Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation ...Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.展开更多
AIM: To investigate the expression of mitogen-activated protein kinases (MAPKs) and its upstream protein kinase in human gastric cancer and to evaluate the relationship between protein levels and clinicopathological p...AIM: To investigate the expression of mitogen-activated protein kinases (MAPKs) and its upstream protein kinase in human gastric cancer and to evaluate the relationship between protein levels and clinicopathological parameters.METHODS: Westem blot was used to measure the expression of extracellular signal-regulated kinase (ERK)-1, ERK-2, ERK-3,p38 and mitogen or ERK activated protein kinaseMEK-1proteins in surgically resected gastric carcinoma, adjacent normal mucosa and metastatic lymph nodes from 42 patients.Immunohistochemistry was employed for their localization.RESULTS: Compared with normal tissues, the protein levels of ERK-1 (integral optical density value 159 526±65 760 vs122 807±65 515, P = 0.001), ERK-2 (168 471±95 051 vs120 469±72 874, P＜0.001), ERK-3 (118 651±71 513 vs70 934±68 058, P＜0.001), P38 (104 776±51 650 vs82 930±40 392, P= 0.048) and MEK-1(116 486±45 725 vs101 434±49 387, P = 0.027) were increased in gastric cancer tissues. Overexpression of ERK-3 was correlated to TNM staging [average ratio of integral optic density (IOD)tumor:IODnormal in TNM Ⅰ, Ⅱ, Ⅲ, Ⅳ tumors was 1.43±0.34,5.08±3.74, 4.99±1.08, 1.44±1.02, n = 42, P = 0.023] and serosa invasion (4.31±4.34 vs 2.00±2.03, P = 0.037). In poorly differentiated cancers (n = 33), the protein levels of ERK-1 and ERK-2 in stage Ⅲ and Ⅳ tumors were higher than those in stage Ⅰ and Ⅱ tumors (2.64±3.01 vs 1.01±0.33,P = 0.022; 2.05±1.54 vs 1.24±0.40, P = 0.030). Gastric cancer tissues with either lymph node involvement (2.49±2.91vs 1.03±0.36, P= 0.023; 1.98±1.49 vs 1.24±0.44, P= 0.036)or serosa invasion (2.39±2.82 vs 1.01±0.35, P = 0.022;1.95±1.44 vs 1.14±0.36, P = 0.015) expressed higher protein levels of ERK-1 and ERK-2. In Borrmann Ⅱ tumors,expression of ERK-2 and ERK-3 was increased compared with Borrmann Ⅲ tumors (2.57±1.86 vs 1.23±0.60, P = 0.022;5.50±5.05 vs 1.83±1.21, P = 0.014). Borrmann Ⅳ tumors expressed higher p38 protein levels. No statistically significant difference in expression of MAPKs was found when stratified to tumor size or histological grade (P＞0.05).Protein levels of ERK-2, ERK-3 and MEK-1 in metastatic lymph nodes were 2-7 folds higher than those in adjacent normal mucosa. The immunohistochemistry demonstrated that ERK-1, ERK-2, ERK-3, p38 and MEK-1 proteins were mainly localized in cytoplasm. The expression of MEK-1 in gastric cancer cells metastasized to lymph nodes was higher than that of the primary site.CONCLUSION: MAPKs, particularly ERK subclass are overexpressed in the majority of gastric cancers.Overexpression of ERKs is correlated to TNM staging, serosa invasion, and lymph node involvement. The overexpression of p38 most likely plays a prominent role in certain morphological subtypes of gastric cancers. MEK-1 is also overexpressed in gastric cancer, particularly in metastatic lymph nodes. Upregulation of MAPK signal transduction pathways may play an important role in tumorigenesis and metastatic potential of gastric cancer.展开更多
Objective:Mitogen-activated protein kinases (MAPKs) are correlated with a more malignant phenotype in many cancers.This study was designed to evaluate the predictive value of the expression of MAPK phosphatase-1 (MKP-...Objective:Mitogen-activated protein kinases (MAPKs) are correlated with a more malignant phenotype in many cancers.This study was designed to evaluate the predictive value of the expression of MAPK phosphatase-1 (MKP-1) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK 1/2),as the key regulatory mechanism of the MAPKs,in lung squamous cell carcinoma (SCC).Methods:We assessed the expressions of MKP-1 and p-ERK 1/2 in twenty subjects at different differentiation degree of SCC and five normal lungs by immunohistochemistry and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis.Results:Immunohistochemistry and real-time RT-PCR assay showed that the expression of MKP-1 was gradually decreased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma,and it was negatively correlated with tumor differentiation (P<0.01).However,the expression of p-ERK 1/2 or ERK 1/2 was gradually increased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma,and it was positively correlated with tumor differentiation (P<0.01).Conclusions:Our data indicates the relevance of MKP-1 and p-ERK 1/2 in SCC as a potential positive and negative prognostic factor.The imbalanced expression of MKP-1 and p-ERK 1/2 may play a role in the development of SCC and these two molecules may be new targets for the therapy and prognosis of SCC.展开更多
