6C 919.9 . a B a...” Q wt ”i mu 3 I: P ,o - ”"5 b. o 0-. .. . o r .. o. n . . . t “:0”.on i . .. o...“ a . an. o u n . r . . ... a .94 o. u... . any... or 2. o . , - Perv“.- fi. To .. 3.... at.“ v..—. 9.».- rtah u E. k. ’5. N Q‘d I‘CI 1.... [HtSlS - T L; ' ~ I I 1'} 1'." N1 it}: i 1 1! 3 m ‘CC K \H‘Emk‘ \_ 2513/ r‘ I M‘ . Au. " ERRATA Page Line Correction Abstract 5 ILP increase correlated with simul- taneOUS'gastric antral facilitation (r=0.98) and gastric body relaxation (r=0.99). ii 9 assistance l2 egpresses 13 assistance The author failed to acknowledge Mrs. Jackie Hellmann for her assis- tance in preparing this final man- uscript, and especially for her willingness to type into the morn- ing hours so a deadline could be met. l8 2 procedure 47 lo Accummulation 48 l2 gastric blood flow 49 23 Alvareg_ 51 6 Ambachg_ 53 6 inhibition ABSTRACT VAGALLY INDUCED GASTRIC MOTILITY FACILITATION AND INHIBITION IN ANESTHETIZED RABBITS by Thomas D. Burns Gastric antral and body contractile activities recorded by extra- luminal force transducers were correlated with intraluminal pressure (ILP) responses elicited by electrical stimulation of the decentral- ized vagus nerves of anesthetized, fasted rabbits weighing 2-4 kg. (r = 0.98) and gastric body relaxation (r = 0.99), having a consis- tent frequency dependent pattern with maximum at 16-32 cps. Corre- lations with r > 0.90 suggest that the amplitude of the ILP post- facilitation inhibition is related to the amplitude of the immediately preceding ILP facilitation. Atropine (l mg/kg, i.v.) converted the vagally induced ILP facilitation to inhibition but failed to abolish the subsequent after-contractions. Barium chloride (4 mg/kg, i.v.) increased pre-stimulation ILP in the atropinized animal, thereby allow- ing an augmented vagally induced ILP and antral inhibition. VAGALLY INDUCED GASTRIC MOTILITY FACILITATION AND INHIBITION IN ANESTHETIZED RABBITS By I .\_\. Thomas D.”Burns A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1969 ACKNOWLEDGEMENTS The author wishes to express his sincere gratitude and apprecia- tion to Dr. David A. Reinke for his most valuable advice, guidance, and financial aid during the course of this study and especially for his persistent demand for a manuscript exhibiting many hours of pre- paration. The author would also like to thank Dr. T. Adams, Dr. 0.0. Chou, Dr. J.M. Dabney, and Mr. J.R. Stiefel for their encouragement, guid- ance, and most valuable critical reading of the manuscript. The technical assitance of Miss M. Taylor in the preparation of figures, calculation of statistical analysis and aid during the ex- periments is gratefully acknowledged. Finally to his wife, Caroll, the author espresses special appre- ciation not only for her assitance in preparing this thesis but also for her love, patience, and understanding. 11' TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ............................ ' ................... ii LIST OF FIGURES ................................................ v INTRODUCTION ................................................... l Methods for Study of Vagally Induced Gastric Inhibition ........................... . ..................... l Vagally Induced Gastric Motility Following Drug Administration ........................................ 8 Postulated Neuromechanisms for Vagally Induced Gastric Inhibition ......................................... 9 Statement of Problem ....................................... 12 EXPERIMENTAL METHODS ........................................... l3 Extraluminal Contractile Force Transducers ................. l3 Preparation of Animal ............................ C .......... l3 Nerve Stimulation and Drug Administration .................. l4 Integrating Intraluminal Pressure (ILP) Recordings ......... 15 RESULTS ........................................................ 20 Effects of Vagal Stimulation on Rabbit Gastric Motility ................................................... 20 Decreased ILP Responses Following Atropine ................. 2l Gastric Motility Following Barium Chloride ................. 2l Table of Contents (cont) Page DISCUSSION ..................................................... 45 Vagally Induced Gastric Facilitation ....................... 45 Postfacilitation Inhibition (PFI)........ .................. 46 Effects of Atropine on Vagally Induced Gastric Motility ................................................... 49 Effects of Barium Chloride on Vagally Induced Gastric Inhibition ......................................... 5l Neuroregulation of Vagally Induced Gastric Inhibition ................................................. 52 SUMMARY ........................................................ 55 REFERENCES ..................................................... 57 iv LIST OF FIGURES Figure Page l. Positioning of monitoring devices ........................ l7 2. High and low sensitivity ILP recordings .................. l9 3. ILP, BCF, and ACF responses, l minute intervals (2-128 cps) ................................ 24 4. ILP, BCF, and ACF responses, 2 minute intervals (2-64 cps) .......................... . ...... 26 5. ILP response to stimulation of the left vagus and both vagi (graph) .......................... 28 6. ILP, BCF, and ACF responses expressed graphically as percent of maximum .................... 30 7. ILP responses following atropine (graph) ................. 32 8. ILP responses before and after atropine .................. 34 9. ILP responses following barium chloride .................. 36 l0. ILPb changes following BaCl2 (graph) ..................... 38 ll. Effects of increased tonus on vagally induced inhibition (graph)................. .......... 40 l2. ILP responses in atropinized animals following barium chloride ............................ 42 l3. ILP, BCF, and ACF responses following atropine and barium chloride ......................... 44 INTRODUCTION Bayliss and Starling (5) reported that the vagus (parasympathetic) and splanchnic (sympathetic) nerves exert reciprocal control on gastro- intestinal motility. Although vagal parasympathetic efferents, which comprise less than 10% of the total cervical vagal trunk fibers (1);. primarily facilitate, they also inhibit gastric motility (5,14,43,44,47). It has been suggested that gastric receptive relaxation, (13,37) occurring primarily in the proximal portion of the stomach, results from excitation of vagal inhibitory fibers (13,14,40). Vagally in- duced gastric inhibition is apparently noncholinergic (5,32,43), and contingent on gastric tonus (pre-stimulatory contractile state) (12,32,40,50). The following review of vagally induced gastric inhibition pre- sents the methods, results, and conclusions of previous investigators. ‘\ Methods for Study of Vagally Induced Gastric Inhibition The existence of vagal inhibitory fibers innervating the aliment- ary canal was first reported by Openchowski in 1883. Since Open- chowski's work, many investigators of gastrointestinal motility have observed vagally induced gastric inhibition (39,44,47,48). Openchowski (51) studied esophageal-cardia motility in anesthetized rabbit, dog and cat, by inserting into the cardiac opening a small elastic bag connected to a Marey's tambour, recording intraluminal pressure changes on a smoked kymograph drum. Dilation of the cardiac orifice was obtained by stimulating a nerve on the lower esophagus which he called "nervus dilator cardiac". Openchowski reported a relationship between stimulating impulse frequency and magnitude 1 2 of excitation and inhibition with increasing frequency depressing excitation and augmenting inhibition. Langley (40) inserted an open tip rubber tube into the rabbit esophagus (positioning the tip at the lower thoracic level) monitor- ing intraluminal pressure changes by a fluid filled graduated ver- ticle column. In the atropinized rabbit (15-20 mg) vagal stimulation, either electrically or mechanically, produced cardia dilation allow- ing the fluid to enter the stomach. Direct visual observation showed relaxation of the esophageal cardia region. According to Langley, passage of fluid from the esophagus to the stomach was the result of cardiac dilation. When minimal effective induction shocks were applied, initial low amplitude esophageal-cardia contractions were recorded which rapidly decreased in amplitude with prolonged stimulation. In- creased stimulating impulse