Neuro-Modulation of Ion Secretion by Inflammatory Mediators" HELEN J. COOKE Department of Physiology 302 Hamilton Hall I645 Neil Avenue Columbus. Ohio 43210
INTRODUCTION The host is protected from the multitude of microorganisms presiding within the intestinal lumen. The integrity of the intestinal epithelium, secretion of mucus (in combination with secretory IgA), the presence of antimicrobial metabolites produced by normal enteric flora, peristalsis and possibly secretion of fluid all impede the entry and dissemination of enteric pathogens. Disruption of the epithelial barrier and uptake of antigen triggers an immune response which results in The antibodies sensitize local mast cells by binding production of IgE or IgG, to Fc receptors on their surface for periods as long as several months.' Mast cells have important immunoregulatory functions at the interface of the intestine with the external environment. Challenge of sensitized intestine with antigen elicits a type I hypersensitivity reaction that results from crosslinking the antibodies occupying Fc receptors on mast cells and the consequent release of mediators. This activates intestinal effector systems resulting in profuse secretion and altered motility patterns which are coordinated to clear the intestinal lumen of its contents with a possible consequence of producing a diarrheal state.4 Preformed and newly synthesized substances are released from mast cells in response to immunologic stimuli. Soluble mediators that affect ion transport include histamine, prostaglandins, leukotrienes, adenosine, 5-hydroxytryptamine and others.2 This paper will focus on the interactions of a few of these mast cell mediators in amplification of secretory responses. Augmentation of secretion can occur at several levels: 1) Mast cell mediators may stimulate different signal transduction pathways and second messengers within epithelial cells to enhance each other's 2) At the level of the epithelial cell, mast cell mediators may augment the actions of other intermediary messengers released from immunocytes or mesenchymal cells within the submucosa/lamina propria;2 3) Mast cell mediators may increase neuronal excitability resulting in release of neurotransmitters which could augment the epithelial responses to the mediators.' To study interactions of mast cell mediators with the enteric nervous system and the epithelium, two models have been used in my laboratory. These include the Trichinella spiralis parasitized host, and bovine milk-sensitized guinea pigs. In the former model, guinea pigs are immunized by oral inoculation with 2 x lo3 Trichinella spiralis larvae and sacrificed 6-8 weeks later.3-4Colonic tissues are challenged in vitro with a somatic protein antigen derived from infective Trichinella .'v2
a This work was supported by National Institutes of Health Grants R01-DK37240 and R01-DK37237 and by the Ohio State University Grant B/A 222157. 346
COOKE: INFLAMMATORY MEDIATORS
347
spiralis larvae. In a second group of animals, immunization to P-lactoglobulin results from oral ingestion of bovine milk which is substituted for water for a threeweek period. Animals are sacrificed and colonic segments are challenged in uitro with P-lactoglobulin.8 In parasitized- or bovine milk-sensitized animals, addition of Trichinella spiralis antigen or p-lactoglobulin, respectively, evokes an increase in short-circuit current reflecting an increase in chloride secretion.,^^ Active transport of chloride into the lumen accompanied by passive flux of sodium provides an osmotic driving force for water movement. The responses to antigenic challenge in sensitized animals are reduced by histamine receptor antagonists, by prostaglandin synthesis inhibitors and leukotriene D, receptor antagonist^.^^^.^ Administration of either Trichinella spiralis or P-lactoglobulin to nonimmunized animals does not elicit a response. The implications of these findings are that histamine, prostaglandins and leukotrienes mediate secretory responses during intestinal anaphylaxis. Mast cell mediators influence colonic secretion, in part, by activating cholinergic neurons in the submucous plexus and by activating epithelial cells d i r e ~ t l y . ~ J ~ J ~ The involvement of cholinergic neurons is substantiated by reports that tetrodotoxin and atropine reduce the secretory response and that P-lactoglobulin stimulates release of a ~ e t y l c h o l i n e .Because ~*~ mast cell mediators can act directly on epithelial cells, or indirectly by releasing chemical messengers from neurons, mesenchymal cells or other immunocytes, there is potential for synergistic interactions of second messenger systems at multiple sites. Amplification of Responses: Action of Mast Cell Mediators on Epithelial Cells
