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Putative mediator(s) of adaptive cytoprotection?
PROSTAGLANDINS
EDITORIAL PUTATIVE MEDIATOR(S) OF ADAPTIVE CYTOPROTECTION? G.L. Kauffman, Jr. Division of General Surgery, Department of Surgery The Milton S. Hershey Medical Center The Pennsylvania State University Hershey, PA 17033
The term "cytoprotection" is used in "gastric mucosa" circles to refer to the property of exogenous prostanoids of the A, E, and F type, given at non-antisecretory doses, to prevent macroscopic gastric mucosal injury produced by alcohol, HCI, NaOH, hypertonic NaCl, and thermal injury (i). The term was initially coined by Andre Robert in collaboration with Eugene Jacobson in the context of protection afforded the distal ileum by exogenous prostanoids against injury produced by oral indomethacin. In this situation, the prostanoid protective effect is independent of its antisecretory effect. The next several years saw a number investigators attempt to define the mechanism(s) of cytoprotection such as increasing mucosal blood flow, stimulating gastric mucus and bicarbonate secretion, and stimulating epithelial proliferation, however, no unified theory emerged. Lacy and Ito (2) studied cytoprotection histologically, demonstrating that surface epithelial cells were not protected, however, the depth of mucosal injury is clearly reduced. Although surface epithelial "cells" are not "protected", cytoprotection as a concept continues to be studied and referred to as "direct cytoprotection". A year after his major description of cytoprotection, Robert, in collaboration with Chaudhury, described the protective effect of exposing the gastric mucosa to a lesser concentration of a compound than that which routinely produces significant mucosal injury (3). In other words, exposure of the gastric mucosa to 0.15 N - 0.35 N HCI, 10% - 25% ethanol, or 5mM taurocholic acid prior to exposing the mucosa to 80mM acidified taurocholic acid was associated with less macroscopic injury than that observed following gastric mucosal exposure to 80mM acidified taurocholic acid alone. Indomethacin blocked this form of protection which suggested that endogenous prostanoids were involved.This "mild irritant"-induced protection is that to which the term "adaptive cytoprotection" is applied.
The following year,"mild irritant-induced" protection of the gastric mucosa was confirmed by Konturek and co-investigators (4) .Using bioassay techniques, they demonstrated an increase in prostaglandin-like activity in gastric mucosal extracts from rats treated with a mild irritant. In a 1983 publication, Robert (5)
MARCH 1991 VOL. 41 NO. 3
201
PROSTAGLANDINS defended the role of endogenous prostanoids as mediators of adaptive cytoprotection as i) this form of protection is blocked by pre-treatment with indomethacin, 2) this form of protection, blocked by indomethacin, can be reinstated by treatment with exogenous prostanoids, and 3) gastric mucosal extracts from rats given a mild irritant form more endogenous prostaglandins than gastric mucosal extracts from control rats. In this issue of PROSTAGLANDINS, Miller and his colleagues present data which suggest that adaptive cytoprotection is not due to increased prostanoid synthesis (6). The mild irritant which they used was 25% ethanol, the major irritant, 100% ethanol, and prostaglandin production was measured in microsomal membrane preparations incubated with 14C arachidonic acid, measuring arachidonio acid metabolites by chromatography and radioimmunoassay. Macroscopic damage, as assessed by planimetry, was reduced by 90% by pre-treatment with the mild irritant in rats with physiological cyclo-oxygenase activity and by 66% in rats to which indomethacin had been given. No differences in concentrations (ng Prost/mg Protein/incubation) of 6-keto PGFI, , P G F ~ , PGE 2, PGD~, PGA 2, and TXB2, were observed comparing t h e micr-osomal-membrane preparation ~rom gastric mucosa exposed to 25% ethanol to control microsomal membranes. Less supportive of their conclusion is the observation that prostanoid synthesis in microsomal preparations from the gastric mucosa of rats treated with 25% ethanol followed by 100% ethanol were greater than prostanoid synthesis in preparations from rats treated with 25% ethanol alone. The former reflects the conditions under which macroscopic protection was actually observed. It is also somewhat disconcerting that the dose of indomethacin given