intensity produced a brief rise in pressure followed by a rapid fall. To Langley, this indicated a struggle be- tween excitatory and inhibitory fibers. The same effects were ob- served whether the vagi were stimulated supra— or sub-diaphragmatically. The most constant visible effect of vagal stimulation was an after- contraction of the cardiac-fundic region upon cessation of stimulation. After-contractions (contractions occurring upon cessation of stimu- lation) were conspicuous even when relaxation was not observed, with the magnitude of these contractions decreasing with prolonged stimu- lation. Bayliss and Starling (5) recorded gastrointestinal motility on a float-type piston recorder, using the enterograph connected to a tam- bour, simultaneously recording intraluminal pressure changes via intra- luminal balloons. Vagally induced gastrointestinal motility in the dog, resulted in both excitation and inhibition suggesting two function- ally different groups of fibers. Cannon and Lieb in 1911 (13) investigated receptive relaxation using thin rubber balloons positioned in the cat's gastric fundus. A cylinder (5.5 cm internal diameter) type float recorder, monitored motility, a glass cannula positioned in the cardiac sphincter resisted any cardiac pressure. Gastric intraluminal changes were transferred to the cylinder through a connecting catheter and recorded by a writing lever secured to a floating cork. The relatively large, fluid surface area in the cylinder permitted the recording of volume changes with little change in intraballoon pressure. Cannon and Lieb observed a relaxation in the gastric fundus 2-3 sec. after the rise of the larynx in deglutition. Even though the food bolus was expelled through an esophageal fistula, relaxation occurred at the time the bolus would have reached the cardia. Bercovitz and Rogers (6) recorded turtle gastric motility kymo- graphically, by inserting through the esophagus intraluminal balloons connected to a water-float manometer with a writing 1eVer. Vagal stimulation elicited gastric relaxation in the turtle. Carlson gt_al, (14) studying gastric motility in the ether anes- thetized cat, positioned a small condom balloon (4-6 cm in length) in the gastric cardia. Intraballoon pressure was monitored by two separ- ated water manometers, one through an esophageal fistula and the other through a gastric fistula allowing the measurement of cardiac tone by recording respiratory excursion through the cardiac end of the balloon. A larger condom (10—15 cm) simultaneously recorded gastric motility. Vagal stimulation with high frequency impulses produced esophageal- cardia excitation when tonus was low and inhibition during conditions of high tonus, which varied at different anesthesia levels. Carlson 4 suggested that the vagus and splanchnics contain both facilitory and inhibitory fibers innervating the esophagus and gastric cardia. Veach (59) investigated vagal inhibition to the cat lower eso- phagus, cardia and gastric body, using intraluminal balloons, obser- ving two distinct responses: 1) excitatory motor responses when low intensities of electrical stimulation were applied, and 2) inhibitory responses when the intensity was increased above a critical frequency level. Initial contractions occurred more rapidly as the frequency or intensity increased with inhibition becoming more pronounced as intensity or frequency increased above that yielding maximum contract- ions. The duration and magnitude of the after-contractions which were observed on cessation of stimulation, seemed to be related to duration of the stimulation period. Inhibition induced by one vagus could be nullified by an excitatory effect of the other. McSwiney and Madge (50) investigating tone-inhibition relation- ships recorded gastric motility with an open tip catheter connected to a float manometer. Anesthetized cats laparotomized with a mfidline in- cision, the duodenum ligated and the abdominal walls retracted to form a warm saline bath. Stimulation of either vagal trunk produced inhibi- tion, but a greater effect occurred when both were stimulated simul- taneously, with initial atony requiring a greater stimulating intensity to produce excitation or inhibition. The results obtained did not show a relationship between reaction and intensity or frequency of stimuli as suggested by Veach, for low or high frequency or intensity produced inhibition when tone was high. McSwiney and Spurrell (49) working with anesthetized cats inserted an open tip catheter through the esophagus, ligated the pylorus and 5 monitored motility with a fluid manometer. McSwiney and Spurrell (49) reported inhibition depending upon the intensity of stimulation, gastric tonus and animal's postsurgical condition. Intracranial (vagal trigone region of medulla) vagal stimulation was as effective in pro- ducing inhibition as intrathoracic vagal stimulation. Harrison working with McSwiney (32), isolated vagally innervated strips from the gastric fundus of cats, suspending them in a bath (125 cc) of oxygenated Ringer-Locke solution maintained at 37°C. Harrison and McSwiney (32) reported that gastric excitation and gastric inhibition were tone dependent. Celander (15), using a rubber intraluminal balloon technique, re- corded intestinal motility in exteriorized intestinal loops filled with Tyrode-solution. A polyethylene tube from the cut end of the loop was connected to a reservoir with a relatively large cross-sectional area maintaining pressure constant even when volume changed markedly. He reported that vagally induced intestinal relaxation differed character- istically from the splanchnically induced inhibitory effect. Splanchnic stimulation at impulse frequencies less than 10 cps had little effect on motility, but at supraphysiological frequencies (greater than 10 cps) vasoconstriction was prompt with intestinal relaxation occurring after a considerable latency. However, vagal stimulation produced prompt gastrointestinal relaxation at 1-10 cps. Celander suggested that in- testinal relaxation following splanchnic stimulation was the result of vasoconstriction coupled with a slight overflow of transmitter, known to occur at high frequencies. Martinson and Muren (45, 46) introduced latex balloons (inflated with 40 cc of air), connected to a water manometer, into the cat 6 stomach vja_the esophagus, recording gastric motility elicited by cervical vagal stimulation. Jansson, working with Martinson (37) using chloralose (60-80 mg/kg) anesthetized cats recorded gastric motility through an open tip pyloric cannula connected to an iso- tonic saline filled volume reservoir. The reservoir was arranged in such a way that intragastric pressure could be set at a desired level. To ascertain an exact transmural pressure across the gastric musculature, the open abdominal cavity was filled with Tyrodes solution. The transmural pressure was equal to the difference be- tween the fluid height in the reservoir and that in the abdominal cavity. Martinson in reviewing vagally induced gastric inhibition (44), described two basic methods: 1. pressure recording system - using fluid or air filled intraluminal balloons connected to a water manometer. 