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Vagal stimulation-induced gastric acid secretion in the anesthetized rat
Journal of the Autonomic Nervous System, 16 (1986) 193-204
193
Elsevier JAN 00533
Vagal stimulation-induced gastric acid secretion in the anesthetized rat Hans-Rudolf Berthoud, Watson B. Laughton and Terry L. Powley The Laboratory of Regulatory Psychobiology, Department of Psychological Sciences, Purdue University, West Lafayette, IN 47907 (U.S.A.)
(Received October 10th, 1985) (Revised version received and accepted January 20th, 1986)
Key words: left cervical vagus - right cervical vagus - burst stimulation - vagal
control of cardiovascular function - on-line acid detection system gastric acid
Abstract
The dynamics of gastric acid secretion induced by electrical stimulation of the cervical vagus in the anesthetized rat were investigated using a continuous collection-titration system permitting high temporal resolution. Stimulation with pulse rates of 2 or 4 impulses/s (pps) produced maximal gastric acid responses with small cardiovascular effects. With continuous stimulation, secretion was sustained for at least 1 h. Frequency-response profiles suggested that the parietal cells are innervated predominantly by fine-caliber C-fibers. Continuous stimulation was 3 times as effective as stimulation in bursts of higher frequencies. The minimal latency for the onset of secretion was 2.6 rain at 4 pps, however, one- and two-rain stimulations still produced proportionate but delayed secretory responses. It is concluded that, with low frequency cervical vagus stimulation, the rat stomach preparation described and employed in the present experiment is a useful model for further studies on the interaction of neural and humoral factors on gastric acid secretion.
Correspondence: H.-R. Berthoud, Laboratory of Regulatory Psychobiology,Department of Psychological
Sciences, Purdue University, West Lafayette, IN 47907, U.S.A. 0165-1838/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
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Introduction
The rat has become the model for a number of studies investigating both the central [13,28,29] and peripheral [2,10,15,22,30,33] neural mechanisms controlling gastric acid secretion. However, several basic questions about both the organization and the dynamics of the neural controls of gastric acid secretion still remain unanswered in the rat (or other species). With the development of new techniques permitting rapid, sensitive monitoring of secretory responses in both anesthetized [8,11,32] and unanesthetized [31] rats, it is now feasible to evaluate some of the issues. In particular, a parametric analysis of the dynamics of gastric secretion in response to electrical stimulation of the vagus nerve can address questions concerning the fiber calibers of the preganglionic motor neurons innervating the parietal cells, the distribution of fibers from each vagal trunk to the stomach, and the mechanism of coupling of the parietal cells by the parasympathetic preganglionic fibers. The aim of the present study is, therefore, to provide a quantitative assessment of gastric acid secretion produced by electrical stimulation of the cervical vagi in the rat. The analysis includes a frequency-response profile and an examination of stimulation in bursts, a technique which has recently received much attention [1,9]. In addition, the analysis also examines onset latencies and the pattern of secretion in response to discrete bouts of stimulation, response profiles with sustained electrical stimulation, and the relative magnitudes of the secretory responses elicited by stimulation of the two cervical vagi individually or both simultaneously. Materials and Methods
Animals. Male Sprague-Dawley rats of approximately 280 g b. wt. purchased from Harlan Industries (Indianapolis) were kept on rat laboratory ehow (Wayne) and water for at least 3 weeks prior to the experiment. Lights were on from 08.00 to 20.00 h and the room temperature varied between 20 and 25°C. Gastric fistula pre-implantation. A gastric drainage fistula was permanently implanted into the rumen of the stomach 3 weeks before the acute experiment in order to minimize the surgical stress at the time of the experiment. The technical aspects and implantation procedure for the cannula are explained in detail elsewhere [31]. Acute surgical procedure. Overnight food-deprived rats with gastric fistulas were initially anesthetized with 60 mg/kg of sodium thiopental (Pentothal, Abbot) and anesthesia was then maintained with a mixture of 30 mg/kg a-chloralose and 300 mg/kg Urethane i.v., supplemented as needed. A tracheal fistula (5 cm length of PE 200 tubing) was implanted to ease respiration and to facilitate connection of the animal to a respirator in the few cases of excessive cardioinhibition associated with vagal stimulation. A catheter (PE50) was introduced into the left femoral artery for continuous monitoring of blood pressure (Statham 23Db pressure transducer) and heart rate
195 (Grass Model 7P4F Tachograph). The right jugular vein was exposed, and catheters for blood withdrawal and i.v. infusions were implanted. Electrical stimulation of the nerve. The left or right or both cervical vagi were exposed low in the neck by displacing the ventrally located tissues, and cuff-type silver electrodes [24] were fitted around the distal stump of the cut nerve. The assembly was suspended in paraffin oil. Trains of rectangular pulses from a Grass $48 stimulator were passed through a stimulus-isolation unit and a 33 kI2 resistor in series with the electrode. The voltage drop across the 33 k~2 resistor was measured with an oscilloscope and expressed in milliamperes current through the electrode loop. In most experiments, the anode was located distally on the nerve, but no difference was found for cathodal current. Current intensity (1.0 mA) and pulse duration (1 ms) were held constant. For bilateral stimulation, the two electrodes were connected in series. The stimulus frequency-response relationship was determined using continuous stimulus trains of 10 rain at 1, 2, 4, 8, and 16 impulses per second (pps). For the continuous vs burst stimulation experiment, the vagus nerve was stimulated in random order with continuous stimulus trains (2400 impulses as 4 pps for 10 min) and with burst stimulation patterns (2400 impulses as (a) 40 pps for one out of 10 s for 10 min, (b) 16 pps for 2.5 out of 10 s for 10 min, or (c) 8 pps for 5 out of 10 s for 10 min). Stimulation sequences were altered, as appropriate. (see below). Experimentalprotocol. After the surgical procedures which took an average of 79 (60-90) min, the animal, with its gastric fistula open, was situated in a prone position and the stomach rumen was irrigated with prewarmed saline (pH = 6.0) by means of a small 'showerhead' device situated at the inner edge of the fistula, at a rate of approx. 30 ml/min. The perfusate then passively drained through the large diameter (6 mm) rumen of the fistula and was;collected into a pH-stat bath set for end-point titration at the pH of the saline (for a full description see ref. 31). At the end of the surgical period, a continuous i.v. infusion of a mixture consisting of glucose, the a-adrenergic receptor blocker phentolamine (Regitine, Ciba-Geigy), and the fl-adrenergic receptor blocker, d,l-propranolol (Sigma) was started. Glucose was infused to counteract hypoglycemia which is caused by the continuous infusion of the adrenergic blockers, and to maintain glucose concentration at a relatively uniform level, since plasma glucose modulates gastric acid secretion [17]. Various glucose infusion rates from 0 - 7 m g - k g -1. m i n - I were used in order to obtain plasma glucose concentrations of approx. 130 m g / 1 0 0 ml. The adrenergic blockers were infused to suppress sympathetic activity produced in response to surgical trauma a n d / o r vagal stimulation. Sympathetic activation has been shown to significantly decrease basal and vagally stimulated gastric acid secretion in the rat [33]. Infusion rates for both a and fl blockers were 45 ~ g - k g -1- rain-1 (15 /~l/min). Infusions of the blocker-plus-glucose solution began approximately 30 min before the first vagal stimulation, allowing heart rate and blood pressure to stabilize. Blood samples were taken before and during stimulation, and plasma glucose concentrations were measured on a Beckman Glucose Analyzer using glucose oxidase. Sequences of stimulations as to frequency or duration or use of the left vs the
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fight vs bilateral vagal nerves were altered as appropriate. This design was chosen because pilot experiments suggested that the acid output in response to the second of two successive 10 rain/8 pps stimulations was significantly larger and that, in some cases, the response of the stomach decreased after several stimulation periods. Data analyses. Analyses of variance were applied for the stimulus frequency-response curves of gastric acid and mean arterial blood pressure, and Student's t-tests were used throughout for comparisons of individual means.