intraperitoneally did not result in near complete inhibition of prostanoid synthesis by these microsomal membranes, as might have been expected. In general, however, the data support the conclusion that adaptive cytoprotection, under these experimental conditions, is not likely to be mediated by endogenous prostanoids. Other investigators have come to similar conclusions. Wallace (7) used 1.0 M NaCl as a mild irritant and 70% ethanol as the major irritant, demonstrating adaptive cytoprotection with a 98% reduction in hemorrhagic mucosal necrosis, which was not blocked by pre-treatment with indomethacin. Physical removal of the "mucoid cap" formed after administration of 1.0 M NaCl, which consists of desquamated cellular debris, mucus, and fibrin, did not affect adaptive cytoprotection in this protocol. Although prostanoid production by the gastric mucosa was not assessed in these studies, this indirect evidence suggests that cyclooxygenase inhibition does not preclude an adaptive cytoprotective effect of the mild irritant. Hawkey and coinvestigators (8) made similar observations using 20% ethanol as the mild irritant and 100% ethanol the major irritant. Macroscopic damage was reduced 71% and microscopic injury 25% in the mild irritant-treated group. Ex vivo generation of PGE 2 of gastric mucosal samples taken from rats to which the mild irritant was given was no greater than control values. Different doses of indomethacin (2.5 and i0.0 mg/kg) inhibited ex vivo PGE 2 generation 55% and
202
MARCH 1991 VOL. 41 NO. 3
PROSTAGLANDINS 80%, respectively. Neither dose of indomethacin affected the adaptive cytoprotective effect of the mild irritant. This was the first study which did not demonstrate enhancement of gastric mucosal prostanoid production after administering a mild irritant. Publishing in the same year, MacNaughton and coinvestigators (9) also found that pre-treatment with the mild irritant 0.25 M HCl reduced the microscopic injury, net cationic flux, and transmucosal potential difference produced by 40% ethanol alone in an ex vivo vascularly perfused gastric chamber preparation. Pre-treatment with indomethacin at a dose which inhibited prostanoid production 88% did not attenuate adaptive cytoprotection. From indirect evidence with the use of indomethacin and from direct evidence with prostanoid production studies, it can be concluded that at least some forms of adaptive cytoprotection do not require an intact cyclo-oxygenase system. Are there other potential mediators of adaptive cytoprotection? The answer is clearly yes. Balaa (i0) found that the gastric luminal fluid from rats given 0.3N HCI as a mild irritant protected the gastric mucosa of other rats to which it was given intragastrically before the intragastric administration of 100% ethanol. This protective effect remained present in the gastric fluid taken from rats pre-treated with indomethacin before intragastric instillation of the mild irritant. Prostaglandin E~ concentrations, as measured by radioimmunoassay were nearly identical in both the control and experimental groups. It was concluded that exposure of the gastric mucosa to a mild irritant causes the intraluminal release of protective product(s), other than prostaglandins. Salivary gland factors appear to influence adaptive cytoprotection. Tepperman and coinvestigators (Ii) found that 10% ethanol, a mild irritant, less effectively protected the gastric mucosa against 100% ethanol in surgically sialadenectomized than sham operated rats. This protective effect could be reinstated by treating the sialadenectomized rats with aqueous salivary gland extracts for three days prior to the experiment. Mild irritant treatment of the gastric mucosa in control rats was not associated with increased capacity of the gastric mucosa to generate PGE 2 or prostacyclin, however, sialadenectomy reduced the capacity of the gastric mucosa to generate these prostanoids in control and mild irritant-treated rats. It was concluded that adaptive cytoprotection is dependent upon intact salivary glands rather than endogenous prostanoids. Endogenous gastric dopamine may also mediate adaptive cytoprotection. MacNaughton and Wallace (12), used the ex vivo vascularly perfused gastric chamber preparation and conscious rats to study the effect of pretreatment with specific receptor antagonists to histamine, acetylcholine, norepinephrine, 5-hydroxytryptamine, dopamine, and platelet activating factor on the adaptive cytoprotective effect of 10% ethanol against the major irritant, 70% ethanol. Of these, only cyproheptadine and haloperidol attenuated the effect of the mild irritant, suggesting that activation of endogenous dopamine receptors are involved. This theory was validated by the use of intraluminal bromocryptine or L-B-3,4-