2. volume recording system - using low intragastric pressure (2-4 cm H2 ). Martinson et_al. (43,44,46) reported inhibition superimposed on ex- citation. Vagal stimulation (4 cps and 10 v) produced a reduction in the gastric excitatory response, an interruption in the initial rising phase of excitation and a prolonged depression of basal tone when duration was as low as 0.02 msec. Gastric inhibition had a higher threshold than gastric excitation, which in turn had a higher thresh- old than cardio—inhibition. Their results did not confirm those of McSwiney and Madge (50) who considered inhibition to be related to tone. The intensity-response data obtained implies Veach's hypo- thesis of frequency dependent, not tone dependent responses; Kewenter (39), investigating the vagal control of gastrointestinal 7 motility in the urethane-chloralose anesthetized cat, recorded motility kymographically yj§_a gastric intraluminal balloon (30-40 cm) and in- testinal open tip catheters. Kewenter reported absence of cervical vagal inhibitory fibers to the small intestine, with inhibition induced by sub-diaphragmatic vagal stimulation resulting from activation of high-threshold fibers. Kewenter suggested that the simultaneous occur- rance of intestinal inhibition, decreased intestinal blood flow and increased blood pressure, are the consequence of antidromic activation of thin afferent fibers exciting the intestino-intestinal inhibitory reflex arc. Campbell (12), studying some pharmacological aspects of vagally induced gastric inhibition, used an jg_vjtrg_preparation of the guinea pig stomach. The stomach, first few centimeters of duodenum, and vagal trunks were placed in a 100 ml bath of Krebs solution bubbled with 95% oxygen and 5% carbon dioxide maintained at 37°C. Gastric responses were recorded vig_a pyloric cannula connected to either a float recorder or a wide bore saline manometer. Using this prepara- tion, Campbell studied the effects of amphetamine, atropine, bretylium and hyoscine on vagally induced gastric inhibition. He observed a more effective vagally induced relaxation than splanchnically induced relaxa- tion at frequencies less than 10 cps. Vagally induced relaxation was a more rapid response (0.8 sec latent period) than the sympathetically induced relaxation (greater than 1.0 sec latentperiod). Bulbring and Gershon (9) extensively studied the effect of drug ad- ministration on vagally induced gastric vagal inhibition using the same in_vitrg_technique used by Campbell (12). Their results are presented in a following section. 8 Vagally Induced Gastric Motility Following Drug_Administration Langley (40) was the first to report the augmentation of vagally in- duced inhibition of the esophagus and gastric cardia following the ad- ministration of atropine. That inhibition was recorded if the dose of atropine was large enough to block excitatory cholinergic responses. Very large doses weakened the stimulation induced effects of both motor and inhibitory fibers. Anticholinergic drugs (atropine or scopolamine) block the muscarinic responses of the stomach (52). It has been shown by use of tetrodotoxin which abolishes nerve mediated responses, that catecholamines like ep- inephrine act directly on the smooth muscle (9). Harrison and McSwiney suggested in 1936 (32) that the high threshold gastric inhibitory re- sponses were adrenergically mediated (atropine abolished the excitatory responses but only slightly suppressed the inhibitory response). How- ever, Campbell (12) observed little change in vagally induced gastric inhibition following both atropine administration and adrenergic block- ade by bretylium (10'6 - 3x10'6g/ml) while gastric inhibition induced by perivascular nerve stimulation was completely abolished. At high con- centrations, bretylium (3x10'Sg/ml) was able to reduce vagally induced gastric inhibitory responses markedly confirming Greeff gt_al, (27). Am- phetamine (10—6-10'5g/ml) failed to antagonize high concentrations of bretylium, therefore indicating some action of bretylium other than the specific blockade reported by Day (18). Burnstock §t_al, (10) suggested that bretylium was acting in some other way than specific neuron block- ade, possibly a ganglionic action. Martinson (44) was able to show this same noncholinergic, nonadrenergic response in atropinized animals by adrenergic blockade with quanethidine (4.5 mg/kg) or pronethalol (5.0 mg/kg) 9 and phenoxybenzamine (0.5, 0.8, 1.0 mg/kg). Greeff gt_al, (27) reported that vagally induced inhibitory responses were mediated by some adrenergic mechanism. However, Martinson (44) abolished sympathetic inhibition with pronethalol, a beta adrenergic blocking drug which only slightly suppressed the vagal response. It has been argued that vagal inhibitory fibers were adrenergic fibers which were resistant to pharmacological blockade in much the same way as some cholinergic fibers were resistant to atropine blockade (34). Bulbring and Gershon (9) reported that vagal electrical stimula- tion (supramaximal voltage, 0.1 msec. and 2-6 cps) or 5-hydroxytryp- tamine (SeHT) administration (lO’Sg/ml), after the administration of scopolamine produced relaxation of the isolated guinea-pig stomach. With '9-10'4g/m1) the gastric excitation increasing concentrations of 5-HT (10 was depressed, only relaxation was observed following 10'4g/m1 of 5-HT. 5-HT, failing to antagonize the acetylcholine (10'7g/m1) response, ap- peared to produce a prolonged stimulation of the gastric inhibitory neu- rons. From these data it was concluded that there are two types of post- synaptic receptors: one sensitive to acetylcholine, and the other sen- sitive to 5-HT. The inhibitory responses were completely abolished on- ly when the competitive block of acetylcholine receptors by scopolamine was combined with desensitization of 5-HT receptors by biguanides or quaternary derivatives of 5-HT (2x10'6g/ml). After the depletion of 5-HT stores by reserpine, 5-HT (10'59/m1) resulted in a greater vagally in- duced gastric inhibitory response. Postulated Neuromechanisms for Vagally Induced Gastric Inhibition Langley (40) in his explanation of gastric vagal inhibition, ack- nowledged that vagal inhibitory fibers exist and may predominate during 10 vagal stimulation. Bayliss and Starling (5) suggested that gastrointestinal inhibition and excitation are regulated by a local nervous system (Auerbach's plex- us) containing long inhibitory pathways and short augmenting pathways which carry impulses from one intramural neuron to another. Cannon and Lieb (13)observinga relaxation of the cardia and fun- dus as a result of deglutition mentioned that receptive relaxation was vagally controlled. .The lowest intragastric pressure was observed at the time the expelled bolus would have been delivered to the stomach indicating a possible correlation with the cephalic phase of digestion. It was Veach (59) who first attempted to explain the vagal-gastric inhibitory effect. The nullifying effect of stimulating one vagus with low intensity impulses on gastric inhibition produced by simul- taneous high intensity stimulation of the contralateral vagus, sug- gested a postganglionic inhibitory site. Finding that the excitatory and inhibitory responses depended on the intensity or frequency of the applied stimulus, Veach suggested that the response was related to the frequency of impulses over the postganglionic fibers. Therefore, vagally induced gastric inhibition was the result of Wedensky inhibi- tion (60) in which the frequency of propagated impulses is such that each impulse travels in a refractory phase of the previous one, result- ing in a decreased conduction within the neuromuscular complex. He suggested that tonus level was the result of propagated disturbances over the peripheral part of the neuromuscular mechanism. Vagal stimu- lation increased the postganglionic frequency first into the excitatory range. If the increase in frequency is continued further, it enters into the inhibitory range. When tone was high the propagated distur- bances would be at a greater frequency, therefore vagal stimulation 11 would result in a more rapid inhibition. Veach suggested that the after-contraction was due to a decrease in impulse frequency over the conduction mechanism from a high inhibitory level, passing through the excitatory range at a lower frequency. McSwiney et_al, (32,49) stated that possibly the vagus contained sympathetic fibers originating in the superior cervical ganglia. Fail- ure of atropine to abolish gastric vagal inhibition caused them to con- clude that inhibition was adrenergically mediated. However, this was contradicted later when adrenergic blocking agents failed to abolish inhibition (12). Burnstock et_al, (10) suggested that the postganglion- ic fibers in the vagal inhibitory pathway were the intramural inhibitory nerves. Martinson and Muren (46) suggested that the vagus contained two distinctly different groups of fibers, with the low threshold excita- tory responses mediated cholinergically and the high threshold res- ponses mediated by some unknown transmitter. Bulbring and Gershon (9) were able to show a possible role of 5- hydroxytryptamine in the inhibitory innervation of the stomach. They concluded that 5-HT and acetylcholine function as neurotransmitters in the inhibitory pathway. Both function preganglionically indicating either two different types of vagal fibers or two functionally differ- ent post-ganglionic receptors. Bulbring and Gershon concluded that cholinergic and 5-hydroxytryptaminergic fibers work synergistically on the same cells. The release of 5-HT during gastric vagal inhibition and the resistance of both 5-HT release and gastric inhibition to anti- cholinergic drugs supports their hypothesis that 5-HT participates as a neurotransmitter. 