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Hepatocyte growth inhibitory factor derived from HTLV-I(+) T cell line is identical to IL-6
Hormones, Transmitters, Growth Factors, and their Receptors Al153
April 1998 retarding gastric emptying. To provide further insight into the impact of GLP-1 on gastrointestinal functions this study compared jejunal motor and endocrine responses to subcutaneous (SC) GLP-1 in the interdigestive state and following solid meals of different caloric contents. The study design mimicked the therapeutical approach of NIDDM in healthy subjects. Methods: 8 healthy male volunteers underwent two 24 hour experiments in random order. In each experiment, a 4 hour interdigestive period was started at 9 a.m. Lunch (303 kcal, 45.7% carbohydrate, 35.6% lipid, 18.7% protein) and dinner (801 kcal, 49.7% carbohydrate, 33.7% lipid, 16.6% protein) were consumed at 1 p.m. and 6 p.m., respectively. 5 rain before the interdigestive period and each meal, saline or 1.0 nmol/kg GLP-1 was SC injected. Jejunal motor (solid state catheter with 6 transducers spaced at 4 cm, orad transducer placed immediately beyond Treitz, MMS software, 15 rain interdigestive segments and postprandial deciles) and endocrine (heated hand to determine glucose in arterialized venous blood) responses were assessed. Results: Mean ± SEM, *: P < 0.05 vs saline. Jejunal motor responses Parameter
Drug
lnterdigestive Lunch
Dinner
Time to next phase III (min)
saline 53.7±13.9 155.8±21.2 322.3±34.6 GLP-I 85.0~20.6 211.2±205" 316.2±18.2 Propagation velocityof saline 9.4±1.4 7.1±1.9 4.8±1.1 next phase III (cm/min) GLP-! 4.4±1.0" 4.9±!.0 45±0.7 Peaks/rain; 1. segment/decile saline 1.41±0.19 4.39±0.29 4.82±026 GLP-I 0.93±0.18" 1.01±0.14" 1.48±0.23" GLP-1 did not alter the incidence of phases III in the interdigestive periods in the morning and after both meals including the night. GLP-1 reduced contraction frequency and motility index in the first 4 interdigestive segments, in the first 3 deciles after lunch, and the first 2 deciles after dinner (p<0.05). Thereafter, it stimulated contraction frequency and motility index in the 6th decile after lunch and in the 4th to 6th deciles after dinner (p<0.05). Parameter
Drug
Endocrine responses lnterdigestive Lunch
Dinner
Blood glucose (AUC over basal; mg/dl • 60 min) Insnlinogenicindex (AUC over basal; mU/mg • 60 min)
saline GLP-I saline GLP-I
ll.l:t4.5 -41.4±5.9" -0.1:t-0.1 3.3±0.8*
197.1±21.6 30.5±14.0" 15.0±2.1 5.5±1.3"
106.4±15.0 -18.5±5.9" 4.9±0.9 1.7±1.6
Conclusions: SC GLP-I markedly reduces interdigestive and postprandial jejunal motility in the 1st hour without abolishing phases III. The subsequent stimulation of postprandial jejunal motility depends on the caloric contents of the meal and is arguably caused by accelerated gastric emptying after cessation of the decelerating effect of GLP-1. GLP-1 prolongs the postprandial pattern of low-caloric meals and decelerates propagation of phase III. Both effects appear to depend on GLP-1 plasma levels. SC GLP-1 is distinctly insulinotropic at euglycemia without inducing hypoglycemia. It clearly reduces postprandial glycemia in the 1st hour after solid meals of different size. There is no stimulation of glucose-related insulin secretion with preprandially injected GLP-1 after solid meals. The importance of insulin secretion for the glycemic response to GLP-1 decreases with increasing caloric contents of the meal. We suggest that the inhibition of glucose-related insulin secretion with GLP-1 after the high-caloric meal is induced by strong diminution of the glucagon response (J Endocrinol 1998;156:177-186). There is no short-term adaptation of motor and endocrine responses to GLP-1.
