. These authors hypothesized that both X-irradiation and TTX produce increased motility by removal of intrinsic neural mechanisms, which have been well studied (l6, 17, 85, 105). This concept of intrinsic inhibitory neural control is further supported by the studies of Hood (130) on isolated cat jejunum. This author reported that atropine (5.0 x 10'5 - 5.0 x 10'4 g/ml), procaine (5.0 x 10'5 - 5.0 x 10-4 g/ml), TTX (5.0 x 10'3 - 7.5 x 10'7 g/ml) and lido- caine (2.5 x 10'6 - 2.5 x 10‘4 g/ml) produced action potentials and large increases in amplitude of circular muscle contractions. Similar increases in frequency and amplitude of phasic contrac- tions elicited by TTX have been reported by Kao (73) to occur in rabbit intestine by an unknown mechanism. Yet Hood (130) has hypothesized a two neuron system within the enteric network, 168 theoretically agreeable with the experimental findings. A spontan- eously active neuron which receives no synaptic input drives a non- spontaneous neuron by a cholinergic synapse. The driven neuron releases a nonadrenergic inhibitory transmitter substance (16, 17) at muscarinic sites on the circular muscle layer. The existence of this type of an intrinsic neural pathway is supported by: 1) direct electrical recordings from enteric nerve cells indicating that one neuron drives another (101); 2) acetylcholine affects the electrical activity of only 21% of the intrinsic neurons (129); 3) atropine affects only a fraction (21%) of the intrinsic neural activity (101); 4) procaine, lidocaine and TTX augments the circu- lar muscle activity to electrical stimulation of intrinsic neurons (131); and 5) transmural electrical stimulation inhibits mechanical and electrical activity produced by atropine, and is sensitive to TTX (130). Atropine appears to produce excitation by removing the driving influence on inhibitory nerves and TTX produces excitation by removing all neural influence thus allowing spontaneous myogenic activity to occur. The ability of TTX to inhibit ongoing spontan- eous activity as was seen in the present study may be related to the cholinergic synapse between the driver neuron and the non- adrenergic inhibitory neuron. If TTX, which has been shown to inhibit acetylcholinesterase at low concentrations (136), prolonged or augmented the effect of the transmitter at the cholinergic synapse spontaneous motility may be inhibited by producing a more efficacious inhibitory influence. 169 Intra-arterial or intra-lumenally administered KCl stimulated motility of both natural and pump-perfused intestinal segments (ileum and jejunum). Like vascular smooth muscle (12, 20, 119) KCl has been reported to have similar direct effects on visceral smooth muscle (18, 26, 49). However, the fact that K+ permeability may differ between these two muscles may be important in the visceral muscle action and thus the indirect vascular responses. In the present study KCl infused locally (elevating calculated serum con- centrations 1-30 mEq/l) or placed intralumenally (elevating serum K+ l.5-10.0 mEq/l) always increased motility as infusion rate was increased (> 1.0 ml/min) or mucosal exposure to a hyperosmotic solution (1500 mOSm/Kg) exceeded 3-4 minutes. These data are in agreement with Dabney gt_§1, (23, 26, 27). However, when KCl was placed in the lumen, venous K1 concentration was well below the vasoconstrictor levels as shown in other tissues, yet increases in motility and vascular resistance occurred (Figure 16 and Table 2). The fact that TTX changes the K+ - induced motility from a nonrhythmical pattern to highly phasic responses may indicate that KCl has a neural as well as non-neural action and TTX removes the neural effect thus demonstrating only myogenic responses synchronous with the basic electrical rhythm of the tissue. However, these data do not rule out the fact that TTX may alter smooth muscle permeability to K+. One must agree that TTX alters the venous concentration of K+. This is explainable by the decreased blood flow but may also indicate either altered K+ flux or cellular loss occurring during increased muscle activity (111). 