MARCH 1991 VOL. 41 NO. 3
203
PROSTAGLANDINS
dihydroxyphenylalanine, specific dopamine receptor agonists, both of which afforded protection against 70% ethanol, the magnitude of which mimicked that observed with the mild irritant pretreatment. Adaptive cytoprotection also requires intact vagal and sympathetic innervation. Using 20% ethanol as the mild irritant and 100% ethanol as the major iritant, Foschi and coinvestigators (13) found that surgical vagotomy or sympathectomy blocked adaptive cytoprotection. Administration of salbutamol, a beta-adrenergic receptor agonist restored adaptive cytoprotection in both vagotomized and sympathetectomized rats. These observations suggest that ethanol, given at mild irritant concentrations activate beta-adrenergic receptors, a requirement for adaptive cytoprotection. It would appear that there is no unifying hypothesis regarding the mediation of adaptive cytoprotection. There is no doubt that exposing the gastric mucosa to a mild irritant prior to a major irritant enhances mucosal defense and reduces the magnitude of the injury by both macroscopic or microscopic assessment. Based upon the variable results using indomethacin and estimating the integrity of the cyclo-oxygenase system, one is forced to conclude that endogenous prostaglandins may be a requirement for adaptive cytoprotection under certain conditions, however, other factors such as the release of some agent into the gastric luminal fluid, the presence of active salivary glands in situ, or the activation of dopaminergic and/or beta-adrenergic receptors in the gastric mucosa have either a primary or permissive role. As is true with most biological systems offering protection against tissue injury, it is reasonable to assume that more than one mediator is involved in adaptive cytoprotection. REFERENCES 1. Robert, A, Nezamis, JE, Lancaster, C, and Hanchar, AJ. Cytoprotection by prostaglandins in rats. Gastroenterology, 7_/7:433, 1979. 2. Robert, A. Cytoprotection by prostaglandins. Gastroenterology, 7_/7:761, 1979. 3. Chaudhury, TK, and Robert, A. Prevention by mild irritants of gastric necrosis produced in rats by sodium taurocholate. Dig. Dis. and Sci., 2_55:830, 1980. 4. Konturek, SJ, Brzozowski, T, Piastucki, I, Radecki, T, Dembinski, A, and Dembinska-Kiec, A. Role of locally generated prostaglandins in adaptive gastric cytoprotection. Dig. Dis. and Sci., 27:967, 1982.
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MARCH 1991 VOL. 41 NO. 3
PROSTAGLANDINS 5. Robert, A, Nezamis, JE, Lancaster, C, Davis, JP, Field, SO, and Hanchar, AJ. Mild irritants prevent gastric necrosis through "adaptive cytoprotection" mediated by prostaglandins. Am. J. Physiol., 245:Gi13, 1983. 6. Smith, GS, Myers, SI, Bartula, LL, and Miller, TA. Adaptive cytoproteotion against alcohol injury in the rat stomach is not due to increased prostanoid synthesis. Prostaglandins. 7. Wallace, JL. Increased resistance of the rat gastric mucosa to hemorrhagic damage after exposure to an irritant. Gastroenterology, 94:22, 1988. 8. Hawkey, CJ, Kemp, RT, Walt, RP, Bhaskar, NK, Davies, J, and Filipowicz, B. Evidence that adaptive cytoprotection in rats is not mediated by prostaglandins. Gastroenterology, 94:948, 1988. 9. MacNaughton, WK, Williamson, TE, and Morris, GP. Adaptive cytoprotection by 0.25 M HCl is truly "cytoprotective" and may not depend upon elevated levels of prostaglandin synthesis. Can. J. Physiol. Pharmacol., 66:1075, 1988. 10. Balaa, MA. Release of protective products, different prostaglandins, by rat stomachs exposed to mild irritant. Dis. and Sci., 34:429, 1989.
from Dig.
ll. Tepperman, BL, Soper, BD, and Morris, GP. Effect of sialoadenectomy on adaptive cytoprotection in the rat. Gastroenterology, 97:123, 1988. 12. MacNaughton, WK, and Wallace, JL. A role for dopamine as an endogenous protective factor in the rat stomach. Gastroenterology, 96:972, 1989. 13. Foschi, D, Castoldi, L, F, Rovati, V, Trabucchi, E, autonomic nervous system on Dig. Dis. and Sci., 34:688,
MARCH 1991 VOL. 41 NO. 3
Del Soldato, P, Musazzi, M, Callioni, and Montorsi, W. Effects of gastric damage by ethanol in the rat. 1989.