Abstract Objective:To explore the effects of γ-irradiation on mitogen-activatedprotein kinases(MAPKs) and role intracellular calcium in this event in intestinal epithelial cell line 6(IEC-6 cells).Methods:After cultu...Abstract Objective:To explore the effects of γ-irradiation on mitogen-activatedprotein kinases(MAPKs) and role intracellular calcium in this event in intestinal epithelial cell line 6(IEC-6 cells).Methods:After cultured rat IIEC-6 cells with or without the pretreatment of intracellular Ca^2+ chelator were exposed to γ-ir-radiation of 6 Gy, the total and phosphorylated MAPKs in the cells were determined with Western blotting and apoptosis was examined with flow cytometry.Activities of Extracellular signal-regulated protein kinase (ERK) and p38 MAPK were determined by using immuoprecipitation followed by Western blotting.Results:In response to γ-irradiation,phosphorylation of ERK was not significantly observed ,while the levels of phos-phorylated c-Jun NH2-terminal kinase(JNK) and p38 MAPK were increased in 30 min and reached the peak 2h after exposure to 6Gy γ-irradiation,though the cell viability was significantly lowered 12h.On the other hand, no obvious changes were seen in the total protein levels of ERK, JNK and p38 MAPK.Chelation of in-tracellular Ca^2+ almost completely suppressed the JNK and p38 MAPK phosphorylation induced by γ-irradiation is a potent activator for JNK and p38 MAPK, and Ca^2+ mobilized from intracellular stores plays an important role in the activation of MAPKs and the induction of apoptosis in IEC-6 cells.展开更多
Objective： To study the effect of salvianolic acid B （SAB） and curcumin, the extracts of Solvie Miltiorrhize and Curcume Longe, on the proliferation and activation of hepatic stellate cell （HSC）, and the extracel...Objective： To study the effect of salvianolic acid B （SAB） and curcumin, the extracts of Solvie Miltiorrhize and Curcume Longe, on the proliferation and activation of hepatic stellate cell （HSC）, and the extracellular signal regulated kinase （ERK） expression in it. Methods： Rat＇s HSC-T6 were cultured and treated by SAB or curcumin. The inhibitory effect on cell proliferation was determined by 3-（4,5-dimthyl-2- 2thiazoly）-2,5-diphenyl-2H-tetrazolium bromide （MTT） colorimetry, and the expression levels of a smooth actin （a-SMA）, collagen type Ⅰ , and ERK were determined by Western blot. Results： SAB and curcumin inhibited the proliferation and activation of rat＇s HSC-T6 in dose-dependent fashion and significantly reduced the expression level of a-SMA （ P〈0.01 ）. Curcumin significantly reduced the expression of collagen type Ⅰ （P〈0.05）. Both SAB and curcumin showed insignificant effect on the ERK expression level, but they could significantly reduce the level of phosphorylated-ERK expression, showing significant difference as compared with that in the control group （ P〈0.01 and P〈0.05 respectively）. Conclusion： SAB and curcumin could significantly inhibit the proliferation, activation of HSC, and the production of type Ⅰ collagen in HSC, the mechanism may be associated with their inhibition on ERK phosphorylation.展开更多
AIM:To explore the role and mechanisms of extracellular signal-regulated protein kinase-mitogen-activated protein kinase(ERK-MAPK) signaling in pentagastrinregulated growth of large intestinal carcinoma.METHODS:HT-29 ...AIM:To explore the role and mechanisms of extracellular signal-regulated protein kinase-mitogen-activated protein kinase(ERK-MAPK) signaling in pentagastrinregulated growth of large intestinal carcinoma.METHODS:HT-29 cells were incubated in different media and divided into the control group,pentagastrin group,proglumide group,and pentagastrin + proglumide group.No reagent was added to the control group,and other groups were incubated with reagent at different concentrations.Changes in proliferation of HT-29 cells were detected by MTT assay,and the optimal concentrations of pentagastrin and proglumide were determined.The changes in proliferation index(PI) and apoptosis rate(AR) of HT-29 cells were detected by Annexin V-fluorescein isothiocyanate flow cytometry.mRNA expression of pentagastrin receptor/cholecystokinin-B receptor(CCK-BR),ERK1/2 and K-ras were detected by reverse transcriptase polymerase chain reaction.The protein and phosphorylation level of ERK1/2 and K-ras were detected by western blotting.All data were analyzed by analysis of variance and SNK-q test.RESULTS:The proliferation of HT-29 cells was stimulated by pentagastrin at a concentration of 6.25-100 mg/L,and the optimal concentration of