Neuromuscular Functions in Esophageal Smooth Muscle of Opossums as Differently Affected by Veratrum Alkaloids

Transcription

1 GASTROENTEROLOGY 1981;81: Neuromuscular Functions in Esophageal Smooth Muscle of Opossums as Differently Affected by Veratrum Alkaloids JAMES CHRISTENSEN and MARWAN ISKANDARANI Research Laboratories of the Division of Gastroenterology-Hepatology, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa Stimulation of intramural nerves in esophageal smooth muscle in vitro reveals that longitudinal muscle receives only excitatory cholinergic nerves and circular muscle receives only nonadrenergic inhibitory nerves. The latter nerves abolish the myogenic tone that characterizes sphincter muscle, but cause only a rebound contraction (the off-response) in esophageal body muscle. The off-response could be myogenic, or it could reflect rebound discharge of a second excitatory neuron. Two mixtures of neurotropic veratrum alkaloids, veratrine and cryptenamine, selectively depressed off-responses at concentrations (up to 3 X 1-6 g/ml) that did not depress the cholinergic contraction of longitudinal muscle and the myogenic tone of sphincter or neurogenic relaxation of sphincter tone. Cryptenamine was less potent than veratrine. Thus, either an interposed excitatory neuron is present that is selectively depressed by veratrum alkaloids, or the mechanism for rebound contraction differs from that involved in cholinergic and tonic contractions. Electrical field stimulation has revealed the neuromuscular properties of esophageal muscle by showing the responses of the muscle to excitation of neurons of the intramural plexuses (1-6). Three different neuromuscular responses can be distinguished in esophageal smooth muscle (Figure 1). The outer longitudinal muscle layer from the Received January 26, Accept ed June 1, Address requests for reprints to: James Christensen, M.D., Pro fessor and Dire ctor, Division of Gastroenterology-Hepatology, Department of Internal Med icin e, University of Iowa Hospitals and Clin ics, Iowa City, Iowa This study was supported by Research Grant AM11242 from th e National Institutes of Health. This paper was presented in part to the annual meeting of the Central Society for Clinical Research in Chicago, November, by the American Gastroenterological Association /81/ $2.5 esophageal body contracts with electrical field stimulation. This is caused by excitatory cholinergic nerves. This contraction is called the "duration response" because it lasts for the full duration of the stimulus to the intramural nerves. The circular muscle layer from the esophageal body contracts after the stimulus has ended. This contraction is prevented by tetrodotoxin but by none of the selective neurohormonal antagonists that act at autonomic receptor sites. Furthermore, this contraction is preceded by a period of inhibition that is not expressed as a relaxation because this muscle has no tone; however, it can be revealed if a drug is used to make the muscle contract during the stimulus. This contraction thus appears to be a consequence of the action of nonadrenergic inhibitory nerves and has been called the "off-response" because it does not occur until after the stimulus is completed. The circular muscle layer from the immediate esophagogastric junction possesses tone and relaxes with electrical field stimulation. The tone of this muscle is reduced by such inhibitory substances as glucagon, dopamine, and isoproterenol, and is associated with a high rate of oxygen consumption. Electrical field stimulation of this muscle induces relaxation that lasts for the full duration of the stimulus. This relaxation is sensitive to tetrodotoxin but not to any recognized selective neurohormonal antagonist acting at autonomic receptor sites. Hence, it represents the action of nonadrenergic inhibitory nerves. This is called the "relaxation response." Both the off-response and the relaxation response are due to the excitation of the nonadrenergic inhibitory nerves. The off-response could be a rebound of the muscle cells to antecedent hyperpolarization. Alternatively, it could represent the action of a second excitatory nerve. These two possibilities will be referred to further as the "one-nerve hypothesis" and the "two-nerve hypothesis."