• G4719 ELUCIDATION OF RECEPTOR DOMAINS OF GASTRINRELEASING PEPTIDE RECEPTOR (GRPR) DETERMINING HIGH SELECTIVITY FOR PEPTIDE ANTAGONISTS. T. Katsuno. T.K. Pradhan, S.A. Mantey, PJ. Donohue, J.F. Battey, NIH, Bethesda, MD; D.H. Coy, Tulane Univ., New Orleans, LA; R.T. Jensen. NIH, Bethesda, MD. In contrast to nonpeptide antagonists, little is known about the receptor domains which are responsible for the selectivity of peptide antagonists for any G protein-coupled receptor. The GRPR differs from most other receptors for hormones in that numerous classes of peptide, but no nonpeptide, antagonists are known. The purpose of the present study was to determine the GRPR domains responsible for the high selectivity (x 50-2,500 fold higher) of 6 classes of GRPR antagonists over the NMBR (neuromedin B receptor). We first constructed 4 chimeric GRPRs by exchanging each of the 4 extracellular domains (ECDs) of the GRPR with the corresponding ECD of the NMBR ([ECD1]-, [ECD2]-, [ECD3]-, and [ECD4]- GRPR) and compared the affinities for 8 representative peptide antagonists. We examined 3 peptides from 2 classes of pseudopeptide antagonists (wl-3); [Leut3,~(CH2NH)13-14, Leul4]Bn(bombesin) (~V1), [DPhe6, Leu 13, w(CH2NH)13-14,CpaI4]Bn(6-14) (~2), [3-PH-Prr,His"/, DAla n, DProt3,~ (CH2NH) 13-14,Prot4]Bn(6-14)NH2 (W3), and 5 peptides from 4 classes of des Met 14 antagonists (dM1-5); [DPhe6]Bn(6-13)NH2 (dM1), [DPher]Bn(6-13)methyl ester (dM2), [DPher]Bn(6-13)hydrazide (dM3), [DPhe6]Bn(6-13)propylamide (dM4), [DPher]Bn(6-13)hexylamide (dM5). Receptors were transiently expressed in BALB/3T3 cells and the affinities were determined using t25I-Bn. The antagonists could be divided into 4 groups depending on their affinities for the GRPR, NMBR and 4 chimeric GRPRs. All groups retained high affinities for [ECD1]-, [ECD2]-, and [ECD3]-GRPR. However, Group 1 (~1 and W2) had low affinities for [ECD4]-GRPR that were equal to those for the NMBR. Group II (dM1-4) had lower affinities for [ECD4]-GRPR which were only 2-8 times higher than those for NMBR. Group III (W3) had 30 times lower affinities for [ECD4]-GRPR than the GRPR which were 90 times higher than that of the NMBR. Group IV (dM5) had only 4 times lower affinities for
[ECD4]-GRPR than the GRPR which were at least 40 times higher than that of the NMBR. To determine which amino acid residues in the 4th ECD were responsible for the large decrease in affinity, we substituted amino acids which differed between the GRPR and the NMBR in the 4th ECD of the GRPR. Three amino acid substitutions, [Thr297pro,Phe3°2Met,Ser3°SThr]GRPR showed almost the same change in affinities as that seen with [ECD4]-GRPR. These results show that not only are different domains of the GRPR responsible for the selectivity of different classes of peptide antagonists, but even within a given class, different peptide antagonists b i n d to different receptor domains. The 4th extracellular domain of the GRPR is particularly important for the selectivity for the group I and II peptide antagonists and Thr297, Phe 3°2, and Ser3°5 in the 4th extracellular domain of the GRPR are the key amino acids for the selectivity. However, extracellular domains of the GRPR are partially responsible for the selectivity of the group III and IV peptide antagonists. • G4720 HEPATOCYTE GROWTH INHIBITORY FACTOR DERIVED FROM HTLV-I(+) T CELL LINE IS IDENTICAL TO IL-6. Y. Kawai. A. Yamauchi, H. Nakamura, T. Inamoto, and Y. Yamaoka, Dept. of Gastroenterol. Surgery, Graduate school of Medicine, Kyoto University, Kyoto, JAPAN. Background and Aim: We previously reported that a factor derivied from a human T cell leukemia virus (HTLV-I) infected-T cell line, ATL-2, has an inhibitory activity on the EGF-stimulated proliferation of primarily cultured rat hepatocytes and is arbitrarily named a hepatocyte growth inhibitory factor (HGI). The purpose of this study was to clarify HGI. Materials and Methods: We purified HGI from the culture supematant of ATL-2 using gel-filtration, heparin