pentagastrin was 25.0 mg/L(F = 31.36,P < 0.05).Proglumide had no obvious effect on the proliferation of HT-29 cells,while it significantly inhibited the proliferation of HT-29 cells stimulated by pentagastrin when the concentration of proglumide was 8.0-128.0 mg/L,and the optimal concentration was 32.0 mg/L(F = 24.31,P < 0.05).The PI of the pentagastrin(25.0 mg/L) group was 37.5% ± 5.2%,which was significantly higher than 27.7% ± 5.0% of the control group and 27.3% ± 5.8% of the pentagastrin(25.0 mg/L) + proglumide(32.0 mg/L) group(Q = 4.56-4.75,P < 0.05).The AR of the pentagastrin(25.0 mg/L) group was 1.9% ± 0.4%,which was significantly lower than 2.5% ± 0.4% of the control group and 2.4% ± 0.3% of the pentagastrin(25.0 mg/L) + proglumide(32.0 mg/L) group(Q = 4.23-4.06,P < 0.05).mRNA expression of CCK-BR was detected in HT-29 cells.The phosphorylation levels of ERK1/2 protein and phosphorylated K-ras protein of the pentagastrin group were 0.43% ± 0.04% and 0.45% ± 0.06%,which were significantly higher than 0.32% ± 0.02% and 0.31% ± 0.05% of the control group(Q = 7.78-4.95,P < 0.05),and 0.36% ± 0.01% and 0.35% ± 0.04% of the pentagastrin + proglumide group(Q = 5.72-4.08,P < 0.05).There were no significant differences in the mRNA and protein expression of ERK1/2 and K-ras among the control,pentagastrin,proglumide and pentagastrin + proglumide groups(F = 0.52,0.72,0.78,0.28;P > 0.05).CONCLUSION:Gastrin stimulates proliferation of HT-29 cells and inhibits apoptosis by upregulating phosphorylation of ERK and K-ras through the Ras-Raf-MEK1/2-ERK1/2 pathway,and this is restrained by proglumide.展开更多
文摘Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.
基金Supported by Technology Foundation of Ministry of Education, China
文摘AIM: To investigate the expression of mitogen-activated protein kinases (MAPKs) and its upstream protein kinase in human gastric cancer and to evaluate the relationship between protein levels and clinicopathological parameters.METHODS: Westem blot was used to measure the expression of extracellular signal-regulated kinase (ERK)-1, ERK-2, ERK-3,p38 and mitogen or ERK activated protein kinaseMEK-1proteins in surgically resected gastric carcinoma, adjacent normal mucosa and metastatic lymph nodes from 42 patients.Immunohistochemistry was employed for their localization.RESULTS: Compared with normal tissues, the protein levels of ERK-1 (integral optical density value 159 526±65 760 vs122 807±65 515, P = 0.001), ERK-2 (168 471±95 051 vs120 469±72 874, P＜0.001), ERK-3 (118 651±71 513 vs70 934±68 058, P＜0.001), P38 (104 776±51 650 vs82 930±40 392, P= 0.048) and MEK-1(116 486±45 725 vs101 434±49 387, P = 0.027) were increased in gastric cancer tissues. Overexpression of ERK-3 was correlated to TNM staging [average ratio of integral optic density (IOD)tumor:IODnormal in TNM Ⅰ, Ⅱ, Ⅲ, Ⅳ tumors was 1.43±0.34,5.08±3.74, 4.99±1.08, 1.44±1.02, n = 42, P = 0.023] and serosa invasion (4.31±4.34 vs 2.00±2.03, P = 0.037). In poorly differentiated cancers (n = 33), the protein levels of ERK-1 and ERK-2 in stage Ⅲ and Ⅳ tumors were higher than those in stage Ⅰ and Ⅱ tumors (2.64±3.01 vs 1.01±0.33,P = 0.022; 2.05±1.54 vs 1.24±0.40, P = 0.030). Gastric cancer tissues with either lymph node involvement (2.49±2.91vs 1.03±0.36, P= 0.023; 1.98±1.49 vs 1.24±0.44, P= 0.036)or serosa invasion (2.39±2.82 vs 1.01±0.35, P = 0.022;1.95±1.44 vs 1.14±0.36, P = 0.015) expressed higher protein levels of ERK-1 and ERK-2. In Borrmann Ⅱ tumors,expression of ERK-2 and ERK-3 was increased compared with Borrmann Ⅲ tumors (2.57±1.86 vs 1.23±0.60, P = 0.022;5.50±5.05 vs 1.83±1.21, P = 0.014). Borrmann Ⅳ tumors expressed higher p38 protein levels. No statistically significant difference in expression of MAPKs was found when stratified to tumor size or histological grade (P＞0.05).Protein levels of ERK-2, ERK-3 and MEK-1 in metastatic lymph nodes were 2-7 folds higher than those in adjacent normal mucosa. The immunohistochemistry demonstrated that ERK-1, ERK-2, ERK-3, p38 and MEK-1 proteins were mainly localized in cytoplasm. The expression of MEK-1 in gastric cancer cells metastasized to lymph nodes was higher than that of the primary site.CONCLUSION: MAPKs, particularly ERK subclass are overexpressed in the majority of gastric cancers.Overexpression of ERKs is correlated to TNM staging, serosa invasion, and lymph node involvement. The overexpression of p38 most likely plays a prominent role in certain morphological subtypes of gastric cancers. MEK-1 is also overexpressed in gastric cancer, particularly in metastatic lymph nodes. Upregulation of MAPK signal transduction pathways may play an important role in tumorigenesis and metastatic potential of gastric cancer.