Mast cell mediators that act directly on epithelial cells can augment each other’s actions by stimulating different signal transduction pathways and second messenger systems. Elevation of either intracellular calcium or cyclic AMP is known to evoke chloride secretion.’S6 Synergistic interactions occur when both intracellular messengers are elevated simultaneously resulting in profuse secretion. Histamine acts at H , receptors on epithelial cells to increase intracellular calcium and chloride secretion.I0,l2Histamine has the potential for enhancing the effects of other mast cell mediators that activate different signal transduction pathways and second messenger systems in epithelial cells. This is evident when intracellular cyclic AMP is increased, because secretory responses to prostaglandins and adenosine are augmented by prior administration of histamine. lo Augmenting Interactions: Release of Intermediary Messengers from Cells in the SubmucosalLamina Propria
Histamine augments the secretory effects of the cholinergic agonist, carbachol. This appears at first to be a paradox, because both histamine and carbachol utilize intracellular calcium as a signal to evoke secretion. However, histamine evokes release of prostaglandins E2 from mesenchymal cells within the submucosa.2,6.10 Consequently, the induction of two different second messenger systems could explain the amplification of the secretory response. Evidence that this is the case comes from observations that augmentation of the carbachol response by exogenous histamine can be prevented by prostaglandin synthesis inhibitors. l o These observations suggest that increased secretion occurs as a result of the combined interaction of elevated intracellular calcium by hista-
348
ANNALS NEW YORK ACADEMY OF SCIENCES
FIGURE 1. Innervation of the epithelium. Sensory neurons which detect the composition of the intestinal lumen or tension in the gut wall activate interneurons and motor neurons regulating epithelial chloride secretion. Stimulation of secretion may result from activation of neural reflex pathways within the submucous plexus or via activation of myenteric neurons that synapse with submucous neurons. Acetylcholine and possibly vasoactive intestinal peptide (VIP)are neurotransmitters at neuro-epithelial junctions. Excitatory or inhibitory input to the submucous motor neurons may arise from either other submucous neurons or from myenteric neurons. Extrinsic sympathetic axons also provide inhibitory input to the submucous ganglia.
mine and cyclic AMP by prostaglandins. This is likely to occur during intestinal anaphylaxis when both histamine and prostaglandins are released simultaneously from mast cells. This illustrates only one example of how secretory responses are amplified when many different chemical messengers are released. Enhanced Secretory Responses: Effects of Activating Enteric Neurons The epithelium is richly innervated by the enteric nervous system consisting of the myenteric plexus lying between the longitudinal and circular muscle and the submucous plexus within the submucosa (FIG.1). Continuous monitoring of the luminal composition or tension in the gut wall by sensory neurons provides excitatory and inhibitory input to interneurons and motor neurons which control the rate of secretion of salts and water into the lumen (FIG.l).I39l4 Acetylcholine and possibly vasoactive intestinal polypeptide (VIP) appear to be the neurotransmitters released from submucous motor neurons at neuroepithelial junctions.13.'4 In bovine milk- or Trichinellu spiralis-sensitized animals, there is a marked increase in the number of submucous neurons that have spontaneousaction potential dis~harge.~~'~-'* In nonimmune animals, only a small percentage of the popula-
COOKE: INFLAMMATORY MEDIATORS
349
tion tested are spontaneously active in contrast to about one third of the neurons in immune animals.17~laThis activity is due to endogenous histamine present in colonic tissues from sensitized animals and is prevented by histamine receptor blockers.la The reason for greater levels of endogenous histamine in immune tissues is unknown. It could result from: 1) an increased number of mast cells that secrete the same basal rate of histamine; 2) an increased basal secretion rate from the same number of mast cells; 3) or a combination of both. Mast cell hyperplasia has been reported for the parasitized host, but not for milk-sensitized animak3J9 The finding that similar numbers of neurons were spontaneously active in Trichinella spiralis- and bovine milk-sensitized animals would suggest factors other than mast cell