affinity column, anion exchange column, reverse phase column and SDS-PAGE. Results: The recovery of activity was 26.7% and the purification degree was about 44 thousands times. Finally, the purified proteins were consisted of two bands of 20kDa and 27kDa in SDS-PAGE analysis. Proteins extracted from the two bands had inhibitory activity. The amino acid analysis of the three peptide fragments prepared from the purified 20 kDa band revealed that 34 amino acids was identical to IL-6. The concentration of IL-6 in the fractions measured by ELISA increased as the purifying proceeded and inhibitory activity of the factor was neutralized by anti IL-6 neutralizing antibody. In Western blot analysis of the factor, anti IL-6 antibody recognized the factor at both 20 kDa and 27 kD bands. Conclusion: These results confirmed that the hepatocyte growth inhibitory factor derived from ATL-2 was IL-6. • G4721 CENTRAL PYY PREVENTS ETHANOL-INDUCED GASTRIC DAMAGE THROUGH THE ACTIVATION OF YI AND/OR PYY-PREFERRING RECEPTORS IN ANESTHETIZED RATS. K. Kawakubo, H. Yang, H. Wong, V. Wu, G. Ohning and Y. Tach6. CURE/Digestive Dis. Res. Center, VA Medical Center, Dept. of Medicine, UCLA, Los Angeles, CA. BACKGROUND: We previously reported that intracisternal (ic) injection of peptide Y Y (PYY) and low-doses of TRH or TRH analogue, RX 77368, increased the resistance of the gastric mucosa against ethanol injury through vagal pathways in urethane-anesthetized rats (Soc. Neurosci. 22:397, 1996, Digestion 57:322, 1996). Six NPY-receptor subtypes have been classified according to the potency order of analogues (Peptide 18: 445, 1997). AIM: To investigate 1) the receptor subtype(s) involved in the protective effect of ic-administered PYY on ethanolinduced gastric damage, and 2) whether the gastric protection of ic PYY against intragastric (ig)-administered ethanol is dependent upon the activation of central TRH receptors. METHODS: Under urethane anesthesia (1.25 g/kg, ip), fasted SD rats (250 - 300 g) were injected ic with saline (10 gl), rat PYY or NPY -related receptor agonists 30 rain before ig administration of 45% ethanol (5 ml/kg). Percentage of corpus mucosa containing lesions was determined by computerized image analyzer 1 hr after ethanol injection. Polyclonal PYY antibody (CURE #9153) was injected 10 min before ic injection of PYY or saline. TRH-antisense and mismatch oligonucleotide (100 lag/rat) were injected ic twice at 48 h and 24 h before experiments. RESULTS: PYY (200 ng, ic) significantly reduced by 67% gastric damage induced by 45% ethanol, which covered 22 4- 3% of the gastric mucosa in ic vehicle urethane-anesthetized rats. [Pro 34] PYY (Yt/PYY-preferring agonist; 50, I00, 200 and 500 ng, ic) decreased dosedependently gastric lesions by 30%, 49% (p<0.05), 61% (p<0.01) and 75% (p<0.01), respectively. PYY3-36, a Y2 agonist, (500 ng, ic), NPY (Y3/Ys/Y1/Y2, 500 ng, ic) and rat PP (Y4, 500 rig, ic) did not influence the formation of ethanol-induced gastric lesion. PYY antibody injected ic (460 lag/20 lal, -10 min) reduced by 46% the protective effect of ic PYY (100 rig) while iv injection (2 mg/kg, - 10 min) did not alter ic PYY action. PYY antibody (ic) alone did not modify the formation of ethanol-induced gastric damage. The TRH antisense inhibited the gastric protective effect of RX 77368 (1.5 ng, ic) on ethanol-induced gastric damage, while it did not reverse the ic PYY (200 ng, ic)-induced gastric protection. Combined ic injections of RX 77368 (1 ng) and PYY (25 ng) reduced by 56% ethanol-induced gastric injury while the peptides given singly, or RX 77368 (1 ng) in combination with PYY3-36 (500 ng) did not alter the formation of erosion induced by ethanol. CONCLUSIONS: These data suggest that 1) ic PYY acts in the brain to prevent ethanol-induced gastric lesion through the activation of YI and/or PYY-preferring receptors; 2) central PYY and TRH represents two independent receptors mediated pathways modulating the resistance of the gastric mucosa to injury.