205
EDITORIAL PUTATIVE MEDIATOR(S) OF ADAPTIVE CYTOPROTECTION? G.L. Kauffman, Jr. Division of General Surgery, Department of Surgery The Milton S. Hershey Medical Center The Pennsylvania State University Hershey, PA 17033
The term "cytoprotection" is used in "gastric mucosa" circles to refer to the property of exogenous prostanoids of the A, E, and F type, given at non-antisecretory doses, to prevent macroscopic gastric mucosal injury produced by alcohol, HCI, NaOH, hypertonic NaCl, and thermal injury (i). The term was initially coined by Andre Robert in collaboration with Eugene Jacobson in the context of protection afforded the distal ileum by exogenous prostanoids against injury produced by oral indomethacin. In this situation, the prostanoid protective effect is independent of its antisecretory effect. The next several years saw a number investigators attempt to define the mechanism(s) of cytoprotection such as increasing mucosal blood flow, stimulating gastric mucus and bicarbonate secretion, and stimulating epithelial proliferation, however, no unified theory emerged. Lacy and Ito (2) studied cytoprotection histologically, demonstrating that surface epithelial cells were not protected, however, the depth of mucosal injury is clearly reduced. Although surface epithelial "cells" are not "protected", cytoprotection as a concept continues to be studied and referred to as "direct cytoprotection". A year after his major description of cytoprotection, Robert, in collaboration with Chaudhury, described the protective effect of exposing the gastric mucosa to a lesser concentration of a compound than that which routinely produces significant mucosal injury (3). In other words, exposure of the gastric mucosa to 0.15 N - 0.35 N HCI, 10% - 25% ethanol, or 5mM taurocholic acid prior to exposing the mucosa to 80mM acidified taurocholic acid was associated with less macroscopic injury than that observed following gastric mucosal exposure to 80mM acidified taurocholic acid alone. Indomethacin blocked this form of protection which suggested that endogenous prostanoids were involved.This "mild irritant"-induced protection is that to which the term "adaptive cytoprotection" is applied.
The following year,"mild irritant-induced" protection of the gastric mucosa was confirmed by Konturek and co-investigators (4) .Using bioassay techniques, they demonstrated an increase in prostaglandin-like activity in gastric mucosal extracts from rats treated with a mild irritant. In a 1983 publication, Robert (5)
MARCH 1991 VOL. 41 NO. 3
201
PROSTAGLANDINS defended the role of endogenous prostanoids as mediators of adaptive cytoprotection as i) this form of protection is blocked by pre-treatment with indomethacin, 2) this form of protection, blocked by indomethacin, can be reinstated by treatment with exogenous prostanoids, and 3) gastric mucosal extracts from rats given a mild irritant form more endogenous prostaglandins than gastric mucosal extracts from control rats. In this issue of PROSTAGLANDINS, Miller and his colleagues present data which suggest that adaptive cytoprotection is not due to increased prostanoid synthesis (6). The mild irritant which they used was 25% ethanol, the major irritant, 100% ethanol, and prostaglandin production was measured in microsomal membrane preparations incubated with 14C arachidonic acid, measuring arachidonio acid metabolites by chromatography and radioimmunoassay. Macroscopic damage, as assessed by planimetry, was reduced by 90% by pre-treatment with the mild irritant in rats with physiological cyclo-oxygenase activity and by 66% in rats to which indomethacin had been given. No differences in concentrations (ng Prost/mg Protein/incubation) of 6-keto PGFI, , P G F ~ , PGE 2, PGD~, PGA 2, and TXB2, were observed comparing t h e micr-osomal-membrane preparation ~rom gastric mucosa exposed to 25% ethanol to control microsomal membranes. Less supportive of their conclusion is the observation that prostanoid synthesis in microsomal preparations from the gastric mucosa of rats treated with 25% ethanol followed by 100% ethanol were greater than prostanoid synthesis in preparations from rats treated with 25% ethanol alone. The former reflects the conditions under which macroscopic protection was actually observed. It is also somewhat disconcerting that the dose of indomethacin given intraperitoneally did not result in near complete inhibition