Involvement of calcium-sensing receptor in ischemia/reperfusion-induced apoptosis in rat cardiomyocytes

The calcium-sensing receptor (CaR) is a seven-transmembrane G-protein coupled receptor, which activates intracellular effectors, for example, it causes inositol phosphate (IP) accumulation to increase the release of intracellular calcium. Although intracellular calcium overload has been implicated in the cardiac ischemia/reperfusion (I/R)-induced apoptosis, the role of CaR in the induction of apoptosis has not been fully understood. This study tested the hypothesis that CaR is involved in I/R cardiomyocyte apoptosis by increasing [Ca2+]i. The isolated rat hearts were subjected to 40-min ischemia followed by 2 h of reperfusion, meanwhile GdCl3 was added to reperfusion solution. The expression of CaR increased at the exposure to GdCl3 during I/R. By laser confocal microscopy, it was observed that the intracellular calcium was significantly increased and exhibited a Deltapsim, as monitored by 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1) during reperfusion with GdCl3. Furthermore, the number of apoptotic cells was significantly increased as shown by TUNEL assay. Typical apoptotic cells were observed with transmission electron microscopy in I/R with GdCl3 but not in the control group. The expression of cytosolic cytochrome c and activated caspase-9 and caspase-3 was significantly increased whereas the expression of mitochondrial cytochrome c significantly decreased in I/R with GdCl3 in comparison to the control. In conclusion, these results suggest that CaR is involved in the induction of cardiomyocyte apoptosis during ischemia/reperfusion through activation of cytochrome c-caspase-3 signaling pathway.     

RNA binding protein QKI contributes to WT1 mRNA and suppresses apoptosis in ST cells

The RNA binding protein quaking (QKI), a key member of the STAR family, as an upstream gene could involve in much process including cell proliferation, apoptosis, differentiation and so on. However, the roles of QKI in germ cell, especially in swine testis (ST) cells, was not clear currently. And apoptosis plays important roles in the growth and development. The purpose of the present study was to clarify the relationship between QKI and apoptosis in ST cells. Firstly, our results showed that pEF1α-QKI and shQKI3 have clear effects on expression levels of QKI. Secondly, we established that QKI directly binds to WT1 3′UTR by binding with QRE-1 (2046–2052 bp, ACTAAC) only. Furthermore, QKI overexpression significantly increased the expression levels of WT1 and Bcl-2. QKI also has the effect on delaying the degradation of WT1 mRNA. In addition, we verified that QKI had a significantly suppressed apoptosis in ST cells. Finally, pBI-WT1 could make up for shQKI3-induced decrease in WT1, Bcl-2 mRNA levels and suppress apoptosis in ST cells. The results demonstrated that QKI was an important regulatory factor that affects apoptosis by targeting WT1 gene.     

Fluvastatin attenuated ischemia/reperfusion-induced autophagy and apoptosis in cardiomyocytes through down-regulation HMGB1/TLR4 signaling pathway

Molecular Biology Reports - Fluvastatin, a traditional fat-decreasing drug, is widely used for curing cardiovascular disease. Previous reports demonstrated that fluvastatin pretreatment protected...     

Epigallocatechin-3-gallate inhibits apoptosis and protects testicular seminiferous tubules from ischemia/reperfusion-induced inflammation

Testicular torsion (TT) is a urologic emergency that may result in future infertility problems. The pathologic process of TT is similar to an ischemia reperfusion injury (IRI). The purpose of this study was to evaluate the effect of epigallocatechin-3-gallate (EGCG) on reversing the damaging consequences of TT-induced IRI by examining its inhibitory effects on the expression of inflammatory and apoptosis mediators in a unilateral TT rat model. Eighteen male Sprague-Dawley rats were divided into 3 groups. Group 1 underwent a sham operation of the left testis under general anesthesia. Group 2 underwent ischemia for 1h followed by 4h reperfusion in the presence of saline. The third group was similar to group 2, however, EGCG (50 mg/kg) was injected i.p. 30 min after ischemia induction. The in vivo protective effect of EGCG was tested by measuring testicular levels of TNF-α, IL-6 and IL-1β by ELISA and mRNA expression of iNOS, MCP-1, p53, Bax, Bcl-2 and survivin by real-time PCR. Also, testicular morphological changes and damage to spermatogenesis were evaluated using H&E staining and Johnsen's scoring system, respectively. EGCG treatment improved testicular structures in the ipsilateral testis, markedly inhibited germ cell apoptosis (GCA) and significantly decreased testicular cytokine levels. In addition, EGCG was able to down regulate the mRNA expression of iNOS, MCP-1 and pro-apoptosis genes in favor of cell survival. For the first time we show that in vivo EGCG treatment rescued the torsed testes from IRI-induced inflammation, GCA and damage to spermatogenesis thus suggesting a new preventive approach to inhibiting the inflammatory and apoptotic consequences of TT-induced IRI.     