April 1998 retarding gastric emptying. To provide further insight into the impact of GLP-1 on gastrointestinal functions this study compared jejunal motor and endocrine responses to subcutaneous (SC) GLP-1 in the interdigestive state and following solid meals of different caloric contents. The study design mimicked the therapeutical approach of NIDDM in healthy subjects. Methods: 8 healthy male volunteers underwent two 24 hour experiments in random order. In each experiment, a 4 hour interdigestive period was started at 9 a.m. Lunch (303 kcal, 45.7% carbohydrate, 35.6% lipid, 18.7% protein) and dinner (801 kcal, 49.7% carbohydrate, 33.7% lipid, 16.6% protein) were consumed at 1 p.m. and 6 p.m., respectively. 5 rain before the interdigestive period and each meal, saline or 1.0 nmol/kg GLP-1 was SC injected. Jejunal motor (solid state catheter with 6 transducers spaced at 4 cm, orad transducer placed immediately beyond Treitz, MMS software, 15 rain interdigestive segments and postprandial deciles) and endocrine (heated hand to determine glucose in arterialized venous blood) responses were assessed. Results: Mean ± SEM, *: P < 0.05 vs saline. Jejunal motor responses Parameter
Drug
lnterdigestive Lunch
Dinner
Time to next phase III (min)
saline 53.7±13.9 155.8±21.2 322.3±34.6 GLP-I 85.0~20.6 211.2±205" 316.2±18.2 Propagation velocityof saline 9.4±1.4 7.1±1.9 4.8±1.1 next phase III (cm/min) GLP-! 4.4±1.0" 4.9±!.0 45±0.7 Peaks/rain; 1. segment/decile saline 1.41±0.19 4.39±0.29 4.82±026 GLP-I 0.93±0.18" 1.01±0.14" 1.48±0.23" GLP-1 did not alter the incidence of phases III in the interdigestive periods in the morning and after both meals including the night. GLP-1 reduced contraction frequency and motility index in the first 4 interdigestive segments, in the first 3 deciles after lunch, and the first 2 deciles after dinner (p<0.05). Thereafter, it stimulated contraction frequency and motility index in the 6th decile after lunch and in the 4th to 6th deciles after dinner (p<0.05). Parameter
Drug
Endocrine responses lnterdigestive Lunch
Dinner
Blood glucose (AUC over basal; mg/dl • 60 min) Insnlinogenicindex (AUC over basal; mU/mg • 60 min)
saline GLP-I saline GLP-I
ll.l:t4.5 -41.4±5.9" -0.1:t-0.1 3.3±0.8*
197.1±21.6 30.5±14.0" 15.0±2.1 5.5±1.3"
106.4±15.0 -18.5±5.9" 4.9±0.9 1.7±1.6
Conclusions: SC GLP-I markedly reduces interdigestive and postprandial jejunal motility in the 1st hour without abolishing phases III. The subsequent stimulation of postprandial jejunal motility depends on the caloric contents of the meal and is arguably caused by accelerated gastric emptying after cessation of the decelerating effect of GLP-1. GLP-1 prolongs the postprandial pattern of low-caloric meals and decelerates propagation of phase III. Both effects appear to depend on GLP-1 plasma levels. SC GLP-1 is distinctly insulinotropic at euglycemia without inducing hypoglycemia. It clearly reduces postprandial glycemia in the 1st hour after solid meals of different size. There is no stimulation of glucose-related insulin secretion with preprandially injected GLP-1 after solid meals. The importance of insulin secretion for the glycemic response to GLP-1 decreases with increasing caloric contents of the meal. We suggest that the inhibition of glucose-related insulin secretion with GLP-1 after the high-caloric meal is induced by strong diminution of the glucagon response (J Endocrinol 1998;156:177-186). There is no short-term adaptation of motor and endocrine responses to GLP-1.