12 Statement of Problem A recently developed method, the extraluminal contractile force [transducer (ECFT) quantitatively samples discrete contractile activi- ty of the longitudinal or circular muscle with uniaxial sensitivity. Jacoby 25.31: (36), reporting the reliability-of the extraluminal force transducer, correlated dog duodenal electrical activity, intraluminal pressure change, and extraluminal contractile force. Reinke gt_al, (53,54) modified the transducer and recorded dog gastrointestinal control patterns of the circular and longitudinal muscle layers dur- ing the digestive and interdigestive periods. Rosenbaum §t_al, (55) more extensively studied the jg_yiy9_force, frequency and velocity of dog gastrointestinal contractile activity. The ECFT has also been used to record uterine contractile activity in unanesthetized rabbits by Dominic and Reinke (20) and Callantine et al. (11) and in dogs by Bass and Callantine (4). The problem of this thesis was the study of vagally induced gastric facilitation and inhibition in the atropinized and non- atropinized rabbit. Vagally induced gastric inhibition has been studied using methods which record only mean gastric motility changes. Due to the limitations of these methods in studying gastric antral and body motility, antral and body ECFT monitored contractile activities were correlated with intraluminal pressure responses elicited by electrical stimulation of the decentralized vagus nerves in anesthetized rabbits. EXPERIMENTAL METHODS Extraluminal Contractile Force Transducers ECFT's were fabricated according to the method described by Reinke et_al, (53,54) and Dominic (19,20). Two etched-foil strain gages1 were bonded, one to the convex and the other to the concave surface of a metal shim stock (12 mm, 5 mm, 0.2 mm)2, in which four suturing holes (1 mm diameter) were drilled. The ECFT and adjacent soldered portions 1, of the lead wires were encapsulated with Dacron mesh reinforced raw silicone elastimer3, then heat-cured at 220°F for 24 hours. Record- ings were made on a curvilinear ink-writing oscillograph (Dynograph ;) Type R)4. ECFT's were calibrated as described by Dominic (19,20) by hanging equal weights on sutures tied one to each end of the supported transducer. Preparation of Animal New Zealand White rabbits (N=9) of either sex, weighing from 2-3 kg, were fasted with water for 18-24 hours in a restraining stock to pre- vent coprophagia. The animals were anesthetized with urethane-allobarbi- 5 tal (.7-1.0ln1/kg) i.p. A pyloric open-tip cannula was connected to a venous pressure transducer (Statham P23-BC)6. The stomach was washed lModel ED-DY-090-DG-350, Micro-Measurements Inc., Romulus, Michigan. 2Berylco 25, The Beryllium Corp., Reading, Pennsylvania. 3Silastic 372, Compliments of Dow Corning Corp., Midland, Michigan. 4Spinco Division, Beckman Instruments, Lincolnwood, Illinois. 5[Dial, Allobarbital (100 mg/ml)-urethane (400 mg/ml)], Veterinary Sales Division, CIBA Pharmaceutical Corp., Summit, New Jersey. 13 14 with five 20 cc aliquots of 370C saline, and then filled with 30 cc of 37° saline. Ligatures were tied around the pylorus and cervical esophagus to prevent loss of fluid. ECFT's were oriented trans- versely on the greater curvatures of the gastric antrum and body (Fig. l) with two Mersiline—OOO7 sutures tied snugly through the suturing holes in the shim stock. The cervical vagi were isolated and cut, the adjacent skin was formed into a well containing mineral oil which served as an electrical insulator. Respiration was main- tained artificially at 40 breaths/min. Nerve Stimulation and Drug_Administration The distal ends of the cut vagus nerve trunks were placed on hooked, dipolar, silver electrodes positioned within the oil-filled well. Electrical impulses were delivered to the electrodes by a square-wave stimulator (Grass S-5)8. Impulse trains (15 sec durat- ion) were applied at l or 2 minute intervals at supramaximal intensi- ty (2 msec duration, 30 volts) while increasing the frequency from 2-128 cps in progressive multiples of 2. I Atropine and barium chloride solutions were administered through a femoral venous cannula. 6Statham Instrument Inc., Oxnard, California. 7Ethicon Inc., Somerville, New Jersey. 8Grass Medical Instruments, Quincy, Massachusetts. 15 Integrating Intraluminal Pressure (ILP) Recordings Facilitory responses to vagal stimulation were recorded at low oscillograph sensitivity (0.2-1.0 mv/cm) and inhibitory responses at high recorder sensitivity (0.05 mv/cm). A composite ILP tracing is shown in Fig. 2. 16 .mcwozvmcmgp mcu op mmcez vamp Loguzvcouummcgp pcmmmgamg N3 ncm F2 .APAV co?mmx Fmow>gwo esp cm vmpmmWF AMV mammsaomm mcp saw: .Amgv macopxq vameF mgp smzoczp vmpcmmcv AHV mpzccmu mczmmmca chwszfimcpcw mg» op coprng 2? mmocmm_uwcpmmm mcp co mcmuzumcmcp mocom w__pumgpcou Amy Anon ucm A saw: mcwomgp mpwmoqeou .u .mugoumc Amcwumgp cosmmvv xpw>wp_mcwm saw; can Amcwumcy vaomv zpw>wpwmcmm zop to mmcwumgp ummoaswcmazm .m .AEU\>E o.F vcm mo.ov mm_pw>wpwmcwm mcwugoumc acmcmmewn ozu gm mpmswcm mEmm any :w zpmpmgwawm umucoumc mmmcoammm .< .Ampmcmszc umpcmFm can mean cowpmcnwrmo _mowpcm> umcmmn an vmpmo_vcw .xpw>wpwmcwm :m_: pm umvcoumcv mwmcoammc 18 xcow_n?;cm acoumpsswpmumoa QJH new Amcmn cowumcnwpmu Pmowpgm> vw_om an umpmu_n:_ .>#w>wuwmcmm 30F pm nwugoumgv mmmcoqmmg QJH zgopwpwumm mcwnaou Op now: mczumomga mzp mo cowpmgcmmmgqmc _mowpmssmgmmwo . N mczmwd _ . . . £558 _ 02645. 33828 _ a . % :1..xlIi. i.::n. Issac mwmzommmm omomoowm ammomgmmmnm mum zommmm omomoomm .333 . . Boom. 33523 535.33 .32 ., 30. RESULTS Effects of Vagal Stimulation on Rabbit Gastric Motility Intraluminal pressure (ILP) responses to vagal stimulation with impulses of supramaximal intensity (30 volts, 2 msec duration) and varied frequency (2-128 cps) have a characteristic pattern, (Figs. 3,4) increasing in magnitude with increasing impulse frequency, with maximum ILP increase recorded at 16-32 cps (Figs 5,6). Stimulation of both vagi resulted in a greater absolute ILP change than stimulation of the left vagus; however, the change expressed as a percent of maxi- mum revealed no statistical difference, therefore maintaining the characteristic pattern (Fig. 5). Upon cessation of stimulation, ILP increased transiently (3-5 sec) then fell to a level below control ILP (Figs. 4,8). Although it is not conspicuous in Figs. 3 or 4, the absolute level of the post-stimu- lation ILP increase appeared to be dependent on the stimulating impulse intensity and the gastric contractile state at cessation of stimulation. Post-facilitation inhibition (PFI) increased as facilitation increased (Figs. 3,4). PFI duration ranged from 30-65 seconds and increased as frequency increased with maxima at 16-32 cps (Fig. 4). Body contractile force (BCF) responses to increasing impulse fre- quency (2-128 cps) increased in amplitude to maximum, then declined (Figs. 3,4, and 6). ILP increases correlated (r=0.99) and coincided with BCF decreases (Figs. 3,6). Antral contractile force (ACF) responses increased to a maximum at stimulation parameters corresponding to and correlating (r=0.98) with ILP and BCF maxima (Fig. 6). Frequently post-stimulation ILP increases coincided with ACF increases and BCF decreases. ACF decreases 20 21 were never observed during post-stimulation ILP increases. Decreased ILP Responses Following Atropine ILP increases were abolished following atropine (1 mg/kg) with subsequent additions of atropine reversing the ILP response to vagal stimulation (Fig. 7). After 2 mg/kg of atropine, vagal stimulation resulted in inhibition (decreased ILP) (Fig. 8). Atropine failed to affect resting ILP in stomachs exhibiting either tonus or atony. Al- I though vagally induced inhibition was recorded, PFI was not recorded from atropinized animals (Fig. 8). Gastric Motility Following Barium Chloride 1 Barium chloride solution (administered to an atropinized animal) produced a gradual increase in resting ILP (Figs. 9,10). Vagal stimu- lation following each dose of barium chloride decreased ILP, with the ILP absolute value approaching that obtained following atropine (Fig. 9). At constant electrical parameters, vagally induced inhibition coincided with increased resting ILP (Fig. 11). Maximum vagally induced inhibi- tion was obtained at frequencies less than 8 cps, with increased fre- quencies not only failing to augment but frequently depressing inhibi- tion (Fig. 12). Vagal stimulation following atropine and progressive doses of BaCl2 (totaling 8.61 mg/kg administered during a 10 min period) resulted in ILP inhibition to pre-drug resting values (0.9 - 1.1 mmHg) (Fig. 12). As frequency increased, post-stimulation ILP response increased. Stimu- lation at 2 cps produced only inhibition with little sign of facilita- tion and only partial recovery upon cessation of stimulation. 