Calcium-sensing receptors induce apoptosis in cultured neonatal rat ventricular cardiomyocytes during simulated ischemia/reperfusion

Calcium-sensing receptors (CaSRs) are G-protein coupled receptors which regulate systemic calcium homeostasis and also participate in cell proliferation, differentiation and apoptosis. We have previously shown that CaSR can induce apoptosis in isolated rat adult hearts and in normal rat neonatal cardiomyocytes. However, no knowledge exists concerning the role of CaSR in apoptosis induced by ischemia and reperfusion in neonatal cardiac myocytes. Therefore, in the present study, we incubated primary neonatal rat ventricular cardiomyocytes in ischemia-mimetic solution for 2h, then re-incubated them in a normal culture medium for 24h to establish a model of simulated ischemia/reperfusion (I/R). We assayed the apoptotic ratio of the cardiomyocytes by flow cytometry; observed morphological alterations by transmission electron microscope; analyzed the expression of caspase-3, Bcl-2, CaSR, extracellular signal-regulated protein kinase (ERK), and Fas/Fas ligand (FasL) by Western blotting; and measured the concentration of intracellular calcium by Laser Confocal Scanning Microscopy. The results showed that simulated I/R increased the expression of CaSR and cardiomyocyte apoptosis. GdCl3, a specific activator of CaSR, further enhanced CaSR expression, along with increases in intracellular calcium and apoptosis in cardiomyocytes during I/R. Activation of CaSR down-regulated Bcl-2 expression, up-regulated caspase-3 and Fas/FasL expression and stimulated ERK1/2 phosphorylation. In summary, CaSR is involved in I/R injury and apoptosis of neonatal rat ventricular cardiomyocytes by inhibiting Bcl-2, inducing calcium overload and activating the Fas/FasL death receptor pathway.     

Effects of polyamines on apoptosis induced by simulated ischemia/reperfusion injury in cultured neonatal rat cardiomyocytes

We incubated neonatal Sprague-Dawley rat cardiomyocytes in primary culture in a medium simulating ischemia (consisting of hypoxia plus serum deprivation) for 2 h, then re-incubated them for 24 h in normal culture medium to establish a model of simulated ischemia/reperfusion (I/R) injury. Apoptotic cell death was measured by MTT assay, TUNEL staining and flow cytometry. Morphological alterations were assessed by transmission electron microscopy, the expression of caspases-3 and -9 and Bcl-2 and the release of cytochrome c by Western blotting, and the intracellular free-calcium concentration ([Ca2+]i) by laser confocal scanning microscopy. The results showed that pretreatment with 10 micromol/l spermine or spermidine significantly inhibited apoptosis in the I/R cells, suppressed the expression of caspases-3 and -9 and cytochrome c release, up-regulated Bcl-2 expression and decreased [Ca2+]i. However, pretreatment with 10 micromol/l putrescine had the opposite effects. Evidence for this "double-edged" effect of polyamines on apoptosis in I/R-injured cardiomyocytes is presented for the first time. It may suggest a novel pharmacological target for preventing and treating cardiac ischemia/reperfusion injury.     

Role of dopamine D2 receptors in ischemia/reperfusion induced apoptosis of cultured neonatal rat cardiomyocytes

Background

Myocardial ischemia/reperfusion injury is the major cause of morbidity and mortality for cardiovascular diseases. Dopamine D₂ receptors are expressed in cardiac tissues. However, the roles of dopamine D₂ receptors in myocardial ischemia/reperfusion injury and cardiomyocyte apoptosis are unclear. Here we investigated the effects of both dopamine D₂ receptors agonist (bromocriptine) and antagonist (haloperidol) on apoptosis of cultured neonatal rat ventricular myocytes induced by ischemia/reperfusion injury.

Methods

Myocardial ischemia/reperfusion injury was simulated by incubating primarily cultured neonatal rat cardiomyocytes in ischemic (hypoxic) buffer solution for 2 h. Thereafter, these cells were incubated for 24 h in normal culture medium.

