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Intrinsic nerves affect gallbladder contraction in the guinea pig
GASTROENTEROLOGY 1990;99:828-830
Intrinsic Nerves Affect Gallbladder Contraction in the Guinea Pig E. A. BROTSCHI,
J. PATTAVINO,
and L. F. WILLIAMS,
Jr.
Department of Surgery. Boston Veterans Administration Medical Center, and Boston University F&o01 of Medicine, Boston, Massachusetts
Muscarinic antagonists block gallbladder contraction induced by cholecystokinin in vivo but have little effect on gallbladder muscle strips. This study examined the effect of neural blockade on cholecystokinin-octapeptide-induced contraction of the intact guinea pig gallbladder in vitro using cholecystokinin-octapeptide applied to the gallbladder serosa, the lumen, or both compartments simultaneously. Simultaneous cholecystokinin stimulation of both the lumen and serosa was the most potent stimulus to contraction, and the responses were significantly inhibited by atropine and tetrodotoxin. Cholecystokinin in the gallbladder lumen alone evoked contraction by a dose-dependent mechanism that was entirely blocked by atropine or tetrodotoxin. Serosal application of cholecystokinin was the least potent, resulting in contractile responses and low sensitivity to neural blockers comparable to effects reported in muscle strips. The results suggest that cholecystokinin can cause gallbladder contraction by stimulating muscle receptors, neural receptors, or both, and combined neural and muscular stimulation is the most potent contractile stimulus.
C
holecystokinin (CCK) has been known for 60 years to cause gallbladder contraction (11, and its importance as the major physiological mediator of postprandial gallbladder emptying has recently been confirmed (2). Because gallbladder smooth muscle contains a high density of high-affinity receptors for CCK (3),the interaction of blood-borne CCK with these receptors has been thought to explain postprandial gallbladder contraction. However, two observations suggest that CCK may stimulate contraction by a more complex mechanism: first, muscarinic antagonists block the contractile effect of CCK in vivo in the gallbladder of humans (4,5)and dogs (6);second, postprandial levels of CCK in serum (7) are much lower than levels required for substantial muscle-strip contraction in vitro.
Because muscarinic antagonists block the effect of exogenous and endogenous CCK on the intact gallbladder in vivo, cholinergic innervation is presumably involved in CCK’s in vivo effect. However, in vitro studies with gallbladder muscle strips show only a small change in contractility with cholinergic blockBecause disruption of gallbladder intrinsic ade (5,8). neurons during strip preparation could produce this discrepancy, we examined the effect of neural blockade on contraction in a gallbladder model with intact intrinsic neural pathways. The intact guinea pig gallbladder in organ bath was used to quantitate the contractile effect of luminal and serosal stimulation by cholecystokinin-octapeptide (CCK-OP) and to assess the role of intrinsic nerves in gallbladder contractile response. Methods Hartley female guinea pigs weighing 300-350 g were fasted overnight then anesthetized with IM ketamine (5 mg/kg) and xylazine (30 mg/kg). Gallbladders were removed in acute terminal experiments, according to the guidelines of the Veterans Administration Animal Care subcommittee. Midline laparotomy was performed, the gallbladder was aspirated, and the fasting volume was measured. Two polyethylene cannulas (0.6 mm ID] were then placed, one in the fundus of the gallbladder through a purse-string suture and one in the cystic duct, and the gallbladder was immediately suspended in a 37’C organ bath of modified Kreb’s buffer (NaCl, 133 mmol/L; NaHCO,, 16 mmol/L; NaH,PO,, 1.2 mmol/L; MgSO,, 0.04 mmol/L; KCl, 4.7 mmol/L; CaCl,, 1.25 mmol/L; and glucose, 7.8 mmol/L) bubbled with 95% 0, and 5% CO, to maintain the pH at 7.35-7.45. The fundic catheter was connected to a saline-filled pressure transducer (Hewlett-Packard 1280) and to a recorder (HP 7702B: Hewlett Packard, Waltham,
Abbreviation used in this paper: CCK-OP, cholecystokininoctapeptide. 0 1990 by the American G&roenterological Association 0018-5085/90/$3.00
September 1990
MA) for continuous monitoring of intraluminal pressure. The second intraluminal catheter was attached to a syringe on a modified Harvard apparatus rodent respirator for timed infusion and withdrawal of a specified volume of fluid from the gallbladder lumen. At the start of each experiment, a luminal volume was determined which resulted in intraluminal pressure of 5-7 cm water when infused into the unstimulated gallbladder over 45 seconds. Our previous studies confirm that this is a physiological pressure in the unstimulated guinea pig gallbladder 191,and this baseline pressure resulted in maximal reproducible responses to contractile stimulation. This initial gallbladder volume (usually about 70% of initial in vivo fasting volume] was used throughout the experiment. A dynamic method was used to measure gallbladder contractility, consisting of continuous luminal pressure recording while the gallbladder was cyclically filled and emptied (10.11). The gallbladder was equilibrated in organ bath during cyclical infusion and withdrawal of the initial volume of luminal buffer (0.67 cycle/min) until the resulting pressure tracing was reproducible in shape and magnitude. As previously described in vivo (11) and in vitro (10,X?), the shape and height of the pressure-volume curve provided a sensitive and reproducible index of the contractile state of the gallbladder. Pressure tracings demonstrated a sine-wave configuration that was dependent on contractile tone and receptive relaxation in the viable gallbladder preparation