numbers must account for these similarities in the two models. Despite hyperactivity of neurons in immune animals, there is little difference in the basal ion transport rates in tissues from immune and nonimmune animals (TABLE1). This is reflected by similar short-circuit currents and tissue conductances in both groups (TABLE1). The reason for this observation is unclear. Possibilities that must be considered include: 1) The hyperactive neurons release neurotransmitters at the interface of effector systems other than the epithelium, i.e., muscularis muscosae or blood vessels; 2) Insufficient numbers of neurons are activated synchronously and fail to produce a change in ion transport. It is likely that basal release of mediators from mast cells of sensitized animals serves to prime the neural circuits by depolarizing them to values near their threshold for action potential discharge. In guinea pigs sensitized to P-lactoglobulin, antigenic challenge releases histamine and prostaglandins among other mediators which may alter the activity of the enteric circuits and thereby indirectly alter secretion rate^.^.^^ The action of histamine at H , receptors on epithelial cells has already been discussed and will not be considered further. Histamine also acts at H2 receptors on submucous neurons to evoke long-lasting, cyclical patterns of secretion in the guinea pig c o l ~ n . H2 ~ ~receptor J~ activation in other cell systems is coupled to elevation of cyclic AMP, and it is likely to be the second messenger system in submucous neurons. An accessory event is suppression of synaptic transmission in the enteric microcircuits by activation of presynaptic H, receptors. The amplitude of the secretory response is regulated by H, receptors on presynaptic neuron^.'^.*^ Activation of H, receptors may provide a mechanism for suppressing activity in some circuits while others continue to be recruited. Prostaglandins, which are released simultaneously with histamine from mast cells, may also affect neuronal activity and intestinal secretion in some species.2 A complex interplay of chemical messengers results when leukotrienes are released from mast cells. Leukotriene D4 stimulates chloride secretion, which can be significantly reduced by tetrodotoxin and a t r ~ p i n e . ~In. ~either ' nonimmune or sensitized guinea pigs, when there is no ongoing neural activity influencing basal 15920
TABLE 1. Basal Electrical Properties of Nonimmune and Trichinella spiralis-
Sensitized Submucosa/Mucosa Preparations from Guinea Pig ColonU Nonimmune Immune Isc G
*
-14 4 8.5 k 0.4
-20 7.4
~~
(I
Isc, short-circuit current; G , tissue conductance.
2 4 pAlcmZ +. 0.3 mS/cm2
350
ANNALS NEW YORK ACADEMY OF SCIENCES
ion transport, the secretory response to leukotriene D4 is small compared to tissues with ongoing neural activity. Comparison of the responses in immune and nonimmune animals suggests that leukotriene D4 evokes greater secretory responses in immune animals, irrespective of the level of ongoing neural activity.*' Leukotrienes enhance the secretory response to carbachol in submucosa/mucosa preparations of guinea pig colon.*' The augmented response is prevented if tetrodotoxin is present prior to addition of leukotrienes. Leukotrienes may not act
SU6MUCOUS NEURONS
FIGURE 2. Interactions of mast cell mediators with the enteric nervous system and the epithelium of guinea pigs. Histamine acts at HI receptors on epithelial cells to evoke a transient secretory response. Histamine acting at H2receptors on submucous neurons evokes a long-lasting, cyclical pattern of secretion. The action of histamine at HIreceptors shuts down synaptic circuits, reduces neurotransmitter release, and acts as a brake to reduce the amplitude of the secretory response. Histamine releases prostaglandins from mesenchymal cells and can act directly on epithelial cells as well as on neurons to evoke secretion. Leukotrienes stimulate secretion predominantly by acting on submucous neurons, but may release intermediary messengers from other cells. Leukotriene activation of neurons that are already in an excitable state gives larger secretory responses than those that are not. Simultaneous elevation of two second messengers by any of these mediators is expected to augment chloride secretion.
directly on epithelial cells to augment basal secretion induced by carbachol, because they have no effect on basal transport properties or carbachol-stimulated secretion in T84 colonic epithelial cells.*' These observations imply that leukotrienes amplify secretory responses to carbachol only if they, or an intermediary messenger they release, can activate submucous neurons projecting to the epithelium.