170 Intra-arterially or luminally placed NaCl or glucose in hyper- osmolar concentrations (1500 m05m/Kg) frequently elicited increases in motility (Figure 18, 33 and 34). These increases were character- ized by both elevations of baseline pressures as well as phasic activity. Although ganglionic blockade with hexamethonium (0.5 mg/min) failed to alter the H-NaCl induced contractions the responses appear to be neurally mediated by their susceptability to TTX (1.0 ug/min). Hyperosmolality has been shown to hyperpolarize different tissues and sodium movement into cells is known to cause loss of membrane potential thus producing action potentials. There- fore hyperosmotic NaCl could possibly affect intestinal muscle by: l) Hyperpolarizing the inhibitory nerves thus removing the previously mentioned inhibitory components, 2) Stimulating excitatory neural components by a different mechanism (i.e. movement of Na+ into the neuron) and 3) Directly stimulating smooth muscle following extra- cellular to intracellular movement of Na‘. If any or all of these mechanisms were true, TTX must block the motility by: l) Removing the inhibitory neural elements thus eliminating the effect of hyper- osmolality, 2) Block depolarization of excitatory nerves by its known effect on Na+ conductance in neural tissue (97), or 3) Block- ing sodium conductance thus depolarization at the muscle cell membrane. Evidence strongly rules against any action of TTX directly on the smooth muscle membrane (5, 39, 80, 86) therefore hyperos- motic NaCl and glucose appears to influence visceral smooth muscle activity through excitatory and/or inhibitory neural mechanisms. 171 Acetylcholine and epinephrine are known to act directly on the smooth muscle membrane. In the present study neither hexamethonium (C5) nor TTX significantly altered the motility responses to these agents (Figure 32). However, ganglionic stimulation with nicotine (5-50 ug, i.a.) (Figure 32) or DMPP produced excitation before 05 and inhibition after 06 (0.5 mg/min). Both of these responses to ganglionic stimulation were blocked by TTX. These data may indicate that the hypothesized cholinergic synapse driving the inhibitory neuron is insensitive to hexamethonium blockade. Thus, if nicotine and DMPP stimulate both excitatory and inhibitory nerves and hexa- methonium only blocked excitatory post-synaptic receptors the inhibitory elements would remain functional. Both of these neural pathways would then be susceptible to TTX thus eliminating both excitatory and inhibitory responses to nicotine or DMPP. li SUMMARY AND CONCLUSIONS In the present work in situ canine preparations of the intestine and gracilis muscle as well as isolated arterial strips were used to study: 1) What effects the neurotoxin tetrodotoxin (TTX) has on vascular smooth muscle, 2) What effects TTX has on intestinal muscle, 3) What doses of TTX block neurogenic responses, 4) The effects of TTX, in neural blocking doses, on the vascular responses to other vasoactive agents (epinephrine, norepinephrine, acetylcholine, ganglionic stimulants, KCl and changes in osmolality). Local infusion of TTX in doses of 0.05-10 ug/min (producing rrr .~ ‘ calculated blood concentrations of 10'5-10"9 g/ml) produced significant increases in blood flow or decreases in vascular resistance of innervated gracilis muscles but has no significant effect on dener- vated gracili. The neural blocking action of TTX was evaluated by its ability to depress and abolish the motor responses to electrical stimulation of the cut nerve trunk and the increases in vascular resistance following carotid occlusion. Doses of TTX as low as 0.1 ug/min i.a. (for 5-15 min) were shown to be effective in depressing neurogenic responses, however, the efficacy of TTX was increased as the dose was elevated. In these neuro-blocking doses, TTX has no effect on the vasodilator action of KCl in acutely denervated gracili muscles, but augmented the response in inner- vated muscles. In cross perfused innervated gracili muscles the vasodilator action of TTX reached a maximum at those doses which abolished vasoconstriction subsequent to carotid occlusion. The vasodilation induced by TTX correlated with the level of initial 172 173 resistance in innervated preparations (r = 0.92) but showed no cor- relation (r = 0.27) in denervated preparations. In innervated intestinal preparations local infusion of TTX never produced increases in venous outflow or decreases in vascular resistance. However, occasionally TTX produced increases in vascular resistance which occurred concomitantly with the TTX-induced increases in motility. In doses (0.05-10.0 ug/min, i.a.; 5-15 min) sufficient to block vagally induced increases in motility or vascu- lar reflex responses to carotid occlusion, TTX abolished the vaso- dilator action of locally infused KCl in