as well as on the flow characteristics of the pump. The parameter used to measure contractility was peak pressure generated in the gallbladder during the third infusion-withdrawal cycle. In a previous study, we separately analyzed the slope and area of the pressure-volume curve and determined that these data paralleled peak pressure response closely (9). The gallbladder was stimulated using a series of buffers with increasing concentrations of CCK-OP (Kinevac; Squibb, New Brunswick, NJ] from lo-l5 to 5 x 10m7 mol/L. The CCK-OP was added to the serosal buffer only, to the luminal buffer only, or to both simultaneously at identical concentrations. Only one route of stimulation was tested in each gallbladder. In some experiments, tetrodotoxin, 10e7 mol/L (Sigma, St. Louis, MO], or atropine, 3 x 1O-6 mol/L (Anpro, Arcadia, CA), was added in conjunction with the CCK-OP. The entire gallbladder pressure response to addition of an agonist was seen immediately on the first infusion-withdrawal cycle, with no perceptible latency, whether the agonist was applied to the lumen, the serosa, or both (Figure 1). At the termination of each experiment a maximal gallbladder contraction was induced by the addition of acetylcholine 5 x 10e4 mol/L [Sigma) to both the luminal and serosal sides of the gallbladder. Either acetylcholine. 5 x 10m4mol/L, or CCK-OP, 5 x 1O-7 mol/L, applied to the luminal and serosal sides of the gallbladder induced a maximal contraction in this preparation, which was not exceeded by stimulation with higher agonist doses. To assess architecture and morphology of the gallbladder preparation, distended gallbladders were transfered into formalin at the end of the experiment, fixed, sectioned, and examined with H&E staining. Data were analyzed by measuring the observed increase in peak gallbladder pressure caused by CCK-OP stimulation and dividing this by the increment in peak gallbladder
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1 min Figure 1.Typicalhdnd pressure tracing showing the effect of various doses of CCK-OP applied simultaneously to the serosa and the lumen of a single gallbladder preparation. A. Pressure tracing in modified Krebs’buffer before stimulation. mol/L CCK-OP. B. Pressure response during stimulation with lo-l5 C. Pressure response during stimulation with lo-” mol/L CCK-OP. D. Pressure response during stimulation with lo-’ mol/L CCK-OP.
pressure during maximal contraction. This ratio was expressed as a percentage of the maximal contractile response. Because gallbladder volume and effective radius were essentially constant for all measurements during an experiment, and the gallbladder was approximately spherical, the measured luminal pressure reflected average wall tension, based on Laplace’s Law [tension = radius x pressure/2). Thus the ratio of the observed pressure response over maximal pressure response reflected the approximate percent of maximal wall tension generated in response to stimulation. Unless otherwise specified, data were evaluated using analysis of variance to compare intragroup and intergroup responses at each concentration of CCK-OP, using Dunnett’s test for significance. Probability to.05 was interpreted as statistically significant. Data are expressed as mean f SE.
Results Microscopic examination of gallbladders used in these experiments showed normal cellular morphology and an intact mucosa. The wall thickness of the distended gallbladder was approximately 100 pm after fixation. The effects on gallbladder contractility of CCK-OP applied to the serosa, the lumen, and to both compartments simultaneously are shown in Figure 2. Combined serosal and luminal application was most potent in contracting the gallbladder, with significant contraction occurring at a CCK-OP concentration of lo-l5
828 BROTSCHI ETAL.
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Figure 3. Effect of CCK-OP applied to the gallbladder serosa, to the lumen, or to both compartments simultaneously on gallbladder contractile response. P values denote sign&ant differences from serosal stimulation: *P < 0.05; **P c 0.01; ***P c 0.001.
mol/L (P < 0.02, baseline vs. stimulated by paired t test]. Combined serosal and luminal stimulation was more potent than either serosal or luminal stimulation alone at each concentration of CCK-OP. The approximate ED,, for combined stimulation was 1.5 x lo-l1 mol/L, compared with 2 x lo-” mol/L for luminal stimulation and 3 x lo-” mol/L for serosal stimulation. Luminal CCK-OP alone produced significant contraction at concentrations of ~10-~’ mol/L and was equally or more potent than serosal stimulation at all but the highest CCK-OP concentrations. Serosal application produced significant contraction at concentrations of ~10-‘~ mol/L. Construction of Eadie-Hofstee plots from the doseresponse data suggested that serosal stimulation was activating primarily a single class of receptors with a Kd of approximately 1.0 x 10-l’ mol/L, as previously described for the gallbladder muscle CCK receptor (13). The data from luminal and combined stimulation gave a nonlinear plot with a variable slope, suggesting a variable population of receptors. The effect of atropine or tetrodotoxin applied simultaneously with CCK-OP to both the serosa and lumen of the gallbladder is illustrated in Figure 3. Atropine inhibited contraction by approximately 50% at lower CCK-OP concentrations. Tetrodotoxin significantly inhibited the response to all CCK-OP concentrations
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Figure 3. Effect of atropine, 3 x la-’ mol/L, or tetrodotoxin, lo-’ mol/L, applied simultaneously with CCK-OP to both the lumen and the serosa of the gallbladder. P values denote significance compared with CCK-OPwithout atropine or tetrodotoxhx *P < 0.05; **p < 0.01; ***p < 0.001.