2 November 1981 VERATRUM ALKALOIDS 867 Sphincter D minute Figure 1. A depiction of the three kinds of response of esophageal smooth muscle to electrical field stimulation. At the left a diagram indicates how the strips of the muscular wall of the esophagus were cut. Arrows indicate the appropriate tracings of tension records taken during electrical field stimulation in 3-s trains indicated below each tracing. As shown, longitudinally oriented strips contract during the train, the duration response; transverse strips cut from the region of the sphincter relax, the relaxation response; transverse strips cut from above the sphincter contract briefly well after the train, the oft-response. In these experiments we sought ways to affect the off-response and the relaxation response differentially. In the case of the one-nerve hypothesis, everything that abolishes the off-response should also abolish the relaxation response. In the case of the two-nerve hypothesis, however, selective suppression of the off-response should be possible. To monitor nonspecific depression of contraction, parallel studies were also carried out to examine both the duration response and the tone of the sphincter muscle. Veratrum alkaloids were examined because of their known action on the nerve membrane to depolarize it and to increase the negative after-potential (7). Because of the cost and limited availability of the pure alkaloids, the alkaloid mixtures veratrine and cryptenamine were used, as has generally been done in studies of these agents (8). Methods Male and female opossums, kg, were anesthetized by intraperitoneal injection of sodium pentobarbital, 5 mg/kg. The stomach and esophagus were removed en bloc, opened lengthwise along the line of the lesser gastric curvature, and pinned flat with the mucosa up, in a bath of modified Krebs solution where the mucosa was then removed by sharp dissection. The modified Krebs solution had the following composition (mm): sodium 139, potassium 4.6, calcium 5., magnesium 2.2, chloride 125, bicarbonate 22, phosphate 3.5, sulfate 2.3, and glucose It was equilibrated with 95% O 2-5% CO 2, A transverse strip of the muscularis propria,.2 X 1.5 em, was cut from the esophagogastric junction in the area that can be recognized as the site of the muscle that constitutes the lower esophageal sphincter. Other transverse strips,.2 X 1.5 em, were cut from 2 to 6 cm above the esophagogastric junction. Longitudinal strips of the same dimensions were also cut from the same segment. All these strips contained only smooth muscle, as evidenced by their response to electrical field stimulation: striated muscle exhibits a characteristic rapid twitch response. Silk threads were tied to both ends of each strip and the strips were transferred to a previously described organ bath (9). In this bath, one end of each strip was fixed while the other was attached to a force-displacement transducer 14 12! 1 III 8 & 6 o 4 ' 2 g no6 Veratrine Cryptenamine -e a " "-:-----!;--'------!;--7-'-----!; Concentration g/ml (log) Figure 2. Effect of veratrine and cryptenamine on the duration response of the longitudinal muscle layer of the opossum esophagus. The ordinate indicates the amplitude of the duration response. The abscissa shows the concentrations of the veratrum mixture used. Observe that both alkaloid mixtures tended to raise the amplitude at lower concentrations and to depress it at higher concentrations.

3 868 CHRISTENSEN AND ISKANDARANI GASTROENTEROLOGY Vol. 81, No.5 (Grass Instrument Company, Quincy, Mass.) whose output led to a strip-chart polygraph recorder (Beckman Instruments, Type R Dynograph). The strip was stretched to 15%of initial length, the length of optimum tension development, at which its thickness was less than 1.5 mm. The muscle bath consists of a small plexiglass block with a central channel in which two muscle strips can be accommodated and through which modified Krebs solution flows from a reservoir. In the reservoir the solution is equilibrated and warmed to maintain temperature in the bath at C. Platinum rings are set into the channel at intervals to allow electrical field stimulation of the strips. Stimuli were delivered by a constant-current stimulator that delivers trains of pulses of 1.-ms duration at 1 Hz in trains up to 1 s long. Amplitude was always made just supramaximal. In each single experiment two such baths were operated simultaneously so that four strips were treated identically. These four strips were always from the same esophagus and included strips of all three kinds. In each single study an initial control period of 1 h elapsed during which the strips were stimulated at 3-s intervals. During this time responses became stable in magnitude. Thereafter, the drug was added at a concentration of 1-8 g/rnl which was then increased in steps to 8 X 1-5 g/ml, without a rinse between increments, each treatment period lasting 2-3 min. Preliminary studies had established this as the effective range of concentrations, had shown the stability of effects during these intervals, and had established the irreversibility of these effects by rinsing. In these studies, also, it was shown that the effect of veratrum is fully developed within 3 min after exposure and that responses are then stable. Two drugs were used, veratrine and cryptenamine (Mallinckrodt Inc., St. Louis, Mo.), because they differ to some degree in their composition of alkaloids. Each set of strips was treated with only one of the two drugs. The drugs were made fresh daily in concentrated stock solutions in.1 N HCl and diluted to their final concentrations in modified Krebs solution. Records were read for duration and amplitude of the duration response, duration and amplitude of off-response, basal tension (or tone) in sphincter strips, and magnitude of the relaxation response. In all cases values were normalized to the values during the initial control period. Data were averaged for all strips. Results The principal effects of both alkaloid mixtures were on the duration response and the off-response. In the duration response, the cholinergic contractions of the longitudinal muscle, the effect was a potentiation that was dose related. With both veratrine and cryptenamine the amplitude of the duration response rose to a peak, at 1-6 g/ml in the case of veratrine and at 3 X 1. 6 g/rnl cryptenamine, above which the amplitudes declined steeply (Figure 2). Duration of contraction also increased at concentrations up to 3 X 1-6 g/rnl, as is evident in Figure 3, and spontaneous contractions sometimes appeared. In respect to the off-response the amplitude declined progressively throughout the range of concentrations used for both alkaloid mixtures. This effect is shown in Figure 4. In respect to the sphincter tone there was no effect up to concentrations of 8 X 1-6 for cryptenamine and 1-6 for veratrine, At higher concentrations tone fell off sharply, as shown in Figure 5. l_ongll\1dinal Strip (Duration Response) 15g v-- L- sj \ \ Transverse Bod Strip (Off-Response) Transverse Sphinc-ter Strip (Relaxation Response) IJ Transverse Body Strip (Off-Response) f Minute A CONTROL --A.- B. VERATRINE, 3xl- 6 g/ml Figure 3. The effect of veratrum on responses of esophageal smooth muscle to electrical field stimulation. As shown, the effect of the veratrum alkaloids on the three responses differ. The off-response of the esophageal body was depressed in amplitude. The duration response of the longitudinally cut strip was prolonged and slightly augmented in magnitude. The sphincter basal tension (tension just before onset of relaxation) was changed little or was slightly depressed, but a prominent after-contraction appeared. Sphincter relaxation itself was unaffected.