• G4719 ELUCIDATION OF RECEPTOR DOMAINS OF GASTRINRELEASING PEPTIDE RECEPTOR (GRPR) DETERMINING HIGH SELECTIVITY FOR PEPTIDE ANTAGONISTS. T. Katsuno. T.K. Pradhan, S.A. Mantey, PJ. Donohue, J.F. Battey, NIH, Bethesda, MD; D.H. Coy, Tulane Univ., New Orleans, LA; R.T. Jensen. NIH, Bethesda, MD. In contrast to nonpeptide antagonists, little is known about the receptor domains which are responsible for the selectivity of peptide antagonists for any G protein-coupled receptor. The GRPR differs from most other receptors for hormones in that numerous classes of peptide, but no nonpeptide, antagonists are known. The purpose of the present study was to determine the GRPR domains responsible for the high selectivity (x 50-2,500 fold higher) of 6 classes of GRPR antagonists over the NMBR (neuromedin B receptor). We first constructed 4 chimeric GRPRs by exchanging each of the 4 extracellular domains (ECDs) of the GRPR with the corresponding ECD of the NMBR ([ECD1]-, [ECD2]-, [ECD3]-, and [ECD4]- GRPR) and compared the affinities for 8 representative peptide antagonists. We examined 3 peptides from 2 classes of pseudopeptide antagonists (wl-3); [Leut3,~(CH2NH)13-14, Leul4]Bn(bombesin) (~V1), [DPhe6, Leu 13, w(CH2NH)13-14,CpaI4]Bn(6-14) (~2), [3-PH-Prr,His"/, DAla n, DProt3,~ (CH2NH) 13-14,Prot4]Bn(6-14)NH2 (W3), and 5 peptides from 4 classes of des Met 14 antagonists (dM1-5); [DPhe6]Bn(6-13)NH2 (dM1), [DPher]Bn(6-13)methyl ester (dM2), [DPher]Bn(6-13)hydrazide (dM3), [DPhe6]Bn(6-13)propylamide (dM4), [DPher]Bn(6-13)hexylamide (dM5). Receptors were transiently expressed in BALB/3T3 cells and the affinities were determined using t25I-Bn. The antagonists could be divided into 4 groups depending on their affinities for the GRPR, NMBR and 4 chimeric GRPRs. All groups retained high affinities for [ECD1]-, [ECD2]-, and [ECD3]-GRPR. However, Group 1 (~1 and W2) had low affinities for [ECD4]-GRPR that were equal to those for the NMBR. Group II (dM1-4) had lower affinities for [ECD4]-GRPR which were only 2-8 times higher than those for NMBR. Group III (W3) had 30 times lower affinities for [ECD4]-GRPR than the GRPR which were 90 times higher than that of the NMBR. Group IV (dM5) had only 4 times lower affinities for
[ECD4]-GRPR than the GRPR which were at least 40 times higher than that of the NMBR. To determine which amino acid residues in the 4th ECD were responsible for the large decrease in affinity, we substituted amino acids which differed between the GRPR and the NMBR in the 4th ECD of the GRPR. Three amino acid substitutions, [Thr297pro,Phe3°2Met,Ser3°SThr]GRPR showed almost the same change in affinities as that seen with [ECD4]-GRPR. These results show that not only are different domains of the GRPR responsible for the selectivity of different classes of peptide antagonists, but even within a given class, different peptide antagonists b i n d to different receptor domains. The 4th extracellular domain of the GRPR is particularly important for the selectivity for the group I and II peptide antagonists and Thr297, Phe 3°2, and Ser3°5 in the 4th extracellular domain of the GRPR are the key amino acids for the selectivity. However, extracellular domains of the GRPR are partially responsible for the selectivity of the group III and IV peptide antagonists. • G4720 HEPATOCYTE GROWTH INHIBITORY FACTOR DERIVED FROM HTLV-I(+) T CELL LINE IS IDENTICAL TO IL-6. Y. Kawai. A. Yamauchi, H. Nakamura, T. Inamoto, and Y. Yamaoka, Dept. of Gastroenterol. Surgery, Graduate school of Medicine, Kyoto University, Kyoto, JAPAN. Background and Aim: We previously reported that a factor derivied from a human T cell leukemia virus (HTLV-I) infected-T cell line, ATL-2, has an inhibitory activity on the EGF-stimulated proliferation of primarily cultured rat hepatocytes and is arbitrarily named a hepatocyte growth inhibitory factor (HGI). The purpose of this study was to clarify HGI. Materials and Methods: We purified HGI from the culture supematant of ATL-2 using gel-filtration, heparin