The effect of atropine or tetrodotoxin applied in conjunction with CCK-OP to the serosal surface is shown in Figure 5. Atropine significantly decreased the contractile response to serosal stimulation at 10m9 mol/L, while tetrodotoxin decreased responses at 10-l’ mol/L and 10e9 mol/L. Discussion These experiments are consistent with prior in vivo observations indicating that intrinsic choline&c nerves are important mediators of CCK’s effect on the intact gallbladder. Serosal application of CCK-OP
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Figure 4. Isffectof atropine, 3 x lo-’ mol/L, or tetrodotoxin, lo-’ mol/L, applied with CCK-OP to the lumen of the gallbladder. P values denote significance compared with CCK-OP without atropine or tetrodotoxim ?? P < 0.05; ?? *P < 0.01; ***P < 0.001.
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CCK-OP Concentration (Molar) Figure 5. Effect of &opine, 3 x 10e6movL, or tetrodotoxin, lo-' mol& applied with CCK-OP to the serosaf surface of the gallbladder. P values denote sign&awe compared with CCK-OP without atropine or tetrodotoxh *P c 0.05; ?? *P < 0.01; *+*P-c0.001.
induced contraction that resembled the contractile response seen by others in muscle strips in dose response and the effect of cholinergic blockade (3). In gallbladder strips, as well as in this model, cholinergitally mediated (scopolamine-sensitive) contraction accounts for a significant fraction of the contractile response at low CCK-OP concentrations (50% at 10-l’ mol/L vs. 20% at lo-* mol/L) (5,14). The apparent further inhibition seen with tetrodotoxin in our study might reflect the contribution of noncholinergic nerves to contraction or might be caused by additional suppression of cholinergic neural responses that were not blocked by the dose of atropine used. Luminal stimulation in our model appeared to evoke a neurally mediated contractile response that has not previously been described. The luminal CCK-OP appeared to stimulate submucosal nerves to produce gallbladder contraction, and inhibition of this response by atropine and tetrodotoxin showed that smooth muscle receptors for CCK were not being activated during luminal stimulation at CCK-OP concentrations
NERVES IN GALLBLADDER
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response curve to the right, and tetrodotoxin shifted it still further. The increased response seen with combined luminal and serosal CCK-OP may indicate that stimulation of an intrinsic neural plexus contributed to a lower threshhold for smooth muscle activation or that smooth muscle binding of CCK-OP enhanced the response to neural stimulation. These possibilities are consistent with previous observations in intact gallbladder models that subthreshhold doses of polypeptide hormones greatly increased the response to transmural electrical stimulation in the isolated perfused guinea pig gallbladder (15). Conversely, electrical stimulation of the guinea pig gallbladder in vivo enhanced the contractile response to CCK-OP over a range of doses (16). The potential for neurally mediated gallbladder contraction is not surprising when gallbladder anatomy is considered. The anatomy of the gallbladder includes a complex innervation, analogous to that in the bowel wall. Two ganglionated nerve plexuses have been identified, located in the subserosal and submucosal layers (17). The intrinsic nerve plexus communicates with extrinsic fibers from the vagus, the celiac ganglion, and the thoracic dorsal root ganglia (18). A number of enteric peptide hormones and other neurotransmitter substances have been identified by immunocytochemistry in nerves and ganglia of the gallbladder, including vasoactive intestinal polypeptide (19), galanin (201, substance P (18), gastrinreleasing hormone (20), and neuropeptide Y (18). Plexuses of nerve bundles and fibers have been identified by histochemical techniques as discrete networks of adrenergic, cholinergic, and “purinergic” nerves in the gallbladder wall (21). Cholecystokinin-octapeptide has been shown to be an important neurotransmitter in the ileum, where its contractile effect is mediated by cholinergic nerves (22). Direct electrical measurements from neurons provide further evidence that CCK-OP functions as a neurotransmitter, causing depolarization of ileal myenteric neurons (23). and discharge from gastric mechanoreceptor neurons in the vagus (24). Although the gallbladder is innervated by the enteric nervous system, most investigators have concluded that CCK’s effect as a cholinergically mediated neurotransmitter in gallbladder muscle strips is relatively unimportant (8). Perhaps the relative paucity of neural elements in the gallbladder wall compared with the ileum (17) contributes to the inability to measure neural effects in gallbladder muscle strips which are easily measured in ileal muscle. Several studies now demonstrate that the intact gallbladder in vivo does require cholinergic innervation to respond to CCK (4-6), and our studies suggest that the effects of CCK in the intact in vitro gallbladder also involve intrinsic nerves. The observation that CCK-OP transiently enhances binding to