COOKE: INFLAMMATORY MEDIATORS
351
Secretory responses during intestinal anaphylaxis result from a complex interplay between multiple mediators that affect several signal transduction pathways and second messenger systems. FIGURE 2 illustrates the potential interactions among only three of the multitude of mediators that are released from mast cells. Additional mediators that are released will add to the complexity of the interactions between the enteric nervous system, the immune system and the epithelium. The end result is induction of secretion, which is coordinated with other effector systems, to rapidly clear material from the lumen with the possibility of inducing a diarrheal state. ACKNOWLEDGMENTS
I am indebted to Dr. Thomas Frieling, Dr. Jeffrey Palmer, Dr. Y.-Z.Wang, Dr. Y.Rikihisa and Ms. Najma Javed for their contributions to the work described in this paper. The technical assistance of Ms. Jeannette Bidinger and Ms. Beth Anthony is appreciated. REFERENCES 1. ROITT,I., J. BROSTOFF & D. MALE. 1985. Immunology. The C.V. Mosby Co. St. Louis, MO. 2. SARTOR, R. B. & D. W. POWELL.1991. Mechanisms of diarrhea in intestinal inflammation and hypersensitivity: immune system modulation of intestinal transport. In Current Topics in Gastroenterology: Diarrheal Diseases. M. Field, Ed. 75-1 14. Elsevier. New York. 3. CASTRO,G. A. 1982. Immunological regulation of epithelial function. Am. J. Physiol. 243: G3214329. 4. WANG,Y.-2.. J. M. PALMER & H. J. COOKE.1990. Neuroimmune regulation of colonic secretion in nematode-infected guinea pigs. Am. J. Physiol. 260: G307-G3 14. 5 . YAJIMA,T, T. SUZUKI & Y. SUZUKI. 1988. Synergism between calcium-mediated and cyclical AMP-mediated activation of chloride secretion in isolated guinea pig distal colon. Jpn. J. Physiol. 38: 427-443. 6. CHANG,E. B. & M. C. RAO. 1991. Intracellular mediators of intestinal electrolyte transport. In Current Topics in Gastroenterology: Diarrheal Diseases. M. Field, Ed. 49-72. Elsevier. New York. 7. WOOD,J. D. 1991. Communication between minibrain in gut and enteric immune system. NIPS 6 64-69. 8. WANG,Y.-Z. & H. J. COOKE.1991. Comparison of effects of /3-lactoglobulin and Nformyl-methionyl-leucyl-phenylalanine(FMLP) on ion transport in the colon of milksensitized guinea pigs. Gastroenterology 100: A708. 9. JAVED,N., Y.-Z. WANG& H. J. COOKE.1991. Mast cell inflammatory mediators stimulate acetylcholine release from submucosal neurons during intestinal anaphylaxis, Gastroenterology 100: A453. 10. WANG,Y.-Z., H. J. COOKE,H.-C. Su & R. FERTEL.1990. Histamine augments colonic secretion in the guinea pig distal colon. Am. J. Physiol. 2 5 8 (34324439. 11. WANG,Y.-Z. & H. J. COOKE.1990. H2 Receptors mediate cyclical chloride secretion in the guinea pig distal colon. Am. J. Physiol. 258: G887-G893. 12. WASSERMAN, S. I., K. E. BARRETT, P. A. HUOTT,G. BEUERLEIN, M. F. KAGNOFF & K. DHARMSATHAPHORN. 1988. Immune-related intestinal C1- secretion. I. Effect of histamine on the T84 cell line. Am. J. Physiol. 254: C53-C61. 13. COOKE,H. J. & H. V. CAREY.1990. Neural regulation of intestinal ion transport. I n Testbook of Secretory Diarrhea. E. Lebenthal & M. Duffey, Eds. 1-14. Raven Press, Ltd. New York.
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J. C. & J. B. FURNESS.1988. Correlated electrophysiological and histo14. BORNSTEIN, chemical studies of submucous neurons and their contribution to understanding enteric neural circuits. J. Auton. Nerv. Syt. 25: 1-13. Y.-Z.WANG& J. D. WOOD.1990. Neuro-immuno-modula15. COOKE,H. J., T. FRIELING, tion of intestinal chloride secretion. In Exocrine Secretion 11. P. Y. D. Wong & J. A. Young, Eds. 39-42. United League Graphics and Printing Co., Ltd. Hong
Kong. T., H. J. COOKE& J. D. 16. FRIELING, 17. 18. 19. 20. 21.