Results
Basal state of the animals. Intravenous infusion of the solution containing the aand fl-adrenergic blockers markedly decreased both mean arterial blood pressure (from 147 -t- 3 to 90 + 2 mm Hg, P < 0.001, n = 45) and heart rate (from 347 _+ 10 to 264 :i: 6 beats per minute (BPM), P < 0.001, n = 36) before and 30 rnin after the start of infusion, respectively. The mean basal gastric acid secretion rate (after blocker infusion) was 0.16 + 0.01 /~Eq/min (121 experiments in 34 rats). The mean basal plasma glucose concentration with adrenergic blockers was 129 _+ 2 m g / 1 0 0 ml (62 experiments in 34 rats). The basal acid secretion was correlated with neither the basal mean arterial blood pressure (r = - 0 . 1 0 , u = 63) nor the basal plasma glucose concentration (r = + 0,15, u = 63). Total 'stimulated acid output' and peak secretion rate. Illustrative records of successive 10-min stimulations of the cervical vagus nerve with varying frequencies for two rats are shown in Fig. 1. It can be seen that, after a latency of up to several minutes, the acid secretory rate rises to a peak and then returns more slowly to baseline levels after the termination of stimulation. Latencies, integrated incremental acid secretion (i.e. total stimulated gastric acid response until return to baseline rate --usually terminated within 25 rain from the start of stimulation), maximal secretion rate, and the occurrence of an off-response (transient increase in secretion rate immediately following the end of stimulation), were all computed for the 13 rats with left and 11 rats with right cervical vagal stimulations. Fig. 2 illustrates the stimulus frequency-response relationship for the total stimulated acid output. As can be seen, stimulations with 2 pps produced a near-maximal gastric acid response. The maximal response occurred at 4 pps. Stimulation with either 8 or 16 pps produced smaller responses. In contrast to this gastric acid response, the mean decrease (over the t0-min stimulation period) of the mean arterial blood pressure was small and not significant with stimulation at 2 pps, increased monotonically with an increase in frequency, and reached the highest value with stimulation at 16 pps (Fig, 2, lower panel). Correlation coefficients between the mean decrease in arterial blood pressure and the total stimulated acid response for the 8- and 16-pps stimulations were low and non-significant (rspps = 0.047, and rl6pp s = 0.083).
For the data summarized in Fig. 2, an analysis of variance (2-way with repeated measures) applied to the incremental gastric acid output revealed that frequency of
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Fig. 1. The effect of successive 10-min electrical stimulations of the left (upper panel) or the fight (lower panel) cervical vagus with varying frequencies (1, 2, 4, 8 or 16 pulses per second) on gastric acid secretion rate of two rats. Acid secretion rate was derived by differentiation of the original cumulative recordings obtained from an on-line perfusion/titration system. The filled circles in the lines indicate the 25-min periods yielding the incremental acid outputs of Fig. 2. For details, see Materials and Methods section.
stimulation had a very reliable effect (F4,88 = 18.82, P < 0.001), and right cervical vagal stimulation was superior to left (F1,22 = 5.00, P < 0.05). The frequency-response profile was the same for both left and right cervical vagi (F4,88 = 1.34, P > 0.1). Frequency of stimulation also had a large effect on mean arterial blood pressure (MABP) (F4,88 = 20.6, P < 0.001), and the right cervical vagus was marginally more effective than the left (Fl,22 = 4.08, P < 0.056). Parallel effects of frequency and the right vs. the left nerve stimulation were also observed for peak gastric acid secretion rates during each 2.5-min interval (data are not shown). Off-response. A brief acceleration of acid secretion was frequently observed immediately after the termination of the stimulation (cf. the response to 1, 4, 8 and 16 pps stimulations in the upper panel of Fig. 1). This off-response pattern was observed in 59% of all left and 42% of all right cervical vagal stimulations. The percentages of occurrence were lowest at 1 pps (40% and 35% for left and right stimulation, respectively) and highest at 16 pps (80% and 67%, respectively). Latency. The latency between the onset of stimulation and the detection of the first 0.1 /tEq of acid above basal secretion was affected by the frequency of stimulation, as illustrated in Fig. 3. Latency decreased gradually with increasing stimulus frequency up to 4 pps, and then showed no further decrease with 8 or 16 pps. The latency at 4 pps was significantly shorter than with 1 pps (2.6 ___0.2 vs 4.1 + 0.4 rain; t = 3.53, df = 40, P < 0.01). Since there was no difference between left and right CV stimulation, the data for the two sides were pooled. Another series of experiments with stimulation train durations of 1, 2, 4 and 10 min was conducted in two rats with left and two rats with right cervical vagal
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Fig. 2. Stimulus frequency-response relationship of right ( i t ) and left (& i ) cervical vagal stimulation-induced gastric acid output and decrease in mean arterial blood pressure (depressor effect) of urethane/chloralose anesthetized rats. The left (n = 13) and the right (n = 11) cervical vagal nerves were stimulated with 1, 2, 4, 8 and 16 pps, for 10 min as in Fig. 1, in balanced order. Each point represents the mean + S.E.M. of 8-13 experiments. The points at zero pps were obtained from an additional 10 animals treated similarly but without turning the stimulator on. Analyses of variance with repeated measures showed that RCV stimulation produced a significantly larger acid acid output than left stimulation (F1.22 = 5.0, P < 0.05) and t-tests showed that this difference was significant at 2 pulses/s (*, P < 0.05). electrodes. T h e t y p i c a l effect of 1 m i n of 4 p p s s t i m u l a t i o n is shown in the inset of the r i g h t h a n d p a n e l in Fig. 3. N o acid was d e t e c t e d during, or for 3 m i n after the 1-min stimulation. T h e r e was a response, however, b e t w e e n 4 a n d 15 m i n after the s t i m u l a t i o n train. T h e p a n e l o n the right of Fig. 3 s u m m a r i z e s relative p e r c e n t a g e increases o f acid o u t p u t as a f u n c t i o n o f s t i m u l a t i o n train d u r a t i o n , w h e n the r e s p o n s e to 2 m i n of s t i m u l a t i o n is t r e a t e d as 100%. T h e m e a n a c i d r e s p o n s e o f 4 a n i m a l s to 1 min o f s t i m u l a t i o n a m o u n t e d to 45.7 + 4.6% of the r e s p o n s e to 2 m i n stimulation. Bilateral nerve stimulation. S t i m u l a t i o n o f b o t h cervical vagi c o n c u r r e n t l y for 10 m i n at 2 p p s in 4 o t h e r rats p r o d u c e d a significantly larger gastric a c i d r e s p o n s e t h a n s t i m u l a t i o n of either t r u n k alone, b u t a m a r g i n a l l y significantly ( P = 0.05, d f = 3) lower r e s p o n s e t h a n the s u m o f i n d e p e n d e n t left a n d right stimulation. I f gastric a c i d o u t p u t is e x p r e s s e d in p e r c e n t a g e o f t h e s u m of i n d e p e n d e n t left a n d f i g h t s t i m u l a tions (100%), b i l a t e r a l s t i m u l a t i o n p r o d u c e d a n a c i d o u t p u t o f 88 + 4% (Fig. 4).