830 BROTSCHI ET AL.
muscarinic receptors in gallbladder tissue (25) suggests that CCK may have a complex role in modulating gallbladder neural responses. It is evident that neither mode of stimulation we used is identical to the circulation of CCK-OP through muscular and submucosal capillary beds. However, it seems likely that blood-borne CCK could stimulate gallbladder intrinsic nerves. Further investigation of the events that stimulate gallbladder neural pathways may increase our understanding of physiological gallbladder contraction. References 1. Ivy AC, Oldberg E. A hormone mechanism for gallbladder contraction and evacuation. Am J Physiol1929;91:338-344. 2. Shiratori K, Watanabe S, Chey WY, Lee KY, Chang T-M. Endogenous cholecystokinin drives gallbladder emptying in dogs. Am J Physiol1986;251:G553-G558. 3. Grider JR, Makhlouf GM. Regional and cellular heterogeneity of cholecystokinin receptors mediating muscle contraction in the gut. Gastroenterology 1987;92:175-180. 4. Fisher RS. Rock E, Malmud LS. Choline@ effects on gallbladder emptying in humans. Gastroenterology 1985;89:716-722. 5.Takahashi T, Yamamura T, Ishikawa Y, Kantoh M. Utsunomiya J. Effects of cholecystokinin-octapeptide on the human gallbladder both in vivo and in vitro. Gastroenterol Jpn 1986;21:49-54. 8.Takahashi I, Suzuki T, Aizawa I, Itoh Z. Comparison of gallbladder contractions induced by motilin and cholecystokinin in dogs. Gastroenterology 1982;82:419-424. JE. Cholecystokinin7. Walsh JH, Lamers CB, Valenzuela octapeptidelike immunoreactivity in human plasma. Gastroenterology 1982;82:438-444. 8. Yau WM. Makhlouf GM, Edwards LE. Farrar JT. Mode of action of cholecystokinin and related peptides on gallbladder muscle. Gastroenterology 1973;65:451-456. 9. Brotschi EA, LaMorte WW, Williams LF Jr. Effect of dietary cholesterol and indomethacin on cholelithiasis and gallbladder motility in the guinea pig. Dig Dis Sci 1984;29:1050-1057. 10.Matsuki Y. Dynamic stiffness of the isolatedguinea-piggallbladder during contraction induced by cholecystokinin. Jpn J Smooth Muscle Res 1985;21:427-438. 11.Schoetz DJ, LaMorte WW, Wise WE, Birkett DH, Williams LF Jr. Mechanical properties of primate gallbladder: description of a dynamic method. Am J Physiol1981;241:G376-G381. 12.Davison JS, Al-Hassani M. The role of noncholinetgic, nonadrenergic nerves in regulating the distensibility of the guinea pig gallbladder. In: Christensen J, ed. Gastrointestinal motility. New York: Raven, 1980:89-95.
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13.Chang RSL, Lotti VJ. Biochemical and pharmacologic characterization of an extremely potent and selective nonpeptide cholecystokinin antagonist. PNAS 1986;83:4923-4926. 14.Yamamura T, Takahashi T, Kusunoki M, Kantoh M, Ishikawa Y, Utsunomiya J. Cholecystokinin octapeptide-evoked [“HIacetylcholine release from guinea pig gallbladder. Neurosci Lett 1986;65:167-170. 15.Al-Hassani MH, Davison JS. Interactions between intrinsic cholinergic nerve stimulation and gastrointestinal polypeptides on the guinea-pig gallbladder. J Physiol (Lond) 1978;285:24P. 16.Pomeranz IS, Davison JS, Friedhandler TM, Shaffer EA. The relative importance of choline&c and hormonal stimuli in in vivo gallbladder contraction (abstr). Gastroenterology 1983;84: Al275. 17.Sutherland SD. The neurons of the gallbladder and gut. J Anat 1967;101:701-709. 18.Mawe GM, Gershon MD. Unique properties of gallbladder ganglia: relationship to the enteric nervous system (ENS) (abstr]. Gastroenterology 1988;94:A292. 19.Sundler F, Alumets J, Hakanson R, Ingemansson S, Fahrenkrug J, Schaffalitzky de Muckadell 0. VIP innervation of the gallbladder. Gastroenterology 1977;72:1375-1377. 20.Padbury RTA, Toouli J, Furness JB. The histochemistry of gallbladder innervation (abstr). Gastroenterology 1988;94:A339. 21.Davison JS, Al-Hassani M, Crowe R, Burnstock G. The nonadrenergic, inhibitory innervation of the guinea-pig gallbladder. Pilugers Arch 1978;377:43-49. 22.Vizi SE, Bertaccini G. Impicciatore M, Knoll J. Evidence that acetylcholine released by gastrin and related polypeptides contributes to their effect on gastrointestinal motility. Gastroenterology 1973;64:268-277. 23.Nemeth PR, Zafirov DH, Wood JD. Effects of cholecystokinin, caerulein, and pentagastrin on electrical behavior of myenteric neurons. Eur J Pharm 1985;116:263-269. 24.Davison JS, Clarke GD. Mechanical properties and sensitivity to CCK of vagal gastric slowly adapting mechanoreceptors. Am J Physiol1988;255:G55-G61. 25.Seidel ER. Modulation of the muscarinic cholinergic receptor of the guinea pig gallbladder by the C-terminal octapeptide of cholecystokinin. Can J Physiol Pharmacol1986;64(Suppl):184.
Received July 5.1989. Accepted March 5,1QQ0. Address requests for reprints to: Erica A. Brotschi, M.D., Department of Surgery, Boston VAMC, 150 South Huntington Avenue, Boston, Massachusetts 02130. This work was supported by the general medical service of the Veterans Administration. The authors thank Dr. Carol Walsh for her critical review of the manuscript.