WOOD.1991. ElectroRhysiological properties of neurons in submucous ganglia of the guinea-pig distal colon. Am. J. Physiol. 260. G835-G841. FRIELING, T., H. J. COOKE& J. D. WOOD. 1991. Synaptic transmission in submucous ganglia of the guinea-pig distal colon. Am. J. Physiol. 261: G37-G44. FRIELING, T., H. J. COOKE,J. D. WOOD& J. M. PALMER.1991. Electrical and synaptic behavior of colonic submucosal neurons in Trichinella spiralis and p-lactoglobulinsensitized guinea pigs. Gastroenterology 9 8 A352. CROWE,S. E. & M. H. PERDUE.1992. Gastrointestinal food hypersensitivity: basic mechanisms and pathology. Gastroenterology. In press. WANG,Y.-Z. & H. J. COOKE.1990. Presynaptic H3receptors regulate cyclical secretion in guinea pig distal colon. Gastroenterology 98: A400. JAVED, N. & H. J. COOKE.1991. Effect of leukotriene D4 on colonic ion transport in p-lactoglobulin-sensitizedguinea pigs. Gastroenterology 100: A708.
INTRODUCTION The host is protected from the multitude of microorganisms presiding within the intestinal lumen. The integrity of the intestinal epithelium, secretion of mucus (in combination with secretory IgA), the presence of antimicrobial metabolites produced by normal enteric flora, peristalsis and possibly secretion of fluid all impede the entry and dissemination of enteric pathogens. Disruption of the epithelial barrier and uptake of antigen triggers an immune response which results in The antibodies sensitize local mast cells by binding production of IgE or IgG, to Fc receptors on their surface for periods as long as several months.' Mast cells have important immunoregulatory functions at the interface of the intestine with the external environment. Challenge of sensitized intestine with antigen elicits a type I hypersensitivity reaction that results from crosslinking the antibodies occupying Fc receptors on mast cells and the consequent release of mediators. This activates intestinal effector systems resulting in profuse secretion and altered motility patterns which are coordinated to clear the intestinal lumen of its contents with a possible consequence of producing a diarrheal state.4 Preformed and newly synthesized substances are released from mast cells in response to immunologic stimuli. Soluble mediators that affect ion transport include histamine, prostaglandins, leukotrienes, adenosine, 5-hydroxytryptamine and others.2 This paper will focus on the interactions of a few of these mast cell mediators in amplification of secretory responses. Augmentation of secretion can occur at several levels: 1) Mast cell mediators may stimulate different signal transduction pathways and second messengers within epithelial cells to enhance each other's 2) At the level of the epithelial cell, mast cell mediators may augment the actions of other intermediary messengers released from immunocytes or mesenchymal cells within the submucosa/lamina propria;2 3) Mast cell mediators may increase neuronal excitability resulting in release of neurotransmitters which could augment the epithelial responses to the mediators.' To study interactions of mast cell mediators with the enteric nervous system and the epithelium, two models have been used in my laboratory. These include the Trichinella spiralis parasitized host, and bovine milk-sensitized guinea pigs. In the former model, guinea pigs are immunized by oral inoculation with 2 x lo3 Trichinella spiralis larvae and sacrificed 6-8 weeks later.3-4Colonic tissues are challenged in vitro with a somatic protein antigen derived from infective Trichinella .'v2
a This work was supported by National Institutes of Health Grants R01-DK37240 and R01-DK37237 and by the Ohio State University Grant B/A 222157. 346
COOKE: INFLAMMATORY MEDIATORS
347
spiralis larvae. In a second group of animals, immunization to P-lactoglobulin results from oral ingestion of bovine milk which is substituted for water for a threeweek period. Animals are sacrificed and colonic segments are challenged in uitro with P-lactoglobulin.8 In parasitized- or bovine milk-sensitized animals, addition of Trichinella spiralis antigen or p-lactoglobulin, respectively, evokes an increase in short-circuit current reflecting an increase in chloride secretion.,^^ Active transport of chloride into the lumen accompanied by passive flux of sodium provides an osmotic driving force for water movement. The responses to antigenic challenge in sensitized animals are reduced by histamine receptor antagonists, by prostaglandin synthesis inhibitors and leukotriene D, receptor antagonist^.^^^.^ Administration of either Trichinella spiralis or P-lactoglobulin to nonimmunized animals does not elicit a response. The implications of these findings are that histamine, prostaglandins and leukotrienes mediate secretory responses during intestinal anaphylaxis. Mast cell mediators influence colonic secretion, in part, by activating cholinergic neurons in the submucous plexus and by activating epithelial cells d i r e ~ t l y . ~ J ~ J ~ The involvement of cholinergic neurons is substantiated by reports that tetrodotoxin and atropine reduce the secretory response and that P-lactoglobulin stimulates release of a ~ e t y l c h o l i n e .Because ~*~ mast cell mediators can act directly on epithelial cells, or indirectly by releasing chemical messengers from neurons, mesenchymal cells or other immunocytes, there is potential for synergistic interactions of second messenger systems at multiple sites. Amplification of Responses: Action of Mast Cell Mediators on Epithelial Cells