of prostanoid synthesis by these microsomal membranes, as might have been expected. In general, however, the data support the conclusion that adaptive cytoprotection, under these experimental conditions, is not likely to be mediated by endogenous prostanoids. Other investigators have come to similar conclusions. Wallace (7) used 1.0 M NaCl as a mild irritant and 70% ethanol as the major irritant, demonstrating adaptive cytoprotection with a 98% reduction in hemorrhagic mucosal necrosis, which was not blocked by pre-treatment with indomethacin. Physical removal of the "mucoid cap" formed after administration of 1.0 M NaCl, which consists of desquamated cellular debris, mucus, and fibrin, did not affect adaptive cytoprotection in this protocol. Although prostanoid production by the gastric mucosa was not assessed in these studies, this indirect evidence suggests that cyclooxygenase inhibition does not preclude an adaptive cytoprotective effect of the mild irritant. Hawkey and coinvestigators (8) made similar observations using 20% ethanol as the mild irritant and 100% ethanol the major irritant. Macroscopic damage was reduced 71% and microscopic injury 25% in the mild irritant-treated group. Ex vivo generation of PGE 2 of gastric mucosal samples taken from rats to which the mild irritant was given was no greater than control values. Different doses of indomethacin (2.5 and i0.0 mg/kg) inhibited ex vivo PGE 2 generation 55% and
202
MARCH 1991 VOL. 41 NO. 3
PROSTAGLANDINS 80%, respectively. Neither dose of indomethacin affected the adaptive cytoprotective effect of the mild irritant. This was the first study which did not demonstrate enhancement of gastric mucosal prostanoid production after administering a mild irritant. Publishing in the same year, MacNaughton and coinvestigators (9) also found that pre-treatment with the mild irritant 0.25 M HCl reduced the microscopic injury, net cationic flux, and transmucosal potential difference produced by 40% ethanol alone in an ex vivo vascularly perfused gastric chamber preparation. Pre-treatment with indomethacin at a dose which inhibited prostanoid production 88% did not attenuate adaptive cytoprotection. From indirect evidence with the use of indomethacin and from direct evidence with prostanoid production studies, it can be concluded that at least some forms of adaptive cytoprotection do not require an intact cyclo-oxygenase system. Are there other potential mediators of adaptive cytoprotection? The answer is clearly yes. Balaa (i0) found that the gastric luminal fluid from rats given 0.3N HCI as a mild irritant protected the gastric mucosa of other rats to which it was given intragastrically before the intragastric administration of 100% ethanol. This protective effect remained present in the gastric fluid taken from rats pre-treated with indomethacin before intragastric instillation of the mild irritant. Prostaglandin E~ concentrations, as measured by radioimmunoassay were nearly identical in both the control and experimental groups. It was concluded that exposure of the gastric mucosa to a mild irritant causes the intraluminal release of protective product(s), other than prostaglandins. Salivary gland factors appear to influence adaptive cytoprotection. Tepperman and coinvestigators (Ii) found that 10% ethanol, a mild irritant, less effectively protected the gastric mucosa against 100% ethanol in surgically sialadenectomized than sham operated rats. This protective effect could be reinstated by treating the sialadenectomized rats with aqueous salivary gland extracts for three days prior to the experiment. Mild irritant treatment of the gastric mucosa in control rats was not associated with increased capacity of the gastric mucosa to generate PGE 2 or prostacyclin, however, sialadenectomy reduced the capacity of the gastric mucosa to generate these prostanoids in control and mild irritant-treated rats. It was concluded that adaptive cytoprotection is dependent upon intact salivary glands rather than endogenous prostanoids. Endogenous gastric dopamine may also mediate adaptive cytoprotection. MacNaughton and Wallace (12), used the ex vivo vascularly perfused gastric chamber preparation and conscious rats to study the effect of pretreatment with specific receptor antagonists to histamine, acetylcholine, norepinephrine, 5-hydroxytryptamine, dopamine, and platelet activating factor on the adaptive cytoprotective effect of 10% ethanol against the major irritant, 70% ethanol. Of these, only cyproheptadine and haloperidol attenuated the effect of the mild irritant, suggesting that activation of endogenous dopamine receptors are involved. This theory was validated by the use of intraluminal bromocryptine or L-B-3,4-