Intrinsic Nerves Affect Gallbladder Contraction in the Guinea Pig E. A. BROTSCHI,
J. PATTAVINO,
and L. F. WILLIAMS,
Jr.
Department of Surgery. Boston Veterans Administration Medical Center, and Boston University F&o01 of Medicine, Boston, Massachusetts
Muscarinic antagonists block gallbladder contraction induced by cholecystokinin in vivo but have little effect on gallbladder muscle strips. This study examined the effect of neural blockade on cholecystokinin-octapeptide-induced contraction of the intact guinea pig gallbladder in vitro using cholecystokinin-octapeptide applied to the gallbladder serosa, the lumen, or both compartments simultaneously. Simultaneous cholecystokinin stimulation of both the lumen and serosa was the most potent stimulus to contraction, and the responses were significantly inhibited by atropine and tetrodotoxin. Cholecystokinin in the gallbladder lumen alone evoked contraction by a dose-dependent mechanism that was entirely blocked by atropine or tetrodotoxin. Serosal application of cholecystokinin was the least potent, resulting in contractile responses and low sensitivity to neural blockers comparable to effects reported in muscle strips. The results suggest that cholecystokinin can cause gallbladder contraction by stimulating muscle receptors, neural receptors, or both, and combined neural and muscular stimulation is the most potent contractile stimulus.
C
holecystokinin (CCK) has been known for 60 years to cause gallbladder contraction (11, and its importance as the major physiological mediator of postprandial gallbladder emptying has recently been confirmed (2). Because gallbladder smooth muscle contains a high density of high-affinity receptors for CCK (3),the interaction of blood-borne CCK with these receptors has been thought to explain postprandial gallbladder contraction. However, two observations suggest that CCK may stimulate contraction by a more complex mechanism: first, muscarinic antagonists block the contractile effect of CCK in vivo in the gallbladder of humans (4,5)and dogs (6);second, postprandial levels of CCK in serum (7) are much lower than levels required for substantial muscle-strip contraction in vitro.
Because muscarinic antagonists block the effect of exogenous and endogenous CCK on the intact gallbladder in vivo, cholinergic innervation is presumably involved in CCK’s in vivo effect. However, in vitro studies with gallbladder muscle strips show only a small change in contractility with cholinergic blockBecause disruption of gallbladder intrinsic ade (5,8). neurons during strip preparation could produce this discrepancy, we examined the effect of neural blockade on contraction in a gallbladder model with intact intrinsic neural pathways. The intact guinea pig gallbladder in organ bath was used to quantitate the contractile effect of luminal and serosal stimulation by cholecystokinin-octapeptide (CCK-OP) and to assess the role of intrinsic nerves in gallbladder contractile response. Methods Hartley female guinea pigs weighing 300-350 g were fasted overnight then anesthetized with IM ketamine (5 mg/kg) and xylazine (30 mg/kg). Gallbladders were removed in acute terminal experiments, according to the guidelines of the Veterans Administration Animal Care subcommittee. Midline laparotomy was performed, the gallbladder was aspirated, and the fasting volume was measured. Two polyethylene cannulas (0.6 mm ID] were then placed, one in the fundus of the gallbladder through a purse-string suture and one in the cystic duct, and the gallbladder was immediately suspended in a 37’C organ bath of modified Kreb’s buffer (NaCl, 133 mmol/L; NaHCO,, 16 mmol/L; NaH,PO,, 1.2 mmol/L; MgSO,, 0.04 mmol/L; KCl, 4.7 mmol/L; CaCl,, 1.25 mmol/L; and glucose, 7.8 mmol/L) bubbled with 95% 0, and 5% CO, to maintain the pH at 7.35-7.45. The fundic catheter was connected to a saline-filled pressure transducer (Hewlett-Packard 1280) and to a recorder (HP 7702B: Hewlett Packard, Waltham,
Abbreviation used in this paper: CCK-OP, cholecystokininoctapeptide. 0 1990 by the American G&roenterological Association 0018-5085/90/$3.00
September 1990
MA) for continuous monitoring of intraluminal pressure. The second intraluminal catheter was attached to a syringe on a modified Harvard apparatus rodent respirator for timed infusion and withdrawal of a specified volume of fluid from the gallbladder lumen. At the start of each experiment, a luminal volume was determined which resulted in intraluminal pressure of 5-7 cm water when infused into the unstimulated gallbladder over 45 seconds. Our previous studies confirm that this is a physiological pressure in the unstimulated guinea pig gallbladder 191,and this baseline pressure resulted in maximal reproducible responses to contractile stimulation. This initial gallbladder volume (usually about 70% of initial in vivo fasting volume] was used throughout the experiment. A dynamic method was used to measure gallbladder contractility, consisting of continuous luminal pressure recording while the gallbladder was cyclically filled and emptied (10.11). The gallbladder was equilibrated in organ bath during cyclical infusion and withdrawal of the initial volume of luminal buffer (0.67 cycle/min) until the resulting pressure tracing was reproducible in shape and magnitude. As previously described in vivo (11) and in vitro (10,X?), the shape and height of the pressure-volume curve provided a sensitive and reproducible index of the contractile state of the gallbladder. Pressure tracings demonstrated a sine-wave configuration that was dependent on contractile tone and receptive relaxation in the viable gallbladder preparation as well as on the flow characteristics of the