Mast cell mediators that act directly on epithelial cells can augment each other’s actions by stimulating different signal transduction pathways and second messenger systems. Elevation of either intracellular calcium or cyclic AMP is known to evoke chloride secretion.’S6 Synergistic interactions occur when both intracellular messengers are elevated simultaneously resulting in profuse secretion. Histamine acts at H , receptors on epithelial cells to increase intracellular calcium and chloride secretion.I0,l2Histamine has the potential for enhancing the effects of other mast cell mediators that activate different signal transduction pathways and second messenger systems in epithelial cells. This is evident when intracellular cyclic AMP is increased, because secretory responses to prostaglandins and adenosine are augmented by prior administration of histamine. lo Augmenting Interactions: Release of Intermediary Messengers from Cells in the SubmucosalLamina Propria
Histamine augments the secretory effects of the cholinergic agonist, carbachol. This appears at first to be a paradox, because both histamine and carbachol utilize intracellular calcium as a signal to evoke secretion. However, histamine evokes release of prostaglandins E2 from mesenchymal cells within the submucosa.2,6.10 Consequently, the induction of two different second messenger systems could explain the amplification of the secretory response. Evidence that this is the case comes from observations that augmentation of the carbachol response by exogenous histamine can be prevented by prostaglandin synthesis inhibitors. l o These observations suggest that increased secretion occurs as a result of the combined interaction of elevated intracellular calcium by hista-
348
ANNALS NEW YORK ACADEMY OF SCIENCES
FIGURE 1. Innervation of the epithelium. Sensory neurons which detect the composition of the intestinal lumen or tension in the gut wall activate interneurons and motor neurons regulating epithelial chloride secretion. Stimulation of secretion may result from activation of neural reflex pathways within the submucous plexus or via activation of myenteric neurons that synapse with submucous neurons. Acetylcholine and possibly vasoactive intestinal peptide (VIP)are neurotransmitters at neuro-epithelial junctions. Excitatory or inhibitory input to the submucous motor neurons may arise from either other submucous neurons or from myenteric neurons. Extrinsic sympathetic axons also provide inhibitory input to the submucous ganglia.
mine and cyclic AMP by prostaglandins. This is likely to occur during intestinal anaphylaxis when both histamine and prostaglandins are released simultaneously from mast cells. This illustrates only one example of how secretory responses are amplified when many different chemical messengers are released. Enhanced Secretory Responses: Effects of Activating Enteric Neurons The epithelium is richly innervated by the enteric nervous system consisting of the myenteric plexus lying between the longitudinal and circular muscle and the submucous plexus within the submucosa (FIG.1). Continuous monitoring of the luminal composition or tension in the gut wall by sensory neurons provides excitatory and inhibitory input to interneurons and motor neurons which control the rate of secretion of salts and water into the lumen (FIG.l).I39l4 Acetylcholine and possibly vasoactive intestinal polypeptide (VIP) appear to be the neurotransmitters released from submucous motor neurons at neuroepithelial junctions.13.'4 In bovine milk- or Trichinellu spiralis-sensitized animals, there is a marked increase in the number of submucous neurons that have spontaneousaction potential dis~harge.~~'~-'* In nonimmune animals, only a small percentage of the popula-
COOKE: INFLAMMATORY MEDIATORS
349