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Fig. 3. Left panel: effect of frequency of cervical vagal stimulation on latency of gastric acid secretion. Since no significant differences were found between right and left stimulation, the data were pooled and each bar represents the mean + S,E.M. of 18-23 experiments. *, P < 0.01, t-test. Right panel: effect of duration of cervical stimulation on gastric acid output. Rats with right (n = 2) and left (n = 2) cervical vagal electrodes were stimulated with 4 pps for 1, 2, 4 and 10 min in an ascending/descending balanced order. Each point is the mean _+S.E.M. of 4 animals. Incremental gastric acid output is expressed as a percentage of the 2-min stimulation. The inset shows the secretion rate of a typical 1-min stimulation experiment; note that acid secretion does not start until approximately 3 min following cessation of stimulation.
Stimulation in bursts. When the number of impulses (as well as pulse duration and pulse amplitude) given to the vagus nerve was kept constant, stimulation in burst patterns was less effective than continuous stimulation with trains at 4 pps in eliciting a gastric response (Fig. 5). There were no significant differences between bursts of 8, 16 or 40 pps. Burst stimulation had the same sharply reduced efficacy for gastric acid secretion with both the left and right vagal stimulation.
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Fig. 5. Comparison of the incremental gastric acid output of anesthetiz~ rats in response to stimulation of the right (RCV) or the left (LCV) cervical vagus at either 4 pps continuously, or in bursts of 8 pps for 5 s, 16 pps for 2.5 s and 40 pps for 1 s at 10-s intervals for 10 min. Note that 2400 simulation pulses were given for each mode of stimulation. The open and closed circles represent the range of acid output of individual determinations of two separate animals for each side of stimulation, while the bars indicate the means of the two rats on each side.
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Prolonged stimulation. With 60-min trains of stimulation of the right or the left cervical vagus (CV) at 4 pps, the maximal rate of gastric acid secretion was reached between 15 and 20 min and was maintained until the end of the 1-h stimulation period (Fig. 6). While the left CV stimulation produced slightly less gastric acid output during the rising phase, the same maximal output levels were reached as with the right CV stimulation. If the right CV, after a pause of approximately 40 min, was stimulated a second time for 60 min, maximal gastric acid output was reached much more rapidly, between 5 and 10 min, and acid output fell to about half maximal during the last 20 min of stimulation (Fig. 6).
Discussion Using electrical stimulation of the vagus nerve, we have presented the first quantitative and parametric analyses of gastric acid secretion in the rat. We have found that electrical stimulation at optimal parameters produced maximal rates of acid secretion that compared well with gastrin- and histamine-stimulated secretion rates [6, and personal observations]. The magnitude and reproducibility of this response suggests that this protocol would be a useful one for tests of vagotomy or other circumstances of impaired vagal function. These stimulus parameters also yield a sustained secretory response in this experimental preparation, thus making the technique a potentially powerful one for assessing the effect of antisecretory agents. The stimulus frequency-response curve for gastric acid output for the rat was characterized by a half-maximal secretory response at approximately 1 pps, a maximal response at 2-8 pps and a below-maximal output at 16 pps. This finding is consistent with the conclusion that gastric parietal cells in the rat are innervated by fine-caliber C-fibers. Such a hypothesis would also be consistent with the decrease in secretory response at 16 pps, since some blocking of nerve impulses would be expected at higher frequencies of stimulation [e.g. ref. 27]. Blocking of the putative C-fiber population would also explain the sharply reduced efficacy of burst pattern stimulation at higher frequencies. Finally, the idea that vagal axons mediating the gastric acid response are of a smaller caliber is in general agreement with the findings that there are only a few ( - 1%) myelinated fibers in the abdominal vagi [7] and that only slow conduction velocities of approximately 1 m / s , corresponding to C-fibers, were found electrophysiologically at this level [25]. In contrast, the cardiac vagal efferents have been found to have conduction velocities in the range of B-fibers in the cat [16] and most probably in all mammals [26]. The inhibition of secretion at higher stimulus frequencies might also be explainable at least in part by a suppressive effect of decreased MABP (with corresponding decrease in gastric mucosal blood flow) on gastric secretion with increasing frequency. This explanation is not supported, however, by the low and insignificant correlations between the magnitude of the depressor effect and acid secretion at both 8 and 16 pps in the present experiment, and by the finding that the two responses are largely dissociable [19]. Alternatively, it is possible that higher frequency stimulation coactivated some other response which had an inhibitory effect on gastric acid
202