pump. The parameter used to measure contractility was peak pressure generated in the gallbladder during the third infusion-withdrawal cycle. In a previous study, we separately analyzed the slope and area of the pressure-volume curve and determined that these data paralleled peak pressure response closely (9). The gallbladder was stimulated using a series of buffers with increasing concentrations of CCK-OP (Kinevac; Squibb, New Brunswick, NJ] from lo-l5 to 5 x 10m7 mol/L. The CCK-OP was added to the serosal buffer only, to the luminal buffer only, or to both simultaneously at identical concentrations. Only one route of stimulation was tested in each gallbladder. In some experiments, tetrodotoxin, 10e7 mol/L (Sigma, St. Louis, MO], or atropine, 3 x 1O-6 mol/L (Anpro, Arcadia, CA), was added in conjunction with the CCK-OP. The entire gallbladder pressure response to addition of an agonist was seen immediately on the first infusion-withdrawal cycle, with no perceptible latency, whether the agonist was applied to the lumen, the serosa, or both (Figure 1). At the termination of each experiment a maximal gallbladder contraction was induced by the addition of acetylcholine 5 x 10e4 mol/L [Sigma) to both the luminal and serosal sides of the gallbladder. Either acetylcholine. 5 x 10m4mol/L, or CCK-OP, 5 x 1O-7 mol/L, applied to the luminal and serosal sides of the gallbladder induced a maximal contraction in this preparation, which was not exceeded by stimulation with higher agonist doses. To assess architecture and morphology of the gallbladder preparation, distended gallbladders were transfered into formalin at the end of the experiment, fixed, sectioned, and examined with H&E staining. Data were analyzed by measuring the observed increase in peak gallbladder pressure caused by CCK-OP stimulation and dividing this by the increment in peak gallbladder
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1 min Figure 1.Typicalhdnd pressure tracing showing the effect of various doses of CCK-OP applied simultaneously to the serosa and the lumen of a single gallbladder preparation. A. Pressure tracing in modified Krebs’buffer before stimulation. mol/L CCK-OP. B. Pressure response during stimulation with lo-l5 C. Pressure response during stimulation with lo-” mol/L CCK-OP. D. Pressure response during stimulation with lo-’ mol/L CCK-OP.
pressure during maximal contraction. This ratio was expressed as a percentage of the maximal contractile response. Because gallbladder volume and effective radius were essentially constant for all measurements during an experiment, and the gallbladder was approximately spherical, the measured luminal pressure reflected average wall tension, based on Laplace’s Law [tension = radius x pressure/2). Thus the ratio of the observed pressure response over maximal pressure response reflected the approximate percent of maximal wall tension generated in response to stimulation. Unless otherwise specified, data were evaluated using analysis of variance to compare intragroup and intergroup responses at each concentration of CCK-OP, using Dunnett’s test for significance. Probability to.05 was interpreted as statistically significant. Data are expressed as mean f SE.
Results Microscopic examination of gallbladders used in these experiments showed normal cellular morphology and an intact mucosa. The wall thickness of the distended gallbladder was approximately 100 pm after fixation. The effects on gallbladder contractility of CCK-OP applied to the serosa, the lumen, and to both compartments simultaneously are shown in Figure 2. Combined serosal and luminal application was most potent in contracting the gallbladder, with significant contraction occurring at a CCK-OP concentration of lo-l5
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Figure 3. Effect of CCK-OP applied to the gallbladder serosa, to the lumen, or to both compartments simultaneously on gallbladder contractile response. P values denote sign&ant differences from serosal stimulation: *P < 0.05; **P c 0.01; ***P c 0.001.
mol/L (P < 0.02, baseline vs. stimulated by paired t test]. Combined serosal and luminal stimulation was more potent than either serosal or luminal stimulation alone at each concentration of CCK-OP. The approximate ED,, for combined stimulation was 1.5 x lo-l1 mol/L, compared with 2 x lo-” mol/L for luminal stimulation and 3 x lo-” mol/L for serosal stimulation. Luminal CCK-OP alone produced significant contraction at concentrations of ~10-~’ mol/L and was equally or more potent than serosal stimulation at all but the highest CCK-OP concentrations. Serosal application produced significant contraction at concentrations of ~10-‘~ mol/L. Construction of Eadie-Hofstee plots from the doseresponse data suggested that serosal stimulation was activating primarily a single class of receptors with a Kd of approximately 1.0 x 10-l’ mol/L, as previously described for the gallbladder muscle CCK receptor (13). The data from luminal and combined stimulation gave a nonlinear plot with a variable slope, suggesting a variable population of receptors. The effect of atropine or tetrodotoxin applied simultaneously with CCK-OP to both the serosa and lumen of the gallbladder is illustrated in Figure 3. Atropine inhibited contraction by approximately 50% at lower CCK-OP concentrations. Tetrodotoxin significantly inhibited the response to all CCK-OP concentrations
10-11
-9
10
Figure 3. Effect of atropine, 3 x la-’ mol/L, or tetrodotoxin, lo-’ mol/L, applied simultaneously with CCK-OP to both the lumen and the serosa of the gallbladder. P values denote significance compared with CCK-OPwithout atropine or tetrodotoxhx *P < 0.05; **p < 0.01; ***p < 0.001.