22 Vagal stimulation following barium chloride (4 mg/kg) and atropine ( 1 mg/kg) resulted in an augmented ILP decrease, with BCF increases and ACF decreases being less conspicuous (Fig. 13). Barium chloride decreased pre-stimulation BCF and increased pre-stimulation ILP. Vagally induced ILP decreases recorded at each impulse frequency never exceeded that recorded at 4 cps; however, the increasing pre-stimu- lation ILP augmented the relative ILP decreases at 32 and 64 cps. 'Post-stimulation ILP increases, indicative of after-contractions, in- creased in amplitude as impulse frequency increased. Maximum post- stimulation ILP increases and pre-drug facilitation occurred at 16-32 cps, with 2 cps resulting in the least conspicuous response (Fig. 13, upper). Post-stimulation ILP increases (after-contractions) Were often recorded when ILP changes failed to occur during stimulation. An additional 1 mg/kg of atropine had little effect on vagally induced ILP decreases, maximum occurring at 4 cps; however, prestimu- lation ILP and ACF increase concomitantly (Fig. 13, middle). Vagal stimulation following an additional 4 mg/kg BaClz resulted in decreased ILP, increased BCF, and decreased ACF with maximums at frequencies of 16 and 32 cps (Fig. 13, lower). 23 .oopo> oopoopoop op Aooppomoov oLoN soot oogop msogo moppcomopoog moon ooppogoppoo mum coo do< cpoo opp: .ocommoco opopomoo opoopoop moon ooppogoppoo opp .mogoopw pcoooomoom oco mpgp op .Amoov oooomm goo mopoxo NNP op N Eocpfidv mopoooooogp mcpmoogocp po mpo>copop opoope ooo po ooco>ppoo ogoz .oopp -oepp Logo: mop to coppgoo >>ooz ogp xo oopoopoop Aoome N u .o .coppogoo ompooep .mppo> om u .> .ooop_o>v mompooep poopcpoopo to mopopp ocooom coopmpd .poo> Foop>coo ooNpFoLpooooo ocp to ooo popmpo ogp to ooppop -osppm poopppumpo op momcoomog Aom po ooco>ppoo moo: AoomE N u a .mp_o> om n >v mcpocp ocooom coopppd .coppo_osppm Pooo> Eocp mopppomog momcoomo» Adu mczmmmga mszOmnm mmmgaxm mcwn co.“ -mgnwfimu .m.m>gmp:w wyzc.e mco gm Achy cowpmgsv ncoumm m_ .o mcwmgu mmpzaEH .Amcwumgu LogoF..>.w mx\me NV wCPQOme gmuwm vcm Amcwumgg Lmaazv mgo.mn mmmcmcu 44H wouzvcw cowpm_:swpm Pmmm> .o mmcwuwgh . w mgsmwu hkU I. 2.. EE 0.. ON; 66' or E... 34 0.. o. H- 0N7... ¢onu «n - .... 3.qu N 2.383 .23 55 m II .3. I ...: OENIO. >OMI> 35 .mmpzcwe m mcwpcmmmcamg xmmgn mcwumxu comm ;p_3 mmcwumgu umpmowccw yo me.“ mgcmmmgqmg m:._ mew. cmaa: .Eo\>E mo.o mo zpw>wwwmcmm camgmonwomo cm pm nmucoumg mwcsmmmga QAH mp:_omnm mpmu.c:. mgmn co.pmgnwrmu .m_m>gmpcw mp::.E _ pm uwgw>WFmv mew: Aumm mfiv m:_mcp mmfizaeH .mxxme Pm.w umquOp Npumm .o mmmou .>.. m>wmmmgmoga .Amao N .ume N .mwpo> omv cowpmpzewgm Pmuwgpumpm mem> Eog. mcwgfismmg mmmcmcu 44H co Am_ummv muwgorcu Ezwxmn mo mpum..m . m mg:m.u .... 3.62... ..._ 3.on n.~ .... 3:2... .... 3.6... ..o .... 3.3... 66 .3 3.3.: ~68. ~68 ~68 ~68 ~68 .5622 hgvlu « ¢ « ¢ .35 Elm «Ll/Kw 7.79.... 1.1.13... 7.9.11. ..... -... oIEE OQONIK 6 . OENIO 3 ~ .. .on.> llllhflflflllllllIIIIIHHFIIIII.Illhflflfllllll.IIIIBHHFIIIIILIllhflflfllllll.IIILHHHIIIII-rIIINHHFIIIIII - . - 5E p 37 .Ammv .oggm cgmucmum w hwy m~:_m> cam: .mFmom mo. 8 co ummmmgaxm Amxxmev mmmov onwgoFgu Ezwgmm .~_umm we :owmz.c. .>.. 50.. m:.ppzmmg ADQAHV mgzmmmgq chwsangp:. Acowpm_:swpmmgav m:_memn :. mmmcmco - 0. 6.3m.. 38 l——0—-l o._. Ex\mE.~_ ~_Uom omen. mo; do l—o—l 5.0 IOI 1.0 .md .md .06 No 8.8...va 8.0 5.0 . ADI 8.5 “3. < 39 .uwnnmg nm~.:.aogum 65p c. muwgo_;u E:.Lmn m:.3o—_ow AQQJHV mgzmmmga FacPE:_mgp:w m:w_mmmn agopmfizewamnmga nmmmmguc. mo co_pu::. 8 mm umppo_a AQAH ummmmgumvv AHV covgwnwscw umu:u:. appmmm> vmmmmgocH - F~ m.:m.m 40 _nd l—o—l 90 90. to ad ~d .6 ,. .. .. . O \m. lo m fl\\\ Mao o . 6:55. W H .46 6. .no ”66 mmflMuZVwm nd 4l .mmpacwe o pcwmmgamg xmmgn mcwomgh .zgwpwpos owgpmmm maomcmycoam mcwugoumc gmp$m wovgma .:.E N. gw>o umgmpmwcwsum mgmz muwgoPco Eswgmn use mcwaoca< .mpm>cmpcw mpzcwe «so an nogm>PFmv mgmz Aomm mpv mcwmgp Azucwzcmgw vmwgm> .omms N .mp~o> omv wmpzasH .Amx.me Fm.m mcwpmpop NFummv mcFLOFSU Ezwgmn mo mmmov w>wmmwgmoga.mcrzohpow Fmsmcm vw~mcmaogpm 65¢ :. AQAHV 6.3mmmga Fm:.E:P -mgucw co Ammo Nm-N n 8v zucmzcmg$ cowpmfisswpm mcwmcmsu we mpommwm - N. 6.36.. .>._ 8.on Ed .3 3.3.... N M... 5&6 d- . Naom 0596.34 0.. or E... m... 43 ..6.\6E .v ~_66m .6 6.66 —6:o.~_uum mcwzoFFO. mmmcoqmmg du< .uom .QAH . wmcwumgu we ajocm .6304 .Amx\ms Fv chQOme mo wmow chowuwnum :6 mcwzo__om mmmcoqmmc uu< .mom .64H - mm:.umg~ mo m:ogm.m_nvwz .A6¥\6e 6V ~.6.m 6:6 A6.\6E.V mcwaogpm mcwzorfiom mmmcoammg mu< .dum .QAH - mmcwomgp mo azogm Lama: .m_6> -Lmuc_ 6p::.E _ p6 umgm>._6u 6.63 A6665 N .mpFo> omv mmm_:aE. F66.L666F6 mo mewmgg vcoumm m. .Npumm new mcwaogpm mcwzoFFO. .zocmzcmL. m:_:e_pm mc.m:6;o mo mpomw66 62p mcwzogm mmcmomcu mu< 6:6 .dum .QAH msoscwpcoo - m. 6.36.. 44 mu< .2. 3:25 2.33: “Ga Sm uuuuuuuuuuuuuuuuuuuuuuuuuu .3. a: ...... ii. n._ . llllll I LI l I .3. 3:06. 5.. 3:060 0:833 ~68 .23 « :7: w I. lllllll II ”a i I, '|’ I, ll, 1",” H E: mU< Nnnn. mUm n:— DISCUSSION Vagally induced facilitation of rabbit gastric motility is mani- fested by antral contractions occurring simultaneously with passive distention of the gastric body (Figs. 3,4,6). PFI appears to be re- lated to the magnitude of the preceding facilitation (Figs. 3,4). Vagally induced gastric inhibition following atropine suggests a non— cholinergic mechanism (Figs. 7,8). Augmentation by barium chloride suggests that inhibition is related to the contractile state (tonus) of the gastric muscle (Figs. 9,ll). Vagally Induced Gastric Facilitation The characteristic pattern of vagally induced gastric motility facilitation (increases in ILP with increasing stimulation impulse frequency to maximum, followed by decreases in ILP) which has been observed in cats (44), guinea pigs (l2) and rabbits (Figs. 3,4), has been interpreted as the response to stimulation of two different classes of vagal fibers: low-threshold excitatory fibers and high— threshold inhibitory fibers (46). This hypothesis was based on the data of other investigators who obtained a more sustained postfacili- tation inhibition (PFI, decrease in ILP following cessation of stimu- lation to a level below prestimulatory ILP) at frequencies producing decreasing facilitation (42-46). Although PFI augmentation was ob- tained in the present study (Fig. 8), the more typical response was a gradual decrease in PFI which corresponded to the decreasing facili- tation (Figs. 3,4). Vagally induced gastric facilitation decreased when the left vagus was stimulated (LVi) and both vagi stimulated (BVS) (Fig. 5), suggesting that the neural mechanism regulating gastric func- tion, influences each contractile unit and stimulation of both vagi recruits more units. When the responses, exhibiting the character- 45 46 istic pattern, were expressed as a percent of maximum for any given series of frequencies, there were no statistical differences between LVS and 8V3; suggesting that the regulating mechanism is consistent for each contractile unit and the quantitative difference observed was indicative of the number of units participating. The characteristic pattern of gastric facilitation observed in these studies suggests that as frequency is increased above some critical level, impulse conduction through the neurocontractile complex (defined here as a complex consisting of pre- and post-gang- lionic fibers and the muscle cell or cells innervated) might be inhibited. The relation of this decreased conduction to the natural refractory period of the neurocontractile complex (60) as suggested by Langley (40) and Veach (59) requires elucidation. Postfacilitation Inhibition (PFI) PFI, which appears to be related to vagally induced gastric motili- ty facilitation, differs characteristically from the results obtained by Martinson and Muren (42-46). Martinson and Muren (42-46) reported an augmented atropine resistant postfacilitation inhibition (PFI) at impulse frequencies greater than that which produced maximum facilita- tion. However, in this study PFI failed to increase in magnitude at high impulse frequencies, and was absent in the atropinized rabbit. If PFI results from high-threshold fiber excitation, one would expect post- facilitation inhibition at high impulse frequencies equal to or greater in magnitude and duration than that obtained following maximum facili- tation. Failure to observe consistently this expected pattern, in this study, suggests either the absence of specific high-threshold inhibitory fibers in the rabbit or decreased efficiency of the neurocontractile 47 mechanism. However, the former explanation is unlikely because the decrease in ILP recorded during vagal stimulation of the atropinized animals indicated the activation of some mechanism capable of relaxing gastric smooth muscle. The appearance of vagally induced gastric in- hibition at 2 cps fails to support a high-threshold frequency concept. Three hypotheses for postfacilitation inhibition in nonatropin- ized animals are: l. Hyperpolarization of the neurocontractile complex following facilitation. 