secretion, such as vagally released somatostatin [2]. Although this issue is not directly addressed, our observations that the large percentage of experiments exhibited an overshoot of gastric acid secretion after the termination of stimulation occurred at 16 pps could be taken as support for such an interpretation. As a choice of optimal parameters for additional experimentation on rat gastric acid secretion, stimulation of the cervical vagus at 2-4 pps would seem ideal. These frequencies minimize the involvement of cardioinhibitory responses and make it feasible to stimulate in the neck, thus avoiding the additional trauma and need for artificial respiration that are required for stimulation of the thoracic vagal nerve. Stimulation with burst patterns at higher frequencies would not seem appropriate where the goal is maximal secretory rates. The fight cervical vagal nerve stimulation was slightly but consistently more effective than the left in producing gastric acid secretion in all experiments. Although we cannot completely rule out the possibility, it seems unlikely that the fistula implantation damaged more parietal cells innervated by the left CV, since the stab wound was made in the midline (greater curvature) of the rumen and at least two weeks recovery were allowed. This significantly greater acid output with the right cervical vagus nerve stimulation could be an indication that the right CV projects to a larger population of parietal cells than the left CV. Consistent with this explanation, there are an estimated 50% more fibers in the posterior gastric branches than in the anterior gastric branches of the vagus [20] and correspondences between the right cervical vagus and the posterior gastric branches and between the left cervical vagus and the anterior gastric branches have been demonstrated both electrophysiologically [25] and anatomically [18]. Species differences seem to exist insofar as in the cat left CV stimulation produced a gastric acid response approximately twice as large as right CV stimulation [3]. Comparisons between unilateral and bilateral CV stimulation address the question of overlap of the peripheral projection fields. If the left and the right CV each innervate separate populations of parietal cells, then one would expect the gastric acid response to bilateral stimulation to be equal to the sum of the left plus right stimulation. The present finding that additivity was 88%, suggests that there is a small population of parietal cells which is innervated by both vagi or, in other words, there is a 12% overlap of innervation. The finding of substantial additivity is consistent with the evidence of Legros and Griffith [14] suggesting that different vagal branches innervate different fields of the rat's gastric mucosa. The present findings with respect to overlap also compare well with the 83% additivity (or 17% overlap) observed in the cat [4]. These innervation patterns, however, contrast with that reported for the dog, in which stimulation of each cervical vagus elicited gastric secretion from the entire fundus and corpus (both sides) of the split stomach preparation [21]. As the present results show, there is a considerable latency of at least 2.6 rain between the start of stimulation (4 pps) and a minimal increase of acid secretion. The latency is clearly a function of the physiological response, since the technical latency of the perfusion/pH star system was approximately 10 s. This slow response contrasts with the fast ( < 1 min) release of insulin into the circulation upon cervical
203 vagal s t i m u l a t i o n [5]. O n e e x p l a n a t i o n for the longer latency of the gastric acid response is that secretion d e p e n d s o n the m e t a b o l i s m - c o u p l e d , e n z y m e - a c t i v a t e d ion exchanges a n d t r a n s f o r m a t i o n of tubulovesicles [23] rather t h a n exocytosis of vesicles from a readily releasable pool. I n addition, the transport a n d diffusion of the secreted acid to the surface of the mucosal rugae, where it can be washed away by the perfusion system, m a y take a significant a m o u n t of time. The fact that the 1-min s t i m u l a t i o n p r o d u c e d a p p r o x i m a t e l y half of the acid o u t p u t of a 2 - m i n s t i m u l a t i o n suggests that it is n o t a threshold a m o u n t of t r a n s m i t t e r released, b u t rather the release a n d t r a n s p o r t m e c h a n i s m itself that is responsible for the 2.6-min m i n i m a l latency to first detection. Finally, the time required for the secretory response to r e t u r n to basal levels after stimulus cessation m u s t reflect other aspects of the physiology of parietal cell secretion. Since vagal activation not only directly activates secretion b u t also potentiates responsiveness to h o r m o n e activation [12], it is conceivable that the time course after the cessation of a stimulus reflects the dissipation of a h o r m o n a l response engaged b y vagal stimulation.
Acknowledgements W e would like to t h a n k E d w a r d Fox, James Prechtl a n d Meredith W a l g r e n for their c o m m e n t s o n a n earlier draft of this paper. This research was s u p p o r t e d by N I H G r a n t AM27627 to T.L.P.
References 1 Adrian, T.E., Bloom, S.R. and Edwards, A.V., Neuroendocrine responses to stimulation of the vagus nerves in bursts in conscious calves, J. Physiol., (London) 344 (1983) 25-35. 2 Aliho, S.F., Garcia, D. and Uvn~s-Moberg, K., On the interaction between intragastric pH and electrical vagal stimulation in causing gastric acid secretion and intraluminal release of gastrin and somatostatin in anesthetized rats, Acta Physiol. Scand., 117 (1983) 491-495. 3 Bauer, R.F. and Brooks, F.P., Acid secretion and gastrin release during electrical vagal stimulation in anesthetized cats, Fed. Proc., Fed. Am. Soc. Exp. Biol., 35 (1976) 217 (Abstract). 4 Bauer, R.F., Electrically induced vagal stimulation of gastric acid secretion. In F.P. Brooks and P.W. Evers (Eds.), Nerves and the Gut, Charles B. Slack, Thorofare, N J, 1977, pp. 86-95. 5 Berthoud, H.-R., Niijima, A., Sauter, J.-F. and Jeanrenaud, B., Evidence for a role of the gastric, coeliac and hepatic branches in vagally stimulated insulin secretion in the rat, J. Auton. Nert,. Syst., 7 (1983) 97-110. 6 Emhs, S., Nylander, G., Wallin, B. and Wallin, C., Inhibition of pentagastrin-stimulated gastric acid secretion by acid perfusion of the duodenum in chronic gastric fistula rats, Scand. J. Gastroenterol., 18 (1983) 685-690. 7 Gabella, G. and Pease, H.L., Number of axons in the abdominal vagus of the rat, Brain Res., 58 (1973) 465-469. 8 Ghosh, M.N. and Schild, H.O., Continuous recording of gastric acid secretion in the rat. Br. J. Pharrnacol., 13 (1958) 54-61. 9 Grundy, D. and Scratcherd, T., Effect of stimulation of the vagus nerve in bursts on gastric acid secretion and motility in the anaesthetized ferret, J. Physiol. (London), 333 (1983) 451-461. 10 Hhkanson, R., Hedenbro, J., Liedberg, G. and Vallgren, S., Effect of vagotomy on gastric acid secretion in the rat, Acta Physiol. Scand., 115 (1982) 135-139.