Results

Treatment of the cardiomyocytes with 10 μM bromocriptine significantly decreased lactate dehydrogenase activity, increased superoxide dismutase activity, and decreased malondialdehyde content in the culture medium. Bromocriptine significantly inhibited the release of cytochrome c, accumulation of [Ca²⁺]_i, and apoptosis induced by ischemia/reperfusion injury. Bromocriptine also down-regulated the expression of caspase-3 and -9, Fas and Fas ligand, and up-regulated Bcl-2 expression. In contrast, haloperidol (10 μM) had no significant effects on the apoptosis of cultured cardiomyocytes under the aforementioned conditions.

Conclusions

These data suggest that activation of dopamine D₂ receptors can inhibit apoptosis of cardiomyocytes encountered during ischemia/reperfusion damage through various pathways.     

E2F1 and RNA binding protein QKI comprise a negative feedback in the cell cycle regulation

pRb/E2F1 activity is coordinately regulated during the cell cycle progression, while the molecular strategies safeguarding this pathway are not fully understood. We have previously shown that RNA binding protein QKI inhibits the cell proliferation and promotes the differentiation of gastrointestinal epithelium, suggesting a role of QKI in cell cycle regulation. Here we found that with the cell entry into S phase, QKI expression increased both at the mRNA and protein levels, which was reminiscent of cyclin E expression. Forced expression of E2F1 increased the endogenous level of QKI. Promoter luciferase assay and ChIP analysis identified that the -542~-538 E2F1 binding site was responsible for the upregulation. Increased QKI expression by E2F1, in turn, reduced the E2F1 activity and delayed S-phase entry, forming a negative feedback. As a gene expression regulator, QKI overexpression increased p27, while it decreased cyclin D1 and c-fos expression. Molecularly, p27 and c-fos were direct targets of QKI, while cyclin D1 reduction might be an indirect effect. Taken together, our results reveal that E2F1 directly transcribes QKI, which, in turn, negatively regulates the cell cycle by targeting multiple cell cycle regulators, forming an E2F1-QKI-pRb/E2F1 negative feedback loop.     

Calcyclin binding protein promotes DNA synthesis and differentiation in rat neonatal cardiomyocytes

During cardiac muscle development, most cardiomyocytes permanently withdraw from the cell cycle. Previously, by suppressive subtractive hybridization, we identified calcyclin-binding protein/Siah-interacting protein (CacyBP/SIP) as one of the candidates being upregulated in the hyperplastic to hypertrophic switch, suggesting an important role of CacyBP/SIP in cardiac development. To show the importance of CacyBP/SIP during myoblast differentiation, we report here that CacyBP/SIP is developmentally regulated in postnatal rat hearts. The overexpression of CacyBP/SIP promotes the differentiation and DNA synthesis of H9C2 cells and primary rat cardiomyocytes, as well as downregulates the expression of beta-catenin. Besides, CacyBP/SIP promotes the formation of myotubes and multinucleation upon differentiation. To investigate the cardioprotective role of CacyBP/SIP in cardiomyocytes, a hypoxia/reoxygenation model was employed. We found that CacyBP/SIP was upregulated during myocardial infarction (MI) and hypoxia/reoxygenation. As a conclusion, CacyBP/SIP may play a role in cardiomyogenic differentiation and possibly protection of cardiomyocytes during hypoxia/reoxygenation injury.     

The role of endogenous nitric oxide in inhibition of ischemia/reperfusion-induced cardiomyocyte apoptosis

The effect of nitric oxide (NO) synthase inhibition on apoptosis of cardiomyocytes during ischemia/reperfusion was investigated. Isolated perfused guinea-pig hearts were subjected to 35 min ischemia (I) followed by 30 min reperfusion (IR) in the presence or absence of NO synthase inhibitors, L-NAME or L-NMMA or a superoxide scavenger, SOD. Apoptosis was assessed by immunohistochemistry (TUNEL assay, Bax protein staining), by spectrophotometric measurement of cytochrome oxidase activity (COX), and by ultrastructural analysis. Inhibition of NOS significantly increased apoptosis with activation of Bax protein and decrease of COX. SOD infusion had a protective effect on these apoptotic markers. The results suggest that endogenous NO synthesis during I/R protects the heart against apoptotic cell death.     