The effect of atropine or tetrodotoxin applied in conjunction with CCK-OP to the serosal surface is shown in Figure 5. Atropine significantly decreased the contractile response to serosal stimulation at 10m9 mol/L, while tetrodotoxin decreased responses at 10-l’ mol/L and 10e9 mol/L. Discussion These experiments are consistent with prior in vivo observations indicating that intrinsic choline&c nerves are important mediators of CCK’s effect on the intact gallbladder. Serosal application of CCK-OP
-
10
-15
Luminal Luminal+A Luminal+T
-8 -13 -11 10 10 10-9 10 CCK-OP Concentration (Molar)
Figure 4. Isffectof atropine, 3 x lo-’ mol/L, or tetrodotoxin, lo-’ mol/L, applied with CCK-OP to the lumen of the gallbladder. P values denote significance compared with CCK-OP without atropine or tetrodotoxim ?? P < 0.05; ?? *P < 0.01; ***P < 0.001.
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00
I
_ 80
-I
Serosal Serosal+A Serosal+T
80
0 10 -15
10 -13
10 -11
10 -9
lo-8
CCK-OP Concentration (Molar) Figure 5. Effect of &opine, 3 x 10e6movL, or tetrodotoxin, lo-' mol& applied with CCK-OP to the serosaf surface of the gallbladder. P values denote sign&awe compared with CCK-OP without atropine or tetrodotoxh *P c 0.05; ?? *P < 0.01; *+*P-c0.001.
induced contraction that resembled the contractile response seen by others in muscle strips in dose response and the effect of cholinergic blockade (3). In gallbladder strips, as well as in this model, cholinergitally mediated (scopolamine-sensitive) contraction accounts for a significant fraction of the contractile response at low CCK-OP concentrations (50% at 10-l’ mol/L vs. 20% at lo-* mol/L) (5,14). The apparent further inhibition seen with tetrodotoxin in our study might reflect the contribution of noncholinergic nerves to contraction or might be caused by additional suppression of cholinergic neural responses that were not blocked by the dose of atropine used. Luminal stimulation in our model appeared to evoke a neurally mediated contractile response that has not previously been described. The luminal CCK-OP appeared to stimulate submucosal nerves to produce gallbladder contraction, and inhibition of this response by atropine and tetrodotoxin showed that smooth muscle receptors for CCK were not being activated during luminal stimulation at CCK-OP concentrations
NERVES IN GALLBLADDER
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829
response curve to the right, and tetrodotoxin shifted it still further. The increased response seen with combined luminal and serosal CCK-OP may indicate that stimulation of an intrinsic neural plexus contributed to a lower threshhold for smooth muscle activation or that smooth muscle binding of CCK-OP enhanced the response to neural stimulation. These possibilities are consistent with previous observations in intact gallbladder models that subthreshhold doses of polypeptide hormones greatly increased the response to transmural electrical stimulation in the isolated perfused guinea pig gallbladder (15). Conversely, electrical stimulation of the guinea pig gallbladder in vivo enhanced the contractile response to CCK-OP over a range of doses (16). The potential for neurally mediated gallbladder contraction is not surprising when gallbladder anatomy is considered. The anatomy of the gallbladder includes a complex innervation, analogous to that in the bowel wall. Two ganglionated nerve plexuses have been identified, located in the subserosal and submucosal layers (17). The intrinsic nerve plexus communicates with extrinsic fibers from the vagus, the celiac ganglion, and the thoracic dorsal root ganglia (18). A number of enteric peptide hormones and other neurotransmitter substances have been identified by immunocytochemistry in nerves and ganglia of the gallbladder, including vasoactive intestinal polypeptide (19), galanin (201, substance P (18), gastrinreleasing hormone (20), and neuropeptide Y (18). Plexuses of nerve bundles and fibers have been identified by histochemical techniques as discrete networks of adrenergic, cholinergic, and “purinergic” nerves in the gallbladder wall (21). Cholecystokinin-octapeptide has been shown to be an important neurotransmitter in the ileum, where its contractile effect is mediated by cholinergic nerves (22). Direct electrical measurements from neurons provide further evidence that CCK-OP functions as a neurotransmitter, causing depolarization of ileal myenteric neurons (23). and discharge from gastric mechanoreceptor neurons in the vagus (24). Although the gallbladder is innervated by the enteric nervous system, most investigators have concluded that CCK’s effect as a cholinergically mediated neurotransmitter in gallbladder muscle strips is relatively unimportant (8). Perhaps the relative paucity of neural elements in the gallbladder wall compared with the ileum (17) contributes to the inability to measure neural effects in gallbladder muscle strips which are easily measured in ileal muscle. Several studies now demonstrate that the intact gallbladder in vivo does require cholinergic innervation to respond to CCK (4-6), and our studies suggest that the effects of CCK in the intact in vitro gallbladder also involve intrinsic nerves. The observation that CCK-OP transiently enhances binding to