204 11 Halter, F., Galleazzi, R., Leuenberger, J. and Smith, G.M., Intragastric titration of acid in the perfused rat stomach preparation following parenteral and intraluminal stimulation, Eur. J. Pharmacol., 43 (1977) 247-252. 12 Hirschowitz, B.I., The vagus and gastric secretion. In F.P. Brooks and P.W. Evers (Eds.), Nerves and the Gut, Charles B. Slack, Thorofare, NJ, 1977, pp. 96-118. 13 Ishikawa, T., Nagata, M. and Osumi, Y., Dual effects of electrical stimulation of ventromedial hypothalamic neurones on gastric acid secretion in rats, Am. J. Physiol., 245 (1983) G265-269. 14 Legros, G. and Griffith, C., The anatomic basis for the variable adequacy of incomplete vagotomy, Ann. Surg., 168 (1968) 1030-1034. 15 Martindale, R., Kaufman, G.L., Levin, S., Walsh, J.H. and Yamada, T., Differential regulation of gastrin and somatostatin secretion from isolated perfused rat stomachs, Gastroenterology, 83 (1982) 240-244. 16 McAllen, R.M. and Spyer, K.M., Two types of vagal preganglionic motorneuronesprojecting to the heart and lungs, J. Physiol. (London), 282 (1978) 353-364. 17 Moore, J.G., Gastric acid suppression by intravenous glucose solutions, Gastroenterology, 64 (1973) 1106-110. 18 Norgren, R. and Smith, G.P., The central distribution of vagal subdiaphragmatic branches in the rat, Proc. Soc. Neurosci., 9 (1983) 9:611 (Abstract). 19 Perry, M.A., Haedicke, G.J., Bulkley, G.B., Kvietys, P.R. and Granger, D.N., Relationship between acid secretion and blood flow in the canine stomach: role of oxygen consumption, Gastroenterology', 85 (1983) 529-534. 20 Powley, T.L., Prechtl, J.C., Fox, E.A. and Berthoud H.-R., Anatomical considerations for surgery of the rat abdominal vagus: distribution, paraganglia and regeneration, J. Auton. Nerv. Syst., 9 (1983) 79-97. 21 Pritchard, G.R., Griffith, C.A. and Harkins, H.N., A physiologic demonstration of the anatomic distribution of the vagal system to the stomach, Surg. Gynecol. Obstet, 126 (1968) 791-798. 22 Rozr, C., Dubrasquet, M., Chariot, J. and Vaille C., Methadone inhibition of vagally induced pancreatic and gastric secretions in rats: central and peripheral sites of action, Eur. J. Pharmacol., 78 (1982) 271-278. 23 Sachs, G., Chang, C.B., Rabon, E., Shackman, R., Saran, H.M. and Saccomani, G., Metabolic and membrane aspects of gastric H + transport, Gastroenterology, 73 (1977) 931-940. 24 Sauter, J.-F., Berthoud, H.-R. and Jean.renand, B., A simple electrode for intact nerve stimulation a n d / o r recording in semi-chronic rats, Pflfigers Arch., 397 (1983) 68-69. 25 Sauter, J.-F., Niijima, A., Berthoud, H.-R. and Jeanrenaud, B., Vagal neurons and pathways to the rat's lower viscera: an electrophysiological study, Brain Res. Bull., 11 (1983) 487-491. 26 Spyer, K.M., Neural organization and control of the baroreceptor reflex, Rev. Physiol. Biochem. Pharmacol., 88 (1981) 23-124. 27 Swett, J.E. and Bourassa, C.M., Electrical stimulation of peripheral nerve. In M.M. Patterson and R.P. Kesner (Eds.), Electrical Stimulation Research Techniques, Academic Press, New York, 1981, pp. 244-298. 28 Tachr, Y., M~ki, W., Rivier, J., Vale, W. and Brown, M., Central nervous system inhibition of gastric secretion in the rat by gastrin-releasing peptide, a mammalian bombesin, Gastroenterology, 81 (1981) 298-302. 29 Tepperman, B.L. and Evered, M.D., Gastrin injected into the lateral hypothalamus stimulates secretion of gastric acid in rats, Science, 209 (1980) 1142-1143. 30 Valgren, S., Ekelund, M. and Hhkanson, R., Mechanism of inhibition of gastric acid secretion by vagal denervation in the rat, Acta Physiol. Scand., 119 (1983) 77-80. 31 Weingarten, H.P. and Powley, T.L., A new technique for the analysis of phasic gastric acid responses in the unanesthetized rat, Lab. Anita. Sci., 30 (1980) 673-680. 32 Winship, D.H. and Shuck, T.J., Continuous recording of gastric acid output in the rat, J. Lab. Clin. Med., 81 (1973) 961-966. 33 Yokotani, K., Muramatsu, I., Fujiwara, M: and Osumi, Y., Effects of the sympathoadrenal system on vagally induced gastric acid secretion and mucosal blood flow in rats, J. PharmacoL Exp. Ther., 224 (1983) 436-442.
193
Elsevier JAN 00533
Vagal stimulation-induced gastric acid secretion in the anesthetized rat Hans-Rudolf Berthoud, Watson B. Laughton and Terry L. Powley The Laboratory of Regulatory Psychobiology, Department of Psychological Sciences, Purdue University, West Lafayette, IN 47907 (U.S.A.)
(Received October 10th, 1985) (Revised version received and accepted January 20th, 1986)
Key words: left cervical vagus - right cervical vagus - burst stimulation - vagal
control of cardiovascular function - on-line acid detection system gastric acid
Abstract
The dynamics of gastric acid secretion induced by electrical stimulation of the cervical vagus in the anesthetized rat were investigated using a continuous collection-titration system permitting high temporal resolution. Stimulation with pulse rates of 2 or 4 impulses/s (pps) produced maximal gastric acid responses with small cardiovascular effects. With continuous stimulation, secretion was sustained for at least 1 h. Frequency-response profiles suggested that the parietal cells are innervated predominantly by fine-caliber C-fibers. Continuous stimulation was 3 times as effective as stimulation in bursts of higher frequencies. The minimal latency for the onset of secretion was 2.6 rain at 4 pps, however, one- and two-rain stimulations still produced proportionate but delayed secretory responses. It is concluded that, with low frequency cervical vagus stimulation, the rat stomach preparation described and employed in the present experiment is a useful model for further studies on the interaction of neural and humoral factors on gastric acid secretion.