4 November 1981 VERATRUM ALKALOIDS "C 12 Ẹ.. 1 Ẓ... en c:: 8 Cl. en a:: 6.J= - 4 "C ::J :t:: C. 2 E «Veratrine N =1 Cryptenamine N = Concentration g/ml (log) Figure 4. The effect of veratrine and cryptenamine on the amplitude of the off-response of the circular layer of smooth muscle from the opossum esophagus. The ordinate shows the amplitude of the off-response. The abscissa shows the concentration of the veratrum mixture used. Note that both alkaloid mixtures reduced the off-response amplitude increasingly throughout the range of concentrations used. In respect to the relaxation response there was no effect on the depth of relaxation up to concentrations of 1-6 for veratrine and 8 X 1-6 for cryptenamine, beyond which concentrations the tone sharply declined (Figure 6). Relaxation, however, still occurred up to the maximal concentrations used. The latency of the off-response and the timing of the relaxation response of the sphincter muscle were both unaffected. The depression of the off-response could not be overcome by increasing the current density used, which had previously been determined to be supramaximal. This was examined in 6 strips in which current strength was doubled, with no resultant increase in amplitude of the off-response. Discussion When veratrum preparations are used in therapy they commonly cause nausea, vomiting, and epigastric and substernal distress, symptoms that might reflect an action on gastrointestinal nerve and muscle. Despite this, the actions of the veratrum alkaloids on gastrointestinal muscle have had very little study. A "spasmogenic" action was suggested... "C Cll Ẹ o e 8 1: Cl. CJ) - 6 c '(i) c 4 I- (ij en 2 Cll m... Veratrine n=6 Cryptenamine n= Concentration, g/ml (log) Figure 5. The effect of veratrine and cryptenamine on basal tension or tone of the muscle from the lower esophageal sphincter. The ordinate indicates tone, the tension measured at the onset of the relaxation induced by electrical field stimulation, relative to zero tension. As shown, both alkaloid mixtures depressed basal tension only at higher concentrations, above 3 X 1-6 g/ml,