830 BROTSCHI ET AL.
muscarinic receptors in gallbladder tissue (25) suggests that CCK may have a complex role in modulating gallbladder neural responses. It is evident that neither mode of stimulation we used is identical to the circulation of CCK-OP through muscular and submucosal capillary beds. However, it seems likely that blood-borne CCK could stimulate gallbladder intrinsic nerves. Further investigation of the events that stimulate gallbladder neural pathways may increase our understanding of physiological gallbladder contraction. References 1. Ivy AC, Oldberg E. A hormone mechanism for gallbladder contraction and evacuation. Am J Physiol1929;91:338-344. 2. Shiratori K, Watanabe S, Chey WY, Lee KY, Chang T-M. Endogenous cholecystokinin drives gallbladder emptying in dogs. Am J Physiol1986;251:G553-G558. 3. Grider JR, Makhlouf GM. Regional and cellular heterogeneity of cholecystokinin receptors mediating muscle contraction in the gut. Gastroenterology 1987;92:175-180. 4. Fisher RS. Rock E, Malmud LS. Choline@ effects on gallbladder emptying in humans. Gastroenterology 1985;89:716-722. 5.Takahashi T, Yamamura T, Ishikawa Y, Kantoh M. Utsunomiya J. Effects of cholecystokinin-octapeptide on the human gallbladder both in vivo and in vitro. Gastroenterol Jpn 1986;21:49-54. 8.Takahashi I, Suzuki T, Aizawa I, Itoh Z. Comparison of gallbladder contractions induced by motilin and cholecystokinin in dogs. Gastroenterology 1982;82:419-424. JE. Cholecystokinin7. Walsh JH, Lamers CB, Valenzuela octapeptidelike immunoreactivity in human plasma. Gastroenterology 1982;82:438-444. 8. Yau WM. Makhlouf GM, Edwards LE. Farrar JT. Mode of action of cholecystokinin and related peptides on gallbladder muscle. Gastroenterology 1973;65:451-456. 9. Brotschi EA, LaMorte WW, Williams LF Jr. Effect of dietary cholesterol and indomethacin on cholelithiasis and gallbladder motility in the guinea pig. Dig Dis Sci 1984;29:1050-1057. 10.Matsuki Y. Dynamic stiffness of the isolatedguinea-piggallbladder during contraction induced by cholecystokinin. Jpn J Smooth Muscle Res 1985;21:427-438. 11.Schoetz DJ, LaMorte WW, Wise WE, Birkett DH, Williams LF Jr. Mechanical properties of primate gallbladder: description of a dynamic method. Am J Physiol1981;241:G376-G381. 12.Davison JS, Al-Hassani M. The role of noncholinetgic, nonadrenergic nerves in regulating the distensibility of the guinea pig gallbladder. In: Christensen J, ed. Gastrointestinal motility. New York: Raven, 1980:89-95.
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13.Chang RSL, Lotti VJ. Biochemical and pharmacologic characterization of an extremely potent and selective nonpeptide cholecystokinin antagonist. PNAS 1986;83:4923-4926. 14.Yamamura T, Takahashi T, Kusunoki M, Kantoh M, Ishikawa Y, Utsunomiya J. Cholecystokinin octapeptide-evoked [“HIacetylcholine release from guinea pig gallbladder. Neurosci Lett 1986;65:167-170. 15.Al-Hassani MH, Davison JS. Interactions between intrinsic cholinergic nerve stimulation and gastrointestinal polypeptides on the guinea-pig gallbladder. J Physiol (Lond) 1978;285:24P. 16.Pomeranz IS, Davison JS, Friedhandler TM, Shaffer EA. The relative importance of choline&c and hormonal stimuli in in vivo gallbladder contraction (abstr). Gastroenterology 1983;84: Al275. 17.Sutherland SD. The neurons of the gallbladder and gut. J Anat 1967;101:701-709. 18.Mawe GM, Gershon MD. Unique properties of gallbladder ganglia: relationship to the enteric nervous system (ENS) (abstr]. Gastroenterology 1988;94:A292. 19.Sundler F, Alumets J, Hakanson R, Ingemansson S, Fahrenkrug J, Schaffalitzky de Muckadell 0. VIP innervation of the gallbladder. Gastroenterology 1977;72:1375-1377. 20.Padbury RTA, Toouli J, Furness JB. The histochemistry of gallbladder innervation (abstr). Gastroenterology 1988;94:A339. 21.Davison JS, Al-Hassani M, Crowe R, Burnstock G. The nonadrenergic, inhibitory innervation of the guinea-pig gallbladder. Pilugers Arch 1978;377:43-49. 22.Vizi SE, Bertaccini G. Impicciatore M, Knoll J. Evidence that acetylcholine released by gastrin and related polypeptides contributes to their effect on gastrointestinal motility. Gastroenterology 1973;64:268-277. 23.Nemeth PR, Zafirov DH, Wood JD. Effects of cholecystokinin, caerulein, and pentagastrin on electrical behavior of myenteric neurons. Eur J Pharm 1985;116:263-269. 24.Davison JS, Clarke GD. Mechanical properties and sensitivity to CCK of vagal gastric slowly adapting mechanoreceptors. Am J Physiol1988;255:G55-G61. 25.Seidel ER. Modulation of the muscarinic cholinergic receptor of the guinea pig gallbladder by the C-terminal octapeptide of cholecystokinin. Can J Physiol Pharmacol1986;64(Suppl):184.
Received July 5.1989. Accepted March 5,1QQ0. Address requests for reprints to: Erica A. Brotschi, M.D., Department of Surgery, Boston VAMC, 150 South Huntington Avenue, Boston, Massachusetts 02130. This work was supported by the general medical service of the Veterans Administration. The authors thank Dr. Carol Walsh for her critical review of the manuscript.