Preconditioning decreases ischemia/reperfusion-induced peroxynitrite formation

The role for peroxynitrite (ONOO(-)) in the mechanism of preconditioning is not known. Therefore, we studied effects of preconditioning and subsequent ischemia/reperfusion on myocardial ONOO(-) formation in isolated rat hearts. Hearts were subjected to a preconditioning protocol (three intermittent periods of global ischemia/reperfusion of 5 min duration each) followed by a test ischemia/reperfusion (30 min global ischemia and 15 min reperfusion). When compared to nonpreconditioned controls, preceding preconditioning improved postischemic cardiac performance and significantly decreased test ischemia/reperfusion-induced formation of free nitrotyrosine measured in the perfusate as a marker for cardiac endogenous ONOO(-) formation. During preconditioning, however, the first period of ischemia/reperfusion increased nitrotyrosine formation, which was attenuated after the third period of ischemia/reperfusion. We conclude that classic preconditioning inhibits ischemia/reperfusion-induced cardiac formation of ONOO(-) and that subsequent periods of ischemia/reperfusion result in a gradual attenuation of ischemia/reperfusion-induced ONOO(-) generation. This mechanism might be involved in ischemic adaptation of the heart.     

Accelerated ischemia/reperfusion-induced injury in autoimmunity-prone mice

Natural Abs have been implicated in initiating mesenteric ischemia/reperfusion (I/R)-induced tissue injury. Autoantibodies have affinity and self-Ag recognition patterns similar to natural Abs. We considered that autoimmunity-prone mice that express high titers of autoantibodies should have enhanced I/R-induced injury. Five-month-old B6.MRL/lpr mice displayed accelerated and enhanced intestinal I/R-induced damage compared with 2-mo-old B6.MRL/lpr and age-matched C57BL/6 mice. Similarly, older autoimmune mice had accelerated remote organ (lung) damage. Infusion of serum IgG derived from 5-mo-old but not 2-mo-old B6.MRL/lpr into I/R resistant Rag-1-/- mice rendered them susceptible to local and remote organ injury. Injection of monoclonal IgG anti-DNA and anti-histone Abs into Rag-1-/- mice effectively reconstituted tissue injury. These data show that like natural Abs, autoantibodies, such as anti-dsDNA and anti-histone Abs, can instigate I/R injury and suggest that they are involved in the development of tissue damage in patients with systemic lupus erythematosus.     

Antagonism of endothelin-1 inhibits hypoxia-induced apoptosis in cardiomyocytes

Apoptosis is well documented to be a common feature of many pathological processes of the heart. Exogenous endothelin-1 (ET-1) has been shown to be proapoptotic or antiapoptotic, depending on ET-1 concentration, cell type, and the ratio of ETA/ETB receptor subtypes. The role of endogenous ET-1 in cardiomyocyte apoptosis, however, is not clarified. This study observed the effects of the ETA-receptor antagonists BQ610 and BQ123 and the ETB-receptor antagonist BQ788 on hypoxia-induced apoptosis in primary cultured neonatal rat cardiomyocytes. Hypoxic apoptosis was induced by incubating cardiomyocytes in serum-free medium under 3% O2 and 5% CO2 for 24 h and evaluated by TUNEL analysis and flow cytometry. TUNEL analysis showed that the apoptotic cardiomyocytes constituted 24.2% +/- 2.2% of the total cells under hypoxic conditions. Treatment with BQ610 (5 micromol/L) significantly reduced the apoptosis rate to 13.2% +/- 3.7% (data from 4 independent experiments, p < 0.01 vs. hypoxia). Flow cytometry showed that the percentage of apoptotic cells positively stained with annexin V and propidium iodide was 42.76% +/- 4.44% (n = 12) in cultures subjected to hypoxia. BQ123 at 0.04, 0.2, and 1.0 micromol/L dose-dependently reduced the apoptosis rate to 34.00% +/- 10.35% (n = 6, p < 0.05), 30.38% +/- 8.28% (n = 6, p < 0.01), and 22.89% +/- 4.19% (n = 6, p < 0.01), respectively. In contrast, BQ788 did not affect hypoxic apoptosis. These findings suggested that endogenous ET-1 contributed to hypoxia-induced apoptosis in cultured cardiomyocytes, which was mediated by ETA receptors, but not by ETB receptors.     