tion tested are spontaneously active in contrast to about one third of the neurons in immune animals.17~laThis activity is due to endogenous histamine present in colonic tissues from sensitized animals and is prevented by histamine receptor blockers.la The reason for greater levels of endogenous histamine in immune tissues is unknown. It could result from: 1) an increased number of mast cells that secrete the same basal rate of histamine; 2) an increased basal secretion rate from the same number of mast cells; 3) or a combination of both. Mast cell hyperplasia has been reported for the parasitized host, but not for milk-sensitized animak3J9 The finding that similar numbers of neurons were spontaneously active in Trichinella spiralis- and bovine milk-sensitized animals would suggest factors other than mast cell numbers must account for these similarities in the two models. Despite hyperactivity of neurons in immune animals, there is little difference in the basal ion transport rates in tissues from immune and nonimmune animals (TABLE1). This is reflected by similar short-circuit currents and tissue conductances in both groups (TABLE1). The reason for this observation is unclear. Possibilities that must be considered include: 1) The hyperactive neurons release neurotransmitters at the interface of effector systems other than the epithelium, i.e., muscularis muscosae or blood vessels; 2) Insufficient numbers of neurons are activated synchronously and fail to produce a change in ion transport. It is likely that basal release of mediators from mast cells of sensitized animals serves to prime the neural circuits by depolarizing them to values near their threshold for action potential discharge. In guinea pigs sensitized to P-lactoglobulin, antigenic challenge releases histamine and prostaglandins among other mediators which may alter the activity of the enteric circuits and thereby indirectly alter secretion rate^.^.^^ The action of histamine at H , receptors on epithelial cells has already been discussed and will not be considered further. Histamine also acts at H2 receptors on submucous neurons to evoke long-lasting, cyclical patterns of secretion in the guinea pig c o l ~ n . H2 ~ ~receptor J~ activation in other cell systems is coupled to elevation of cyclic AMP, and it is likely to be the second messenger system in submucous neurons. An accessory event is suppression of synaptic transmission in the enteric microcircuits by activation of presynaptic H, receptors. The amplitude of the secretory response is regulated by H, receptors on presynaptic neuron^.'^.*^ Activation of H, receptors may provide a mechanism for suppressing activity in some circuits while others continue to be recruited. Prostaglandins, which are released simultaneously with histamine from mast cells, may also affect neuronal activity and intestinal secretion in some species.2 A complex interplay of chemical messengers results when leukotrienes are released from mast cells. Leukotriene D4 stimulates chloride secretion, which can be significantly reduced by tetrodotoxin and a t r ~ p i n e . ~In. ~either ' nonimmune or sensitized guinea pigs, when there is no ongoing neural activity influencing basal 15920
TABLE 1. Basal Electrical Properties of Nonimmune and Trichinella spiralis-
Sensitized Submucosa/Mucosa Preparations from Guinea Pig ColonU Nonimmune Immune Isc G
*
-14 4 8.5 k 0.4
-20 7.4
~~
(I
Isc, short-circuit current; G , tissue conductance.
2 4 pAlcmZ +. 0.3 mS/cm2
350
ANNALS NEW YORK ACADEMY OF SCIENCES
ion transport, the secretory response to leukotriene D4 is small compared to tissues with ongoing neural activity. Comparison of the responses in immune and nonimmune animals suggests that leukotriene D4 evokes greater secretory responses in immune animals, irrespective of the level of ongoing neural activity.*' Leukotrienes enhance the secretory response to carbachol in submucosa/mucosa preparations of guinea pig colon.*' The augmented response is prevented if tetrodotoxin is present prior to addition of leukotrienes. Leukotrienes may not act
SU6MUCOUS NEURONS
FIGURE 2. Interactions of mast cell mediators with the enteric nervous system and the epithelium of guinea pigs. Histamine acts at HI receptors on epithelial cells to evoke a transient secretory response. Histamine acting at H2receptors on submucous neurons evokes a long-lasting, cyclical pattern of secretion. The action of histamine at HIreceptors shuts down synaptic circuits, reduces neurotransmitter release, and acts as a brake to reduce the amplitude of the secretory response. Histamine releases prostaglandins from mesenchymal cells and can act directly on epithelial cells as well as on neurons to evoke secretion. Leukotrienes stimulate secretion predominantly by acting on submucous neurons, but may release intermediary messengers from other cells. Leukotriene activation of neurons that are already in an excitable state gives larger secretory responses than those that are not. Simultaneous elevation of two second messengers by any of these mediators is expected to augment chloride secretion.
directly on epithelial cells to augment basal secretion induced by carbachol, because they have no effect on basal transport properties or carbachol-stimulated secretion in T84 colonic epithelial cells.*' These observations imply that leukotrienes amplify secretory responses to carbachol only if they, or an intermediary messenger they release, can activate submucous neurons projecting to the epithelium.