MARCH 1991 VOL. 41 NO. 3
203
PROSTAGLANDINS
dihydroxyphenylalanine, specific dopamine receptor agonists, both of which afforded protection against 70% ethanol, the magnitude of which mimicked that observed with the mild irritant pretreatment. Adaptive cytoprotection also requires intact vagal and sympathetic innervation. Using 20% ethanol as the mild irritant and 100% ethanol as the major iritant, Foschi and coinvestigators (13) found that surgical vagotomy or sympathectomy blocked adaptive cytoprotection. Administration of salbutamol, a beta-adrenergic receptor agonist restored adaptive cytoprotection in both vagotomized and sympathetectomized rats. These observations suggest that ethanol, given at mild irritant concentrations activate beta-adrenergic receptors, a requirement for adaptive cytoprotection. It would appear that there is no unifying hypothesis regarding the mediation of adaptive cytoprotection. There is no doubt that exposing the gastric mucosa to a mild irritant prior to a major irritant enhances mucosal defense and reduces the magnitude of the injury by both macroscopic or microscopic assessment. Based upon the variable results using indomethacin and estimating the integrity of the cyclo-oxygenase system, one is forced to conclude that endogenous prostaglandins may be a requirement for adaptive cytoprotection under certain conditions, however, other factors such as the release of some agent into the gastric luminal fluid, the presence of active salivary glands in situ, or the activation of dopaminergic and/or beta-adrenergic receptors in the gastric mucosa have either a primary or permissive role. As is true with most biological systems offering protection against tissue injury, it is reasonable to assume that more than one mediator is involved in adaptive cytoprotection. REFERENCES 1. Robert, A, Nezamis, JE, Lancaster, C, and Hanchar, AJ. Cytoprotection by prostaglandins in rats. Gastroenterology, 7_/7:433, 1979. 2. Robert, A. Cytoprotection by prostaglandins. Gastroenterology, 7_/7:761, 1979. 3. Chaudhury, TK, and Robert, A. Prevention by mild irritants of gastric necrosis produced in rats by sodium taurocholate. Dig. Dis. and Sci., 2_55:830, 1980. 4. Konturek, SJ, Brzozowski, T, Piastucki, I, Radecki, T, Dembinski, A, and Dembinska-Kiec, A. Role of locally generated prostaglandins in adaptive gastric cytoprotection. Dig. Dis. and Sci., 27:967, 1982.
204
MARCH 1991 VOL. 41 NO. 3
PROSTAGLANDINS 5. Robert, A, Nezamis, JE, Lancaster, C, Davis, JP, Field, SO, and Hanchar, AJ. Mild irritants prevent gastric necrosis through "adaptive cytoprotection" mediated by prostaglandins. Am. J. Physiol., 245:Gi13, 1983. 6. Smith, GS, Myers, SI, Bartula, LL, and Miller, TA. Adaptive cytoproteotion against alcohol injury in the rat stomach is not due to increased prostanoid synthesis. Prostaglandins. 7. Wallace, JL. Increased resistance of the rat gastric mucosa to hemorrhagic damage after exposure to an irritant. Gastroenterology, 94:22, 1988. 8. Hawkey, CJ, Kemp, RT, Walt, RP, Bhaskar, NK, Davies, J, and Filipowicz, B. Evidence that adaptive cytoprotection in rats is not mediated by prostaglandins. Gastroenterology, 94:948, 1988. 9. MacNaughton, WK, Williamson, TE, and Morris, GP. Adaptive cytoprotection by 0.25 M HCl is truly "cytoprotective" and may not depend upon elevated levels of prostaglandin synthesis. Can. J. Physiol. Pharmacol., 66:1075, 1988. 10. Balaa, MA. Release of protective products, different prostaglandins, by rat stomachs exposed to mild irritant. Dis. and Sci., 34:429, 1989.
from Dig.
ll. Tepperman, BL, Soper, BD, and Morris, GP. Effect of sialoadenectomy on adaptive cytoprotection in the rat. Gastroenterology, 97:123, 1988. 12. MacNaughton, WK, and Wallace, JL. A role for dopamine as an endogenous protective factor in the rat stomach. Gastroenterology, 96:972, 1989. 13. Foschi, D, Castoldi, L, F, Rovati, V, Trabucchi, E, autonomic nervous system on Dig. Dis. and Sci., 34:688,
MARCH 1991 VOL. 41 NO. 3
Del Soldato, P, Musazzi, M, Callioni, and Montorsi, W. Effects of gastric damage by ethanol in the rat. 1989.
205