2. accummulation of biological substances capable of relaxing smooth muscle. 3. Activation of specific fibers, thereby releasing an inhibitory transmitter. Eccles (22), Hughes (33) and Lloyd (41), discussing post- tetanic potentiation, reported that repetitive stimulation caused hyperpolarization of presynaptic fibers. Postganglionic firing resulting from repetitive preganglionic impulses (6l) might result in subsequent hyperpolarization of the postganglionic neurocontractile complex, thereby decreasing tonic postganglionic firing by increasing the relative depolarization required to maintain basal contractile tone. The local accummulation of physiologically active agents has been suggested by Grundfest (28), explaining postactivation potentials, and Scott §t_gl, (56), explaining local regulation of vasodilation. During gastric facilitation, the increased metabolic demands may result in a similar accummulation of metabolites which upon cessation of stimulation produces relaxation either by direct action on the gastric smooth muscle or indirectly vja_nerve fibers. It is well 48 documented that certain substances (carbon dioxide, potassium ions, hydrogen ions, adenosine, adenine nucleotides, and Kreb's metabolites) are capable of relaxing smooth muscle (l7,29,30). One or more of these agents may be involved in the PFI phenomenon; for example, efflux of potassium from the guinea-pig taenia coli has been reported by Bulbring (8) to be high when contractile tension is high and low when contractile tension is low. Eccles (22) suggests that after-hyperpolarization, which is abolished when considerable intracellular potasSium is re- placed by sodium or intercellular potassium becomes depleted, is produced entirely by the net outward movement of K+ ions. Martinson (44) reported that excitation of high-threshold vagal inhibitory fibers resulted in an increased gastric flow suggesting a decrease in cat gastric vascular resistance occurring concomitantly with increased gastric HCl and pepsinogen secretion. The decrease in gastric vas- cular resistance suggests the relaxation of vascular smooth muscle, however this relaxation may be a functional response to increased metabolic demands or passive vasodilation (16) resulting from dimin- ished gastric tone. The third explanation of PFI, specific inhibitory fibers, gains its greatest support from the work reported by Martinson and Muren (42- 46). Although specific vagal inhibitory fibers to the stomach have , not been histologically or pharmacologically identified, vagal stimu- lation of the isolated atropinized guinea-pig stomach results in a more efficient relaxation than that produced by perivascular nerve stimulation (12). Campbell (l2) suggested that the post-ganglionic fibers in the vagal inhibitory pathway were similar to the intramural inhibitory fibers reported by Burnstock §t_a1, (l0). The specific 49 chemical transmitter in this inhibitory pathway has so far elluded investigators of the vagal inhibitory fibers; however, Bulbring and Gershon (9) have shown that 5-hydroxytryptamine is capable of character- istically duplicating vagally induced gastric inhibition in the isolated scopolaminized guinea pig stomach. From their study they concluded that S-HT participates preganglionically in this inhibitory pathway. The postganglionic mechanism still requires ellucidation. Effects of Atropine on Vagallinnduced Gastric Motility Although gastric facilitation is mediated through vagal preganglionic cholinergic fibers (38), increasing doses of atropine gradually converted vagally induced gastric facilitation to inhibition (Figs. 7,8). The important tone-response relationship of gastric motility (40,49) forces consideration of the effects vagotomy might have on basal tone. Past investigators fail to report consistent results of the effect of vagotomy on postoperative gastric tone. However, re- ferences to the importance of gastric tone on motility appear fre- quently (40,45,49). Harper et_al, (3l) and Martinson (44) report increased tone (as a result of bilateral vagotomy in anesthetized cats) which they interpret as evidence for a tonic central inhibition. Thomas and Komarov (58) report hypoactivity and hyposecretion in the unanesthetized dog, following vagotomy, which they interpreted as loss of tonic excitation. Although Alvarex gt_al, (2) report little change in rabbit gastric tone following unilateral vagotomy, bilateral vagotomy resulted in dilatation, diminution of tone, slow and weak peristalsis, and delayed emptying. In the present study, it was assumed that gastric atony (decreased resting and spontaneous contrac- 50 tile activity) existed in those animals in which atropine administration failed to yield vagally induced gastric inhibition. Vagally induced gastric inhibition could be produced in those animals exhibiting gastric atony by increasing resting tone with barium chloride. The failure of atropine administration to affect the level of basal tone in vagally decentralized stomach preparation subjected to low transmural pressure, confirms reports by Martinson (44) who suggested that the failure of atropine to lower basal tone indicated that basal tone was not maintained by continuous nerve discharge. Martinson (45) produced an increase in cat gastric volume (relaxation) only if relaxation was initiated from a state of high tone (low volume). This finding was similar to that of Langley (40) who obtained relaxation in rabbits with high gastric tone and facilitation when gastric tone was low. Existence of atropine resistant cholinergic fibers (34) may play an important role in gastric function. Atropine resistant after-contractions recorded in the present study, confirm those reported by Langley (40) and indicated the possible existence of atropine resistant cholinergic fibers, non-cholinergic gastric facilitory fibers, or a transient overshoot of a recovery response within the hyperpolarized neurocontractile complex. In light of findings that rabbits possess the ability to hydrolyze belladona alkaloids thereby making this species less susceptible to atropine blockade than some other, the validity of the present results along with other data collected from rabbits, may be questionable. However, not only is there species variation of atropinesterase, but enzymatic activity within species varies (3). Ambache et al. (3) report that only 45% of the animals tested exhibited 51 atropinesterase activity and in those animals having the most potent activity, lmg/kg of atropine was effective in abolishing the light reflex for at least 45 minutes. In this study, atropine dosage at least equaled l mg/kg, and all subsequent data was collected in less than 45 minutes. Therefore the use of these data appear to be justifiable in light of the findings of Ambach gt_al, (3). Effects of Barium Chloride on Vagally Induced Gastric Inhibition Vagally induced gastric inhibition in the atropinized animal is more easily obtained with presence of an elevated gastric tone (Figs. 9,12). Relaxation amplitude did not increase appreciably with increased frequency (Fig. 12), suggesting that under these conditions maximum activation of vagally induced inhibitory fibers was obtained at 2 cps. and