COOKE: INFLAMMATORY MEDIATORS
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Secretory responses during intestinal anaphylaxis result from a complex interplay between multiple mediators that affect several signal transduction pathways and second messenger systems. FIGURE 2 illustrates the potential interactions among only three of the multitude of mediators that are released from mast cells. Additional mediators that are released will add to the complexity of the interactions between the enteric nervous system, the immune system and the epithelium. The end result is induction of secretion, which is coordinated with other effector systems, to rapidly clear material from the lumen with the possibility of inducing a diarrheal state. ACKNOWLEDGMENTS
I am indebted to Dr. Thomas Frieling, Dr. Jeffrey Palmer, Dr. Y.-Z.Wang, Dr. Y.Rikihisa and Ms. Najma Javed for their contributions to the work described in this paper. The technical assistance of Ms. Jeannette Bidinger and Ms. Beth Anthony is appreciated. REFERENCES 1. ROITT,I., J. BROSTOFF & D. MALE. 1985. Immunology. The C.V. Mosby Co. St. Louis, MO. 2. SARTOR, R. B. & D. W. POWELL.1991. Mechanisms of diarrhea in intestinal inflammation and hypersensitivity: immune system modulation of intestinal transport. In Current Topics in Gastroenterology: Diarrheal Diseases. M. Field, Ed. 75-1 14. Elsevier. New York. 3. CASTRO,G. A. 1982. Immunological regulation of epithelial function. Am. J. Physiol. 243: G3214329. 4. WANG,Y.-2.. J. M. PALMER & H. J. COOKE.1990. Neuroimmune regulation of colonic secretion in nematode-infected guinea pigs. Am. J. Physiol. 260: G307-G3 14. 5 . YAJIMA,T, T. SUZUKI & Y. SUZUKI. 1988. Synergism between calcium-mediated and cyclical AMP-mediated activation of chloride secretion in isolated guinea pig distal colon. Jpn. J. Physiol. 38: 427-443. 6. CHANG,E. B. & M. C. RAO. 1991. Intracellular mediators of intestinal electrolyte transport. In Current Topics in Gastroenterology: Diarrheal Diseases. M. Field, Ed. 49-72. Elsevier. New York. 7. WOOD,J. D. 1991. Communication between minibrain in gut and enteric immune system. NIPS 6 64-69. 8. WANG,Y.-Z. & H. J. COOKE.1991. Comparison of effects of /3-lactoglobulin and Nformyl-methionyl-leucyl-phenylalanine(FMLP) on ion transport in the colon of milksensitized guinea pigs. Gastroenterology 100: A708. 9. JAVED,N., Y.-Z. WANG& H. J. COOKE.1991. Mast cell inflammatory mediators stimulate acetylcholine release from submucosal neurons during intestinal anaphylaxis, Gastroenterology 100: A453. 10. WANG,Y.-Z., H. J. COOKE,H.-C. Su & R. FERTEL.1990. Histamine augments colonic secretion in the guinea pig distal colon. Am. J. Physiol. 2 5 8 (34324439. 11. WANG,Y.-Z. & H. J. COOKE.1990. H2 Receptors mediate cyclical chloride secretion in the guinea pig distal colon. Am. J. Physiol. 258: G887-G893. 12. WASSERMAN, S. I., K. E. BARRETT, P. A. HUOTT,G. BEUERLEIN, M. F. KAGNOFF & K. DHARMSATHAPHORN. 1988. Immune-related intestinal C1- secretion. I. Effect of histamine on the T84 cell line. Am. J. Physiol. 254: C53-C61. 13. COOKE,H. J. & H. V. CAREY.1990. Neural regulation of intestinal ion transport. I n Testbook of Secretory Diarrhea. E. Lebenthal & M. Duffey, Eds. 1-14. Raven Press, Ltd. New York.
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