5 87 CHRISTENSEN AND ISKANDARANI GASTROENTEROLOGY Vol. 81, No.5 14 Figure 6. The effect of veratrine and cryptenamine upon the relaxation response of smooth muscle from the esophagogastric sphincter. The ordinate indicates the depth to which tension fell in those strips normalized for the depth in the initial control period. The abscissa shows concentrations of the two veratrum extracts. Observe that veratrine has no effect up to 3 X 1-6 g/rnl, -"C 12 COE1 ẕ c: ; CO >< 8 J2 6 a: - 4 "C :t::. E 2 «...Veratrine n=5 Cryptenamine n= Concentration, g/ml (log) long ago, but accurate definition of such responses was difficult because of the spontaneous contractions that occur in most gastrointestinal muscle. Gourzis and Bauer (1) examined the effect of veratrum alkaloids on the small intestine of rabbit in vitro and dog in vivo. They were mainly concerned with the possibility of developing a bioassay that could be usec.as a guide to therapy. In the isolated rabbit intestii.. they observed that the agents caused a rise in the force of spontaneous rhythmic contractions of the longitudinal muscle. An increase of tone was seen at higher concentrations. The fact that the effect was not blocked by atropine was interpreted as indicating that the excitatory effect is exerted directly upon the muscle rather than indirectly through cholinergic nerves. They saw no effect on dog intestine in vivo both in normal animals and in those with Thiry-Vella loops. They seem not to have considered actions that might have been mediated through nerves other than cholinergic excitatory nerves. Full interpretation of the results of these experiments on esophageal muscle is limited by the ignorance of the nature of the nonadrenergic inhibitory nerves responsible for the off-response and the relaxation response, by ignorance of the way in which these nerves affect the muscle, by ignorance of the genesis of tone in sphincter strips, and by the mixed composition of the alkaloid mixtures used. The enhancement of the duration response, both in amplitude and duration, is consistent with the previous suggestion of a "veratrine response" in smooth muscle like that in striated muscle (8). The fact that the relaxation response of the sphincter was unaffected by the alkaloids indicates that this presumed neurotropic action of the alkaloids is not a general action on nerves and may, instead, be selective for cholinergic nerves. The mechanism of the off-response, the central concern of this study, is not fully illuminated by these studies. The one-nerve hypothesis, as outlined earlier in this paper, proposes that the off-response is a rebound contraction of the muscle after a period of inhibition. The two-nerve hypothesis proposes that it is the rebound of a second excitatory nerve linked in series to the inhibitory nonadrenergic nerve. The fact that the off-response is selectively depressed could be explained in three ways. First, it could represent suppression of the nonadrenergic inhibitory nerves by the drugs, but this possibility seems very unlikely because of the fact that the action of these nerves, as observed in the sphincter strips, is not impaired. Second, the depression could represent selective depression of the excitatory nerves that are proposed to exist in the two-nerve hypothesis. If this is the case, that action is highly selective in view of the fact that the alkaloids enhance the action of cholinergic excitatory nerves and do not affect the action of the nonadrenergic inhibitory nerves. Third, the depression of the off-response could represent a depression of one of the processes in the muscle itself that lead to the off-response. This seems unlikely in view of the fact that the cholinergic contractions of the longitudinal layer are enhanced and that the tone of the sphincter

6 November 1981 VERATRUM ALKALOIDS 871 strips is unaffected. There may be, however, differences in the muscular processes by which these different kinds of contractions come about. The present evidence is most easily interpreted in terms of the second explanation given above. The evidence supports the two-nerve hypothesis. These studies indicate that the actions of the veratrine alkaloids on the action of gut smooth muscle are more complex than heretofore thought. Enhancement of contractions, on the present evidence, seems most likely to be due to selective potentiation of cholinergic nerve effects. Depression of contractions is most likely to reflect a depression of those neural and muscular mechanisms that are unique to the off-response. In the giant axon of squid and crayfish the depolarizing action of veratridine is a result mainly of a selective increase in resting nerve membrane permeability to sodium ions (7). Other of these alkaloids can block action potentials without depolarizing nerves. Still others block peak transient sodium and steady-state potassium conductances. The detailed means by which these latter two effects are achieved remain unknown. In view of these multiple actions, no specific mechanism can be proposed as to the way in which the alkaloid mixtures used here selectively depress the off-response. References 1. Christensen J. Lund GF. Esophageal responses to distension and electrical stimulation. J Clin Invest 1969;48: Lund GF. Christensen J. Electrical stimulation of esophageal smooth muscle and effects of antagonists. Am I Physiol 1969;217: Christensen J. Patterns and origin of some esophageal responses to stretch and electrical stimulation. Gastroenterology 197 ;59: Weisbrodt NW. Christensen I. Gradients of contractions in the opossum esophagus. Gastroenterology 1972 ;62: Christensen J. Freeman BW. Miller IK. Some physiological characteristics of the esophagogastric junction in the opossum. Gastroenterology 1973;64: Christensen /. Conklin IL. Freeman BW. Physiologic specialization at esophagogastric junction in three species. Am J Physiol 1973;225: Ohta M. Narahashi T. Keeler RF. Effects of veratrum alkaloids on membrane potential and conductance of squid and crayfish giant axons. I Pharmacol Exp Ther 1973 ;184: Krayer O. Acheson GH. The pharmacology of the veratrum alkaloids. Physiol Rev 1946;26: De Carle OJ. Christensen I. A dopamine receptor in esophageal smooth muscle of the opossum. Gastroenterology 1976; 7: Gourzis JT. Bauer RO. The effect of veratrum derivatives on the isolated intestine of the rabbit and on the intact intestine of the trained unanesthetized dog. J Pharmacol Exp Ther 1951 ;13:471-8.