Correspondence: H.-R. Berthoud, Laboratory of Regulatory Psychobiology,Department of Psychological
Sciences, Purdue University, West Lafayette, IN 47907, U.S.A. 0165-1838/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
194
Introduction
The rat has become the model for a number of studies investigating both the central [13,28,29] and peripheral [2,10,15,22,30,33] neural mechanisms controlling gastric acid secretion. However, several basic questions about both the organization and the dynamics of the neural controls of gastric acid secretion still remain unanswered in the rat (or other species). With the development of new techniques permitting rapid, sensitive monitoring of secretory responses in both anesthetized [8,11,32] and unanesthetized [31] rats, it is now feasible to evaluate some of the issues. In particular, a parametric analysis of the dynamics of gastric secretion in response to electrical stimulation of the vagus nerve can address questions concerning the fiber calibers of the preganglionic motor neurons innervating the parietal cells, the distribution of fibers from each vagal trunk to the stomach, and the mechanism of coupling of the parietal cells by the parasympathetic preganglionic fibers. The aim of the present study is, therefore, to provide a quantitative assessment of gastric acid secretion produced by electrical stimulation of the cervical vagi in the rat. The analysis includes a frequency-response profile and an examination of stimulation in bursts, a technique which has recently received much attention [1,9]. In addition, the analysis also examines onset latencies and the pattern of secretion in response to discrete bouts of stimulation, response profiles with sustained electrical stimulation, and the relative magnitudes of the secretory responses elicited by stimulation of the two cervical vagi individually or both simultaneously. Materials and Methods
Animals. Male Sprague-Dawley rats of approximately 280 g b. wt. purchased from Harlan Industries (Indianapolis) were kept on rat laboratory ehow (Wayne) and water for at least 3 weeks prior to the experiment. Lights were on from 08.00 to 20.00 h and the room temperature varied between 20 and 25°C. Gastric fistula pre-implantation. A gastric drainage fistula was permanently implanted into the rumen of the stomach 3 weeks before the acute experiment in order to minimize the surgical stress at the time of the experiment. The technical aspects and implantation procedure for the cannula are explained in detail elsewhere [31]. Acute surgical procedure. Overnight food-deprived rats with gastric fistulas were initially anesthetized with 60 mg/kg of sodium thiopental (Pentothal, Abbot) and anesthesia was then maintained with a mixture of 30 mg/kg a-chloralose and 300 mg/kg Urethane i.v., supplemented as needed. A tracheal fistula (5 cm length of PE 200 tubing) was implanted to ease respiration and to facilitate connection of the animal to a respirator in the few cases of excessive cardioinhibition associated with vagal stimulation. A catheter (PE50) was introduced into the left femoral artery for continuous monitoring of blood pressure (Statham 23Db pressure transducer) and heart rate
195 (Grass Model 7P4F Tachograph). The right jugular vein was exposed, and catheters for blood withdrawal and i.v. infusions were implanted. Electrical stimulation of the nerve. The left or right or both cervical vagi were exposed low in the neck by displacing the ventrally located tissues, and cuff-type silver electrodes [24] were fitted around the distal stump of the cut nerve. The assembly was suspended in paraffin oil. Trains of rectangular pulses from a Grass $48 stimulator were passed through a stimulus-isolation unit and a 33 kI2 resistor in series with the electrode. The voltage drop across the 33 k~2 resistor was measured with an oscilloscope and expressed in milliamperes current through the electrode loop. In most experiments, the anode was located distally on the nerve, but no difference was found for cathodal current. Current intensity (1.0 mA) and pulse duration (1 ms) were held constant. For bilateral stimulation, the two electrodes were connected in series. The stimulus frequency-response relationship was determined using continuous stimulus trains of 10 rain at 1, 2, 4, 8, and 16 impulses per second (pps). For the continuous vs burst stimulation experiment, the vagus nerve was stimulated in random order with continuous stimulus trains (2400 impulses as 4 pps for 10 min) and with burst stimulation patterns (2400 impulses as (a) 40 pps for one out of 10 s for 10 min, (b) 16 pps for 2.5 out of 10 s for 10 min, or (c) 8 pps for 5 out of 10 s for 10 min). Stimulation sequences were altered, as appropriate. (see below). Experimentalprotocol. After the surgical procedures which took an average of 79 (60-90) min, the animal, with its gastric fistula open, was situated in a prone position and the stomach rumen was irrigated with prewarmed saline (pH = 6.0) by means of a small 'showerhead' device situated at the inner edge of the fistula, at a rate of approx. 30 ml/min. The perfusate then passively drained through the large diameter (6 mm) rumen of the fistula and was;collected into a pH-stat bath set for end-point titration at the pH of the saline (for a full description see ref. 31). At the end of the surgical period, a continuous i.v. infusion of a mixture consisting of glucose, the a-adrenergic receptor blocker phentolamine (Regitine, Ciba-Geigy), and the fl-adrenergic receptor blocker, d,l-propranolol (Sigma) was started. Glucose was infused to counteract hypoglycemia which is caused by the continuous infusion of the adrenergic blockers, and to maintain glucose concentration at a relatively uniform level, since plasma glucose modulates gastric acid secretion [17]. Various glucose infusion rates from 0 - 7 m g - k g -1. m i n - I were used in order to obtain plasma glucose concentrations of approx. 130 m g / 1 0 0 ml. The adrenergic blockers were infused to suppress sympathetic activity produced in response to surgical trauma a n d / o r vagal stimulation. Sympathetic activation has been shown to significantly decrease basal and vagally stimulated gastric acid secretion in the rat [33]. Infusion rates for both a and fl blockers were 45 ~ g - k g -1- rain-1 (15 /~l/min). Infusions of the blocker-plus-glucose solution began approximately 30 min before the first vagal stimulation, allowing heart rate and blood pressure to stabilize. Blood samples were taken before and during stimulation, and plasma glucose concentrations were measured on a Beckman Glucose Analyzer using glucose oxidase. Sequences of stimulations as to frequency or duration or use of the left vs the
196
fight vs bilateral vagal nerves were altered as appropriate. This design was chosen because pilot experiments suggested that the acid output in response to the second of two successive 10 rain/8 pps stimulations was significantly larger and that, in some cases, the response of the stomach decreased after several stimulation periods. Data analyses. Analyses of variance were applied for the stimulus frequency-response curves of gastric acid and mean arterial blood pressure, and Student's t-tests were used throughout for comparisons of individual means.
Results
Basal state of the animals. Intravenous infusion of the solution containing the aand fl-adrenergic blockers markedly decreased both mean arterial blood pressure (from 147 -t- 3 to 90 + 2 mm Hg, P < 0.001, n = 45) and heart rate (from 347 _+ 10 to 264 :i: 6 beats per minute (BPM), P < 0.001, n = 36) before and 30 rnin after the start of infusion, respectively. The mean basal gastric acid secretion rate (after blocker infusion) was 0.16 + 0.01 /~Eq/min (121 experiments in 34 rats). The mean basal plasma glucose concentration with adrenergic blockers was 129 _+ 2 m g / 1 0 0 ml (62 experiments in 34 rats). The basal acid secretion was correlated with neither the basal mean arterial blood pressure (r = - 0 . 1 0 , u = 63) nor the basal plasma glucose concentration (r = + 0,15, u = 63). Total 'stimulated acid output' and peak secretion rate. Illustrative records of successive 10-min stimulations of the cervical vagus nerve with varying frequencies for two rats are shown in Fig. 1. It can be seen that, after a latency of up to several minutes, the acid secretory rate rises to a peak and then returns more slowly to baseline levels after the termination of stimulation. Latencies, integrated incremental acid secretion (i.e. total stimulated gastric acid response until return to baseline rate --usually terminated within 25 rain from the start of stimulation), maximal secretion rate, and the occurrence of an off-response (transient increase in secretion rate immediately following the end of stimulation), were all computed for the 13 rats with left and 11 rats with right cervical vagal stimulations. Fig. 2 illustrates the stimulus frequency-response relationship for the total stimulated acid output. As can be seen, stimulations with 2 pps produced a near-maximal gastric acid response. The maximal response occurred at 4 pps. Stimulation with either 8 or 16 pps produced smaller responses. In contrast to this gastric acid response, the mean decrease (over the t0-min stimulation period) of the mean arterial blood pressure was small and not significant with stimulation at 2 pps, increased monotonically with an increase in frequency, and reached the highest value with stimulation at 16 pps (Fig, 2, lower panel). Correlation coefficients between the mean decrease in arterial blood pressure and the total stimulated acid response for the 8- and 16-pps stimulations were low and non-significant (rspps = 0.047, and rl6pp s = 0.083).