affinity column, anion exchange column, reverse phase column and SDS-PAGE. Results: The recovery of activity was 26.7% and the purification degree was about 44 thousands times. Finally, the purified proteins were consisted of two bands of 20kDa and 27kDa in SDS-PAGE analysis. Proteins extracted from the two bands had inhibitory activity. The amino acid analysis of the three peptide fragments prepared from the purified 20 kDa band revealed that 34 amino acids was identical to IL-6. The concentration of IL-6 in the fractions measured by ELISA increased as the purifying proceeded and inhibitory activity of the factor was neutralized by anti IL-6 neutralizing antibody. In Western blot analysis of the factor, anti IL-6 antibody recognized the factor at both 20 kDa and 27 kD bands. Conclusion: These results confirmed that the hepatocyte growth inhibitory factor derived from ATL-2 was IL-6. • G4721 CENTRAL PYY PREVENTS ETHANOL-INDUCED GASTRIC DAMAGE THROUGH THE ACTIVATION OF YI AND/OR PYY-PREFERRING RECEPTORS IN ANESTHETIZED RATS. K. Kawakubo, H. Yang, H. Wong, V. Wu, G. Ohning and Y. Tach6. CURE/Digestive Dis. Res. Center, VA Medical Center, Dept. of Medicine, UCLA, Los Angeles, CA. BACKGROUND: We previously reported that intracisternal (ic) injection of peptide Y Y (PYY) and low-doses of TRH or TRH analogue, RX 77368, increased the resistance of the gastric mucosa against ethanol injury through vagal pathways in urethane-anesthetized rats (Soc. Neurosci. 22:397, 1996, Digestion 57:322, 1996). Six NPY-receptor subtypes have been classified according to the potency order of analogues (Peptide 18: 445, 1997). AIM: To investigate 1) the receptor subtype(s) involved in the protective effect of ic-administered PYY on ethanolinduced gastric damage, and 2) whether the gastric protection of ic PYY against intragastric (ig)-administered ethanol is dependent upon the activation of central TRH receptors. METHODS: Under urethane anesthesia (1.25 g/kg, ip), fasted SD rats (250 - 300 g) were injected ic with saline (10 gl), rat PYY or NPY -related receptor agonists 30 rain before ig administration of 45% ethanol (5 ml/kg). Percentage of corpus mucosa containing lesions was determined by computerized image analyzer 1 hr after ethanol injection. Polyclonal PYY antibody (CURE #9153) was injected 10 min before ic injection of PYY or saline. TRH-antisense and mismatch oligonucleotide (100 lag/rat) were injected ic twice at 48 h and 24 h before experiments. RESULTS: PYY (200 ng, ic) significantly reduced by 67% gastric damage induced by 45% ethanol, which covered 22 4- 3% of the gastric mucosa in ic vehicle urethane-anesthetized rats. [Pro 34] PYY (Yt/PYY-preferring agonist; 50, I00, 200 and 500 ng, ic) decreased dosedependently gastric lesions by 30%, 49% (p<0.05), 61% (p<0.01) and 75% (p<0.01), respectively. PYY3-36, a Y2 agonist, (500 ng, ic), NPY (Y3/Ys/Y1/Y2, 500 ng, ic) and rat PP (Y4, 500 rig, ic) did not influence the formation of ethanol-induced gastric lesion. PYY antibody injected ic (460 lag/20 lal, -10 min) reduced by 46% the protective effect of ic PYY (100 rig) while iv injection (2 mg/kg, - 10 min) did not alter ic PYY action. PYY antibody (ic) alone did not modify the formation of ethanol-induced gastric damage. The TRH antisense inhibited the gastric protective effect of RX 77368 (1.5 ng, ic) on ethanol-induced gastric damage, while it did not reverse the ic PYY (200 ng, ic)-induced gastric protection. Combined ic injections of RX 77368 (1 ng) and PYY (25 ng) reduced by 56% ethanol-induced gastric injury while the peptides given singly, or RX 77368 (1 ng) in combination with PYY3-36 (500 ng) did not alter the formation of erosion induced by ethanol. CONCLUSIONS: These data suggest that 1) ic PYY acts in the brain to prevent ethanol-induced gastric lesion through the activation of YI and/or PYY-preferring receptors; 2) central PYY and TRH represents two independent receptors mediated pathways modulating the resistance of the gastric mucosa to injury.