Growth hormone signalling and apoptosis in neonatal rat cardiomyocytes

Growth hormone (GH) has been reported to be useful to treat heart failure. To elucidate whether GH has direct beneficial effects on the heart, we examined effects of GH on oxidative stress-induced apoptosis in cardiac myocytes. TUNEL staining and DNA ladder analysis revealed that hydrogen peroxide (H₂O₂)-induced apoptosis of cardiomyocytes was significantly suppressed by the pretreatment with GH. GH strongly activated extracellular signal-regulated kinases (ERKs) in cardiac myocytes and the cardioprotective effect of GH was abolished by inhibition of ERKs. Overexpression of dominant negative mutant Ras suppressed GH-stimulated ERK activation. Overexpression of Csk that inactivates Src family tyrosine kinases also inhibited ERK activation evoked by GH. A broad-spectrum inhibitor of protein tyrosine kinases (PTKs), genistein, strongly suppressed GH-induced ERK activation and the cardioprotective effect of GH against apoptotic cell death. GH induced tyrosine phosphorylation of EGF receptor and JAK2 in cardiac myocytes, and an EGF receptor inhibitor tyrphostin AG1478 and a JAK2 inhibitor tyrphostin B42 completely inhibited GH-induced ERK activation. Tyrphostin B42 also suppressed the phosphorylation of EGF receptor stimulated by GH. These findings suggest that GH has a direct protective effect on cardiac myocytes against apoptosis and that the effect of GH is attributed at least in part to the activation of ERKs through Ras and PTKs including JAK2, Src, and EGF receptor tyrosine kinase.     

Hypothermia attenuates ischemia/reperfusion-induced endothelial cell apoptosis via alterations in apoptotic pathways and JNK signaling

Hypothermia is the most effective means of protecting the brain, heart and other organs during ischemia/reperfusion (I/R) injury. However, the precise mechanisms for hypothermia to inhibit I/R-induced endothelial cell apoptosis are not fully understood. In the present study, human umbilical endothelial cells (HUVECs) were exposed to ischemia followed by reperfusion under normothermia (37 °C) or hypothermia (33 °C). Our results showed that hypothermia markedly reduced I/R-induced endothelial cell apoptosis, the expression of cleaved caspase-3 and PARP. Moreover, hypothermia markedly reversed I/R-induced activation of Fas/caspase-8, the increase of Bax and decrease of Bcl-2. Furthermore, hypothermia inhibited JNK1/2 activation via MKP-1 induction. Together, these data demonstrate that hypothermia represses I/R-induced endothelial cell apoptosis by inhibiting both extrinsic- and intrinsic-dependent apoptotic pathways and activation of JNK1/2.     

Inhibition of the Rac1 GTPase protects against nonlethal ischemia/reperfusion-induced necrosis and apoptosis in vivo.

Reperfusion of ischemic tissue results in the generation of reactive oxygen species that contribute to tissue injury. The sources of reactive oxygen species in reperfused tissue are not fully characterized. We hypothesized that the small GTPase Rac1 mediates the oxidative burst in reperfused tissue and thereby contributes to reperfusion injury. In an in vivo model of mouse hepatic ischemia/reperfusion injury, recombinant adenoviral expression of a dominant negative Rac1 (Rac1N17) completely suppressed the ischemia/reperfusion-induced production of reactive oxygen species and lipid peroxides, activation of nuclear factor-kappa B, and resulted in a significant reduction of acute liver necrosis. Expression of Rac1N17 also suppressed ischemia/reperfusion-induced acute apoptosis. The protection offered by Rac1N17 was also evident in knockout mice deficient for the gp91phox component of the phagocyte NADPH oxidase. This work demonstrates the crucial role of a Rac1-regulated oxidase in mediating the production of injurious reactive oxygen species, which contribute to acute necrotic and apoptotic cell death induced by ischemia/reperfusion in vivo. Targeted inhibition of this oxidase, which is distinct from the phagocyte NADPH oxidase, should provide a new avenue for in vivo therapy aimed at protecting organs at risk from ischemia/reperfusion injury.-Ozaki, M., Deshpande, S. S., Angkeow, P., Bellan, J., Lowenstein, C. J., Dinauer, M. C., Goldschmidt-Clermont, P. J., Irani, K. Inhibition of the Rac1 GTPase protects against nonlethal ischemia/reperfusion-induced necrosis and apoptosis in vivo.     