that the inhibition was not a frequency dependent response. The frequency response curves, at submaximum intensities, reported by Jansson and Martinson (37) may be criticized on the point that stimulation of an entire nerve trunk at submaximum intensities fails to assure activation of every nerve fiber, therefore, in their preparations increasing frequency may be facil- itating recruitment by increasing intraneural current density. However, if supramaximum intensities are used, increasing frequency would elicit only frequency dependent responses. The failure of high impulse frequency to augment vagally induced gastric inhibition supports the suggestion that the neurocontractile mechanism may be hindered (40,59) by these supraphysiological frequencies (greater than 10 cps) (23,24, 25). The apparent effects of barium chloride on gastric motility, in- creased tonus and facilitation of spontaneous motility, may be due to 52 the ability of barium ions to substitute for sodium ions, thereby producing prolonged action potentials in B and C fibers (26), and stinnflating the release of acetylcholine from cholinergic nerves (21). Barium chloride at doses greater than 4 mg/kg often produced erratic spontaneous gastric motility. Acknowledging that muscle cells are most active at low membrane potentials (39,57) and that barium ions potentiate depolarization of certain nerve fibers (26), barium chloride might be affecting the muscle cell by lowering membrane potential. Gastric inhibition occurred not only in the gastric body, but also in the gastric antrum (Fig. 13). At increased tone levels, decreased ILP occurred simultaneously with relaxation in the gastric antrum and contraction in the gastric body (Fig. 13). Blair gt_§l, (7) report- ed small contractions superimposed on a background of decreased gastric tone of etherized cats. This superimposed pattern was also observed in this study, although infrequently. Neuroregulation of Vagally Induced Gastric Inhibition Little evidence has been reported ellucidating the neuro- regulatory mechanism for vagally induced gastric inhibition, or supporting those mechanisms hypothesized by past investigators. However, vagally induced gastric inhibition, recorded in the present study, occurred well within the physiological frequency range of less than 10 cps reported by Folkow (23) and Garry and GilleSpie (25). Campbell (12), working with in_vitrg_guinea pig stomachs, recorded maximum gastric relaxation at about 30 cps. The fact that 65% of maximum was reached at 3 cps strengthens the concept that autonomic control of gastrointestinal effector cells is maintained by low frequency impulses (23,24,25,39). In the present study impulse 53 frequencies greater than 32 cps depressed facilitation and impulses less than 10 cps produced inhibition in the atropinized rabbit. Vagally induced gastric inhibition was postulated to be adrenergically mediated because cholinergic blocking drugs abolished vagally induced gastric facilitation but had little effect on vagally induced gastric inhibiton (32,52); however, Bulbring and Gershon (9), confirming Martinson (43) and Campbell (12) abolished splanchnically induced relaxation with propranolol (lo-Sg/ml) and phenoxybenzamine (10'7g/m1), having little effect on vagally induced relaxation. Adrenergic inhibition of the cat's gastrointestinal tract has been reported to be postganglionic (39), but Martinson (44) suggests vagally induced gastric inhibition in the cat to be controlled by preganglionic fibers regulating the intrinsic nerve plexus and differing from adrenergically mediated inhibition (42,43,44). In the present study atropine failed to abolish vagally induced gastric inhibition suggesting a noncholinergic muscarinic site and possibly a ganglionic action; however, atropine given in doses 4-8 times greater than those abolishing facilitation failed to abolish inhibition suggesting that a noncholinergic ganglionic transmitter is involved. The indication of a noncholinergic ganglionic transmitter has been re- ported by Bulbring and Gershon (9). Using an ifi_vjtrg_preparation of the guinea pig stomach, they were able to obtain evidence indicating the possible complimentary role of acetylcholine (ACh) and 5-hydroxytryp- tamine (5-HT) in the vagal inhibitory pathway. Addition of 5-HT (10'sg/m1) to the bath medium produced a sustained gastric relaxation indicating that 5-HT is capable of activating an inhibitory mechanism. Chemical analysis of the bath medium revealed the release of 5-HT 54 during gastric vagal inhibition. Tetrodotoxin abolished the release suggesting that 5-HT originates from a neural tissue. When vagal induced gastric inhibition was abolished following cholinergic blockade combined with desensitization of 5-HT receptors, Bulbring and Gershon hypothesized that ACh and 5-HT participated synergistically as preganglionic transmitters with separate postganglionic receptors. SUMMARY Vagal influence on gastric motility was studied in anesthetized rabbits by means of cervical vagal stimulation with graded im- pulse frequencies (2-128 cps) at supramaximum intensities. Gastric antral and body contractile activities, monitored by extraluminal contractile force transducers, were correlated with gastric intraluminal pressure changes. Noncholinergic gastric inhibition elicited by vagal stimulation was studied following atropine administration, i.v., and tonus related effects of vagal stimulation were studied following varied doses of barium chloride. On the basis of the data obtained in the present investigation the following conclusions seem justified: a. Gastric facilitation (ILP increases, BCF decreases, and ACF increases, recorded simultaneously) had a consistent characteristic pattern, increasing in amplitude with increasing impulse frequencies, and had maxima at 16- 32 cps. Depression of gastric facilitation at impulse frequencies greater than those producing maximum facili- tation, suggests a decrease in nerve impulse conduction. b. Gastric postfacilitation inhibition increased in amplitude and duration at increasing impulse frequencies with a 55 56 pattern coinciding to vagally induced gastric facilitation, suggesting a relationship to the previous facilitory response. This postfacilitation inhibition, which failed to occur in the atropinized rabbit, may be the result of hyperpolarization of the neuromuscular complex, or_a neuromuscular inhibiting substance released during and related to the metabolic pro- CESS. Atropine converted the vagally induced gastric facilitation to inhibition, with impulse frequency of 2 cps often pro- ducing near maximum inhibition, suggesting a noncholinergic intensity-dependent, not frequency-dependent pathway. Barium chloride administration increased prestimulatory gastric tone and augmented vagally induced gastric in- hibition with the absolute inhibition obtained never exceed- ing postatropine level. The inhibition which was recorded in ILP and ACF tracings appeared to decrease at frequencies greater than 10 cps, suggesting that conduction through the neuromuscular inhibitory pathway is also hindered and that the facilitory and inhibitory pathways may have a common component. 10. REFERENCES Agostoni, E.J., H.E. Chinnock, D.M. Deburgh, and J.F. Murray. Functional and histological studies of the vagus nerve and its branches to the heart, lungs, and abdominal viscera in the cat. J. Physiol., London, 135: 182-205, 1957. Alvarez, w.c., K. Hosoi, A. Overgard, and H. Ascanio. The effects of degenerative section of the vagi and the splanchnics on the digestive tracts. Amer. J. Physiol., 90: 631-655, 1929. Ambache, N., L. Kavanaugh, and D.M. Shapiro. A rapid procedure for selection of atropinesterase-free rabbits. J. Physiol., London, 171: 1P-2P, 1964. Bass, P. and M.R. Callantine. Simultaneous recording of electrical and mechanical activity of the uterus in the unanesthetized animal. Nature, London, 203: 1365, 1964. Bayliss, N.M. and E.H. Starling. The movements and innervation of the small intestine. J. Physiol., London, 24: 99-143, 1899. Bercovitz, A. and F.T. Rogers. The influence of the vagi on gastric tonus and motility in the turtle. Amer. J. Physiol., 55: 323-338, 1921. Blair, E.L., A.A. Harper, C. Kidd, and T. Scratcherd. Post-activation potentiation of gastric and intestinal contractions in response to stimulation of the vagus nerves. J. Physiol., London, 148: 437-449, 1959. Bulbring, E. Smooth muscle of the alimentary tract. Modern Trends in Gastroenterology., Hoeber Medical Division, Harper and Row, New York: 1-11, 1958. Bulbring, E. and M.D. Gershon. 