natural-flow experiments but not in constant-flow intestinal preparations. Luminal placement *' of KCl significantly decreased vascular resistance and increased venous osmolality and KT concentration, and volume recovered from the lumen. TTX augmented the changes in venous osmolality and potassium but converted vasodilation to vasoconstriction. TTX altered the responses in motility induced by KCl from a pattern showing little phasic activity to one exhibiting large phasic responses. Intra-arterial or luminal placement of hyperosmotic NaCl produced vasodilation followed by vasoconstriction which was accompanied by increased motility. Following TTX, motility responses were abolished with only vasodilation occurring. Isolated arterial strips responded to vasoconstricting agents by an increase in contractile tension and vasodilator agents by a decrease in tension. Responses to all agents studied, were pro- gressively augmented as pre-load was increased (1, 2, 4 and 6 gms). TTX (10‘9-10“6 g/ml) had no significant effect on strips before or 174 after 24 hrs of cold storage. TTX blocked the increases in tension to low doses of nicotine or DMPP (5 x 10"6 g/ml) but had no effect on the responses to norepinephrine, acetylcholine, epinephrine, tyramine, KCl or hyper and hypo-osmotic solutions. Increasing extracellular K+ (+ 1.5, 4.5, 7.5 and 15.0 mEq/l) above control (4.7 mEq/l) generally produced only increases in tension in vascular strips subjected to different pre-load tensions; however, in the presence of active tension, produced by BaClz, increasing bath K+ to levels below 12.2 mEq/l produced relaxation while concentrations greater than 12.2 mEq/l produced contractions. Increasing bath osmolality (50 mOSm/Kg) above control (300 m05m/Kg) frequently 5“ produced contractions of passively loaded strips. After actively contracting the strips with BaClz hyperosmolality (310-390 :t 10 m05m/Kg) always produced relaxation; however, decreasing osmolality frequently produced increases in tension before or after BaClz. The responses to increasing extracellular K7, or changing bath osmolality were all augmented in strips equilibrated at higher pre-loads with relaxation appearing to be related to the level of active tension produced by BaClz. Neither the responses to change in bath K'+ or osmolality were altered by TTX, adrenergic or cholinergic blockade. Based on the present study the author makes the following con- clusions: 1) Local infusion of TTX in doses producing blood concentrations of 10"9 g/ml has no vascular action in denervated gracilis muscles but demonstrates a vasodilator effect in innervated gracili III‘ [I ill-ll Ill Ill-'51 I'll '1 Ill 175 with the level of dilation directly related (r = 0.92) to initial resistance. 2) TTX in doses as low as 0.1 pg/min (10"9 g/ml of blood) for 5-15 min exhibits neural blocking action in skeletal and intestinal muscle with the efficacy of neural blockade increased at higher doses . f“ "" 3) TTX in neural blocking doses (10'9-10"6 g/ml, 5-15 min) has - no effect on the vascular action of vasoconstrictor or vasodilator agents which act directly on vascular muscle. ’_ 4) TTX in bath concentrations of 10"9-10"6 g/ml has no effect fly on the tension of fresh arterial strips or following cold storage. 5) TTX (10-7-10-5 g/ml) and cold storage (24 hrs) abolishes the responses of isolated arterial strips to neural stimulating agents which work through Na+ related action potentials. 6) Equilibrating isolated arterial strips under tension aug- ments the responses to vasoconstrictor and vasodilator agents. 7) In isolated arterial strips, actively contracted with BaClz. elevating extracellular K+ produces responses similar to those seen 1Q_vivg_with relaxations at [KSJO <112 mEq/l and contractions at [KTJO >»12 mEq/l. 0uabain (10'5 g/ml) significantly attenuates the relaxing action of K+. 8) In the presense of active tension hyperosmolality (+10-100 mOsm/Kg) produces a decrease in tension of arterial strips and hyposmolality (-10-50 mOSm/Kg) produces increases in tension. 9) TTX, adrenergic and cholinergic blocking agents have no effect on responses produced by K+ or hyperosmolality or hyposmolality. |\ 176 10) Local infusion of TTX may inhibit spontaneous intestinal activity in low doses but generally stimulates motility in neural blocking doses. 11) TTX frequently alters the intestinal vasodilator action of i.a. or luminally placed KCl. 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