Vasopressin accelerates protein synthesis in neonatal rat cardiomyocytes

Arginine vasopressin (AVP) has been shown to promote vascular smooth muscle cell hypertrophy and hyperplasia of fibroblasts. The present study examines the effect of AVP and endothelin-1 (ET-1) on protein, DNA, and RNA synthesis in primary cultures of serum deprived neonatal rat cardiomyocytes (RC) as assessed by changes in [3H] phenylalanine, [3H] thymidine, and [14C] uridine incorporation respectively. Both AVP and ET-1 evoked significant increases in protein synthesis in RC of 36 ± 12% (p < 0.05) and 53 ± 22% (p < 0.01) respectively. The stimulating action of AVP on [3H] phenylalanine incorporation was abolished by pretreatment with 2-nitro-4carboxyphenyl-N, N-diphenylcarbamate (NCDC), a phospholipase C (PLC) inhibitor. [14C] uridine incorporation was significantly higher in cells incubated with ET-1 (95 ± 12%) but not AVP (9 ± 11%). Neither AVP nor ET-1 significantly affected cell number or [3H] thymidine incorporation, suggesting a lack of a hyperplastic effect. AVP evoked an increase in [Ca2+]i levels (162 ± 12 nmol/L from a basal value of 77 ± 6 nmol/L) which was completely abolished by pretreatment with either NCDC or cyclopiazonic acid (sarcoplasmic reticulum (SR) Ca2+ pump inhibitor) but unaffected by ryanodine (ryanodine sensitive SR Ca2+ store depletor). Taken together, these data suggest that AVP, in a PLC dependent manner, stimulates both protein synthesis and augments [Ca2+]i release in RC from ryanodine insensitive (IP3 sensitive) Ca2+ stores. Thus, AVP may promote cardiac hypertrophy via direct effects on cardiomyocyte protein synthesis secondary to IP3 mediated [Ca2+]i release.     

Taurine prevents the ischemia-induced apoptosis in cultured neonatal rat cardiomyocytes through Akt/caspase-9 pathway

Activated Akt kinase has been proposed as a central role in suppressing apoptosis by modulating the activities of Bcl-2 family proteins and/or caspase-9. To study the mechanism underlying the anti-apoptotic effect of taurine, the interaction between taurine and Akt/caspase-9 pathway was examined using a simulated ischemia model with cultured rat neonatal cardiomyocytes sealed in closed flasks. Taurine (20mM) treatment attenuated simulated ischemia-induced decline in the activity of Akt. Although taurine treatment had no effect on the expression of Bcl-2 in mitochondria and the level of cytosolic cytochrome c, it inhibited ischemia-induced cleavage of caspases 9 and 3. Moreover, adenovirus transfer of the dominant negative form of Akt objected taurine-mediated anti-apoptotic effects, cancelling the suppression of caspase-9 and caspase-3 activities by taurine. These findings provide the first evidence that taurine inhibits ischemia-induced apoptosis in cardiac myocytes with the increase in Akt activities, by inactivating caspase-9.     

Pathogenic importance of pepsin in ischemia/reperfusion-induced gastric injury

We investigated the role of pepsin in the development of ischemia/reperfusion (I/R)-induced gastric lesions in rats. Under urethane anesthesia, the pylorus was ligated, the celiac artery was clamped, and 1 ml of HCl (50-150 mM) was instilled in the stomach. Then, reperfusion was established 15 min later by removing the clamp, and 2 h later the stomach was assessed for gross mucosal damage. Pepstatin (a specific pepsin inhibitor) or pepsin was given i.g. after the pylorus was ligated while cimetidine, omeprazole, or atropine was given s.c. 30 min before the ligation. I/R produced hemorrhagic gastric injury, with a concomitant increase in the amount of pepsin secreted, and the degree of both these responses was dependent on the concentration of HCl. The formation of lesions by IR in the presence of 100 mM HCl was significantly prevented by atropine or bilateral vagotomy, but neither omeprazole nor cimetidine had any effect. Intragastric administration of pepstatin dose-dependently reduced the severity of the I/R-induced gastric lesions, the effect being significant even at 0.1 mg/kg, while that of pepsin markedly aggravated these lesions. The increased pepsin output during I/R was associated with luminal acid loss and significantly inhibited by bilateral vagotomy or pretreatment with atropine but not cimetidine or omeprazole, while pepstatin significantly inhibited the pepsin activity. In conclusion, we suggest that pepsin plays a pivotal role in the pathogenesis of I/R-induced gastric lesions, and pepsin secretion is increased during I/R, the process being associated with acid back-diffusion and mediated through a vagal-cholinergic pathway.