For the data summarized in Fig. 2, an analysis of variance (2-way with repeated measures) applied to the incremental gastric acid output revealed that frequency of
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stimulation had a very reliable effect (F4,88 = 18.82, P < 0.001), and right cervical vagal stimulation was superior to left (F1,22 = 5.00, P < 0.05). The frequency-response profile was the same for both left and right cervical vagi (F4,88 = 1.34, P > 0.1). Frequency of stimulation also had a large effect on mean arterial blood pressure (MABP) (F4,88 = 20.6, P < 0.001), and the right cervical vagus was marginally more effective than the left (Fl,22 = 4.08, P < 0.056). Parallel effects of frequency and the right vs. the left nerve stimulation were also observed for peak gastric acid secretion rates during each 2.5-min interval (data are not shown). Off-response. A brief acceleration of acid secretion was frequently observed immediately after the termination of the stimulation (cf. the response to 1, 4, 8 and 16 pps stimulations in the upper panel of Fig. 1). This off-response pattern was observed in 59% of all left and 42% of all right cervical vagal stimulations. The percentages of occurrence were lowest at 1 pps (40% and 35% for left and right stimulation, respectively) and highest at 16 pps (80% and 67%, respectively). Latency. The latency between the onset of stimulation and the detection of the first 0.1 /tEq of acid above basal secretion was affected by the frequency of stimulation, as illustrated in Fig. 3. Latency decreased gradually with increasing stimulus frequency up to 4 pps, and then showed no further decrease with 8 or 16 pps. The latency at 4 pps was significantly shorter than with 1 pps (2.6 ___0.2 vs 4.1 + 0.4 rain; t = 3.53, df = 40, P < 0.01). Since there was no difference between left and right CV stimulation, the data for the two sides were pooled. Another series of experiments with stimulation train durations of 1, 2, 4 and 10 min was conducted in two rats with left and two rats with right cervical vagal
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Fig. 2. Stimulus frequency-response relationship of right ( i t ) and left (& i ) cervical vagal stimulation-induced gastric acid output and decrease in mean arterial blood pressure (depressor effect) of urethane/chloralose anesthetized rats. The left (n = 13) and the right (n = 11) cervical vagal nerves were stimulated with 1, 2, 4, 8 and 16 pps, for 10 min as in Fig. 1, in balanced order. Each point represents the mean + S.E.M. of 8-13 experiments. The points at zero pps were obtained from an additional 10 animals treated similarly but without turning the stimulator on. Analyses of variance with repeated measures showed that RCV stimulation produced a significantly larger acid acid output than left stimulation (F1.22 = 5.0, P < 0.05) and t-tests showed that this difference was significant at 2 pulses/s (*, P < 0.05). electrodes. T h e t y p i c a l effect of 1 m i n of 4 p p s s t i m u l a t i o n is shown in the inset of the r i g h t h a n d p a n e l in Fig. 3. N o acid was d e t e c t e d during, or for 3 m i n after the 1-min stimulation. T h e r e was a response, however, b e t w e e n 4 a n d 15 m i n after the s t i m u l a t i o n train. T h e p a n e l o n the right of Fig. 3 s u m m a r i z e s relative p e r c e n t a g e increases o f acid o u t p u t as a f u n c t i o n o f s t i m u l a t i o n train d u r a t i o n , w h e n the r e s p o n s e to 2 m i n of s t i m u l a t i o n is t r e a t e d as 100%. T h e m e a n a c i d r e s p o n s e o f 4 a n i m a l s to 1 min o f s t i m u l a t i o n a m o u n t e d to 45.7 + 4.6% of the r e s p o n s e to 2 m i n stimulation. Bilateral nerve stimulation. S t i m u l a t i o n o f b o t h cervical vagi c o n c u r r e n t l y for 10 m i n at 2 p p s in 4 o t h e r rats p r o d u c e d a significantly larger gastric a c i d r e s p o n s e t h a n s t i m u l a t i o n of either t r u n k alone, b u t a m a r g i n a l l y significantly ( P = 0.05, d f = 3) lower r e s p o n s e t h a n the s u m o f i n d e p e n d e n t left a n d right stimulation. I f gastric a c i d o u t p u t is e x p r e s s e d in p e r c e n t a g e o f t h e s u m of i n d e p e n d e n t left a n d f i g h t s t i m u l a tions (100%), b i l a t e r a l s t i m u l a t i o n p r o d u c e d a n a c i d o u t p u t o f 88 + 4% (Fig. 4).
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Fig. 3. Left panel: effect of frequency of cervical vagal stimulation on latency of gastric acid secretion. Since no significant differences were found between right and left stimulation, the data were pooled and each bar represents the mean + S,E.M. of 18-23 experiments. *, P < 0.01, t-test. Right panel: effect of duration of cervical stimulation on gastric acid output. Rats with right (n = 2) and left (n = 2) cervical vagal electrodes were stimulated with 4 pps for 1, 2, 4 and 10 min in an ascending/descending balanced order. Each point is the mean _+S.E.M. of 4 animals. Incremental gastric acid output is expressed as a percentage of the 2-min stimulation. The inset shows the secretion rate of a typical 1-min stimulation experiment; note that acid secretion does not start until approximately 3 min following cessation of stimulation.
Stimulation in bursts. When the number of impulses (as well as pulse duration and pulse amplitude) given to the vagus nerve was kept constant, stimulation in burst patterns was less effective than continuous stimulation with trains at 4 pps in eliciting a gastric response (Fig. 5). There were no significant differences between bursts of 8, 16 or 40 pps. Burst stimulation had the same sharply reduced efficacy for gastric acid secretion with both the left and right vagal stimulation.
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201
Prolonged stimulation. With 60-min trains of stimulation of the right or the left cervical vagus (CV) at 4 pps, the maximal rate of gastric acid secretion was reached between 15 and 20 min and was maintained until the end of the 1-h stimulation period (Fig. 6). While the left CV stimulation produced slightly less gastric acid output during the rising phase, the same maximal output levels were reached as with the right CV stimulation. If the right CV, after a pause of approximately 40 min, was stimulated a second time for 60 min, maximal gastric acid output was reached much more rapidly, between 5 and 10 min, and acid output fell to about half maximal during the last 20 min of stimulation (Fig. 6).