5-hydroxytryptamine participation in the vagal inhibitory innervation of the stomach. J. Physiol., London, 192: 823-846, 1967. Burnstock, G., G. Campbell, and M.J. Rand. The inhibitory innervation of the taenia of the guinea-pig caecum. J. Physiol., London, 182: 504-526, 1966. 57 ll. 12. 13. 14. 15. 16. 17. l8. 19. 20. 21. 58 Callantine, M.R., 0.P. O'Brien, B.L. Hindson, and R.J. Brown. Inhibition of uterine contractions in_vivo in the unanesthetized rabbit. Nature, London, 213: 507, 1967. Campbell, G. The inhibitory nerve fibers in the vagal supply to the guinea-pig stomach. J. Physiol., London, 185: 600-612, 1966. Cannon, H.E. and C.H. Lieb. The receptive relaxation of the stomach. Amer. J. Physiol. 27: XIII, 1911. Carlson, A.J., T.E. Boyd, and J.F. Pearcy. Studies on the visceral sensory nervous system. Amer. J. Physiol., 61: 14-41, 1922. Celander, 0. Are there any centrally controlled sympathetic inhibitory fibers to the musculature of the intestines? Acta Physiol. Scand., 47: 299-309, 1959. Chou, C.C. and J.M. Dabney. Interrelation of ileal wall compliance and vascular resistance. Amer. J. of Dig. Dis., 12: 1198-1208, 1967. Daugherty, R.M., J.B. Scott, J.M. Dabney, and F.J. Haddy. Local effects of 0 and CO2 on limb, renal, and coronary vascular resistancg. Amer. J. Physiol. 213: 1102-1110, 1967. Day, M.D. _ Effect of sympathomimetic amines on the blocking action of quanethidine, bretylium and xylocholine. Br. J. Pharmac. Chemother. 18: 421—439, 1962. Dominic, J.A. Contractile Activity of the Rabbit Uterus Monitored In Vivo with Extraluminal Force Transducers. Thesis for the Degree of M.S., Michigan State University, 1967. Dominic, J.A. and D.A. Reinke. . Extraluminal force transducer for in_vivo measurement of rabbit uterine contractile activity. Fertility and Sterility, 19: 945-953, 1968. Douglas, w.w. and J.M. Ritchie. 0n excitation of non-medullated afferent fibers in the vagus and aortic nerves by pharmacological agents. J. Physiol., London, 138: 31—43, 1957. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 59 Eccles, J.C. The Physiology of Nerve Cells. London: Oxford'University Press: 76-83, 206-211, 1957. Folkow, B. Impulse frequency in sympathetic vasomotor fibers correlated to the release and elimination of the transmitter. ‘ Acta Physiol. Scand., 25: 49-76, 1952. Folkow, B. Nervous control of the blood vessels. Physiol. Rev., 35: 629-663, 1955. Garry, R.C. and Gillespie, J.S. The responses of the musculature of the colon of the rabbit to stimulation, in vitro of parasympathetic and of the sympathetic outflows. J. Physiol., London, 128: 557-576, 1955. Goodman, L.S. and A. Gilman. The Pharmacological Basis of Therapeutics. 3rd ed., New York; The Macmillan Company, 804, 1965. Greeff, K., H. Kasperat, and W. Osswald. Paradoxe Wirkungen der elektriSchen Vagusreizun am isolierten Magen- und Herzvorhofpraparat des Meerschweinchens sowie deren Beeinflussung durch Ganglienblocker, Sympathi- colyticia, Reserpin and Cocain. Naunyn-Schmiedegerg's Arch. exp. Path. Pharmak. 243: 528- 545, 1962. [cited by Martinson, 1965 (44)] Grundfest, H. Excitation triggers in post-junctional cells. Physiological Triggers and Discontinuous Rate Processes. Ed. - Bullock, T.H., Baltimore Waverly Press, 1957. Haddy, F.J., C.C. Chou, J.B. Scott, and J.M. Dabney. Intestinal vascular responses to naturally occurring vasoactive substances. Gastroenterology, 52: 444-451, 1967. Haddy, F.J., H.W. Overbeck, and R.M. Daugherty. Peripheral vascular resistance. Annual Rev. of Med., 19: 167-194, 1968. Harper, A.A., C. Kidd, and T. Scratcherd. Vago-vagal reflex effects of gastric and pancreatic secretion and gastrointestinal motility. J. Physiol., London, 148: 417-443, 1959. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 60 Harrison, J.S. and B.A. McSwiney. The chemical transmitter of motor impulses to the stomach. J. Physiol., London, 87: 79-86, 1936. Hughes, J.R. Post-tetanic potentiation. Physiol. Rev., 38: 91-113, 1958. Hukovic, S., M.J. Rand and S. Vanov. Observations on an innervated preparation of rat urinary bladder. Br. J. Pharmac. Chemoth., 24: 178-188, 1965. Jacoby, H.I. An Extraluminal Gastrointestinal Smooth Muscle Contractile Force Transducer for Intact UnanesthetiZed Dogs. Doctoral Dissertation. University of Michigan, Ann Arbor, Michigan, 1963. Jacoby, H.I., F. Bass, and D.R. Bennett. In_vivo extraluminal contractile force transducer for gastr01ntestinal muscle. J. Appl. Physiology., 18: 658-665, 1963. Jansson, G. and J. Martinson. Some quantitative considerations on vagally induced relaxation of the gastric smooth muscle in the cat. Acta Physiol. Scand., 63: 351-357, 1965. Kay, A.W. and A.N. Smith. The effect of the ganglion-blocking methonium salts on gastric secretion and motility. Gastroenterology, 18: 503-517, 1951. Kewenter, J. The vagal control of jejunal and ileal motility and blood flow. Acta Physiol. Scand. Suppl., 251: 1-68, 1965. Langley, J.N. On inhibitory fibers in the vagus for the end of the oesophagus and the stomach. J. Physiol., London, 23: 407-414, 1898. Lloyd, D.P. Post-tetanic potentiation of response in monosynaptic reflex pathways of the spinal cord. J. Amer. Physiol., 33: 147-170, 1949. Martinson, J. The effect of graded stimulation of efferent vagal nerve fibers on gastric motility. Acta Physiol. Scand., 62: 256-262, 1965. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 61 Martinson, J. Vagal relaxation of the stomach. Experimental re-investigation of the concept of the transmission mechanism. Acta Physiol. Scand., 64: 453-462, 1965. Martinson, J. Studies on the efferent vagal control of the stomach. Acta Physiol. Scand., 65, Suppl. 255: 1-14, 1965. Martinson, J. and A. Muren. Studies on vagal excitation and inhibition of gastric motility. Acta Physiol. Scand. Suppl., 175: 103-104, 1960. Martinson, J. and A. Muren. Excitatory and inhibitory effects of vagus stimulation on gastric motility in the cat. Acta Physiol. Scand., 57: 309-316, 1963. May, W.F. The innervation of the sphincters and musculature of the stomach. J. Physiol., London, 31: 260-261, 1904, (Cited by McSwiney, 1931 . McSwiney, B.A. Innervation of the stomach. Physiol. Rev., 11: 478-514, 1931. McSwiney, B.A. and W.B. Spurrell. The gastric fibers of the vagus nerve. J. Physiol., London, 77: 447-458, 1933. McSwiney, B.A. and W.J. Wadge. Effects of variations in intensity and frequency on the contractions of the stomach obtained by stimulation of the vagus nerve. J. Physiol., London, 65: 350-356, 1928. Openchowski. , Centralbl f. d. med Wissensch, 31: 546, 1883. Zentralbl f. physiol. 3, 1, 1889. (Cited by Langley 1898, and Veach, 1925). Paton, W.D. and J.R. Vane. An analysis of the response of the isolated stomach to electrical stimulation and to drugs. J. Physiol., London, 165: 10-46, 1963. Reinke, D.A. Patterns and Parameters of Dog Gastrointestinal Contractile Activity Monitored by Extraluminal Force Transducers. Doctoral Dissertation. University of Michigan, Ann Arbor, Michigan, 1964. 54. 55. 56. 57. 58. 59. 60. 61. 62 Reinke, D.A., A.H. Rosenbaum, and D.R. Bennett. Patterns of dog gastrointestinal contractile activity monitored in vivo with extraluminal force transducers. Amer. J. D762 Dis., 12: 113-141, 1967. Rosenbaum, A.H., D.A. Reinke, and D.R. Bennett. In-vivo force, frequency, and velocity of dog gastrointestinal Eontractile activity. _ Amer. J. Dig. Dis., 12: 142-153, 1967. Scott, J.B., R.M. Daugherty, J.M. Dabney, and F. J. Haddy. Role of chemical factors in regulation of flow through kidney, hindlimb, and heart. Amer. J. Physiol., 208: 813-824, 1965. Texter, E.C., Jr. Motility in the gastrointestinal tract. J. Amer. Med. Assoc.,184: 640-646, 1965. Thomas, J.E. and S.A. Komarov. Physiological aspects of vagotomy. Gastroenterology, 11: 413-418, 1948. Veach, H.O. Studies of the innervation of smooth muscle. Amer. J. Physiol., 71: 229-264, 1925. Wedensky, W.N. Dans quella partie de 1'appareil neuromusculaire se produit 1' inhibition. Compt. Red. Acad. Sci. CXIII, 805-807, 1891. (Cited by Veach, 1925 . ' Wilson, V.J. Post-tetanic potentiation of polysynaptic reflexes of the spinal cord. J. Gen. Physiol., 39: 197-206, 1955. .. 3 111111111111 111111111 I5 '5 5 5 '5 I 5 5 .5 l 5 '- ”.9 “‘fufig- ~04.» - *1.- iv- .../yo —- ‘,.~