Discussion Using electrical stimulation of the vagus nerve, we have presented the first quantitative and parametric analyses of gastric acid secretion in the rat. We have found that electrical stimulation at optimal parameters produced maximal rates of acid secretion that compared well with gastrin- and histamine-stimulated secretion rates [6, and personal observations]. The magnitude and reproducibility of this response suggests that this protocol would be a useful one for tests of vagotomy or other circumstances of impaired vagal function. These stimulus parameters also yield a sustained secretory response in this experimental preparation, thus making the technique a potentially powerful one for assessing the effect of antisecretory agents. The stimulus frequency-response curve for gastric acid output for the rat was characterized by a half-maximal secretory response at approximately 1 pps, a maximal response at 2-8 pps and a below-maximal output at 16 pps. This finding is consistent with the conclusion that gastric parietal cells in the rat are innervated by fine-caliber C-fibers. Such a hypothesis would also be consistent with the decrease in secretory response at 16 pps, since some blocking of nerve impulses would be expected at higher frequencies of stimulation [e.g. ref. 27]. Blocking of the putative C-fiber population would also explain the sharply reduced efficacy of burst pattern stimulation at higher frequencies. Finally, the idea that vagal axons mediating the gastric acid response are of a smaller caliber is in general agreement with the findings that there are only a few ( - 1%) myelinated fibers in the abdominal vagi [7] and that only slow conduction velocities of approximately 1 m / s , corresponding to C-fibers, were found electrophysiologically at this level [25]. In contrast, the cardiac vagal efferents have been found to have conduction velocities in the range of B-fibers in the cat [16] and most probably in all mammals [26]. The inhibition of secretion at higher stimulus frequencies might also be explainable at least in part by a suppressive effect of decreased MABP (with corresponding decrease in gastric mucosal blood flow) on gastric secretion with increasing frequency. This explanation is not supported, however, by the low and insignificant correlations between the magnitude of the depressor effect and acid secretion at both 8 and 16 pps in the present experiment, and by the finding that the two responses are largely dissociable [19]. Alternatively, it is possible that higher frequency stimulation coactivated some other response which had an inhibitory effect on gastric acid
202
secretion, such as vagally released somatostatin [2]. Although this issue is not directly addressed, our observations that the large percentage of experiments exhibited an overshoot of gastric acid secretion after the termination of stimulation occurred at 16 pps could be taken as support for such an interpretation. As a choice of optimal parameters for additional experimentation on rat gastric acid secretion, stimulation of the cervical vagus at 2-4 pps would seem ideal. These frequencies minimize the involvement of cardioinhibitory responses and make it feasible to stimulate in the neck, thus avoiding the additional trauma and need for artificial respiration that are required for stimulation of the thoracic vagal nerve. Stimulation with burst patterns at higher frequencies would not seem appropriate where the goal is maximal secretory rates. The fight cervical vagal nerve stimulation was slightly but consistently more effective than the left in producing gastric acid secretion in all experiments. Although we cannot completely rule out the possibility, it seems unlikely that the fistula implantation damaged more parietal cells innervated by the left CV, since the stab wound was made in the midline (greater curvature) of the rumen and at least two weeks recovery were allowed. This significantly greater acid output with the right cervical vagus nerve stimulation could be an indication that the right CV projects to a larger population of parietal cells than the left CV. Consistent with this explanation, there are an estimated 50% more fibers in the posterior gastric branches than in the anterior gastric branches of the vagus [20] and correspondences between the right cervical vagus and the posterior gastric branches and between the left cervical vagus and the anterior gastric branches have been demonstrated both electrophysiologically [25] and anatomically [18]. Species differences seem to exist insofar as in the cat left CV stimulation produced a gastric acid response approximately twice as large as right CV stimulation [3]. Comparisons between unilateral and bilateral CV stimulation address the question of overlap of the peripheral projection fields. If the left and the right CV each innervate separate populations of parietal cells, then one would expect the gastric acid response to bilateral stimulation to be equal to the sum of the left plus right stimulation. The present finding that additivity was 88%, suggests that there is a small population of parietal cells which is innervated by both vagi or, in other words, there is a 12% overlap of innervation. The finding of substantial additivity is consistent with the evidence of Legros and Griffith [14] suggesting that different vagal branches innervate different fields of the rat's gastric mucosa. The present findings with respect to overlap also compare well with the 83% additivity (or 17% overlap) observed in the cat [4]. These innervation patterns, however, contrast with that reported for the dog, in which stimulation of each cervical vagus elicited gastric secretion from the entire fundus and corpus (both sides) of the split stomach preparation [21]. As the present results show, there is a considerable latency of at least 2.6 rain between the start of stimulation (4 pps) and a minimal increase of acid secretion. The latency is clearly a function of the physiological response, since the technical latency of the perfusion/pH star system was approximately 10 s. This slow response contrasts with the fast ( < 1 min) release of insulin into the circulation upon cervical
203 vagal s t i m u l a t i o n [5]. O n e e x p l a n a t i o n for the longer latency of the gastric acid response is that secretion d e p e n d s o n the m e t a b o l i s m - c o u p l e d , e n z y m e - a c t i v a t e d ion exchanges a n d t r a n s f o r m a t i o n of tubulovesicles [23] rather t h a n exocytosis of vesicles from a readily releasable pool. I n addition, the transport a n d diffusion of the secreted acid to the surface of the mucosal rugae, where it can be washed away by the perfusion system, m a y take a significant a m o u n t of time. The fact that the 1-min s t i m u l a t i o n p r o d u c e d a p p r o x i m a t e l y half of the acid o u t p u t of a 2 - m i n s t i m u l a t i o n suggests that it is n o t a threshold a m o u n t of t r a n s m i t t e r released, b u t rather the release a n d t r a n s p o r t m e c h a n i s m itself that is responsible for the 2.6-min m i n i m a l latency to first detection. Finally, the time required for the secretory response to r e t u r n to basal levels after stimulus cessation m u s t reflect other aspects of the physiology of parietal cell secretion. Since vagal activation not only directly activates secretion b u t also potentiates responsiveness to h o r m o n e activation [12], it is conceivable that the time course after the cessation of a stimulus reflects the dissipation of a h o r m o n a l response engaged b y vagal stimulation.
Acknowledgements W e would like to t h a n k E d w a r d Fox, James Prechtl a n d Meredith W a l g r e n for their c o m m e n t s o n a n earlier draft of this paper. This research was s u p p o r t e d by N I H G r a n t AM27627 to T.L.P.
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