基金supported by the National Natural Science Foundation of China (No. 30900654)the Science and Technology Department of Zhejiang Province (No. 2009R10031)the Health Bureau of Zhejiang Province (No. 2009QN010), China
文摘Objective:Mitogen-activated protein kinases (MAPKs) are correlated with a more malignant phenotype in many cancers.This study was designed to evaluate the predictive value of the expression of MAPK phosphatase-1 (MKP-1) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK 1/2),as the key regulatory mechanism of the MAPKs,in lung squamous cell carcinoma (SCC).Methods:We assessed the expressions of MKP-1 and p-ERK 1/2 in twenty subjects at different differentiation degree of SCC and five normal lungs by immunohistochemistry and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis.Results:Immunohistochemistry and real-time RT-PCR assay showed that the expression of MKP-1 was gradually decreased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma,and it was negatively correlated with tumor differentiation (P<0.01).However,the expression of p-ERK 1/2 or ERK 1/2 was gradually increased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma,and it was positively correlated with tumor differentiation (P<0.01).Conclusions:Our data indicates the relevance of MKP-1 and p-ERK 1/2 in SCC as a potential positive and negative prognostic factor.The imbalanced expression of MKP-1 and p-ERK 1/2 may play a role in the development of SCC and these two molecules may be new targets for the therapy and prognosis of SCC.
基金in part by Natural Sciences Foundation of China (No. 39870239)by the Sasagawa Fellowship,Japan.
文摘Abstract Objective:To explore the effects of γ-irradiation on mitogen-activatedprotein kinases(MAPKs) and role intracellular calcium in this event in intestinal epithelial cell line 6(IEC-6 cells).Methods:After cultured rat IIEC-6 cells with or without the pretreatment of intracellular Ca^2+ chelator were exposed to γ-ir-radiation of 6 Gy, the total and phosphorylated MAPKs in the cells were determined with Western blotting and apoptosis was examined with flow cytometry.Activities of Extracellular signal-regulated protein kinase (ERK) and p38 MAPK were determined by using immuoprecipitation followed by Western blotting.Results:In response to γ-irradiation,phosphorylation of ERK was not significantly observed ,while the levels of phos-phorylated c-Jun NH2-terminal kinase(JNK) and p38 MAPK were increased in 30 min and reached the peak 2h after exposure to 6Gy γ-irradiation,though the cell viability was significantly lowered 12h.On the other hand, no obvious changes were seen in the total protein levels of ERK, JNK and p38 MAPK.Chelation of in-tracellular Ca^2+ almost completely suppressed the JNK and p38 MAPK phosphorylation induced by γ-irradiation is a potent activator for JNK and p38 MAPK, and Ca^2+ mobilized from intracellular stores plays an important role in the activation of MAPKs and the induction of apoptosis in IEC-6 cells.
基金Supported by the National Natural Science Foundation of China (No. 30300458)
文摘Objective： To study the effect of salvianolic acid B （SAB） and curcumin, the extracts of Solvie Miltiorrhize and Curcume Longe, on the proliferation and activation of hepatic stellate cell （HSC）, and the extracellular signal regulated kinase （ERK） expression in it. Methods： Rat＇s HSC-T6 were cultured and treated by SAB or curcumin. The inhibitory effect on cell proliferation was determined by 3-（4,5-dimthyl-2- 2thiazoly）-2,5-diphenyl-2H-tetrazolium bromide （MTT） colorimetry, and the expression levels of a smooth actin （a-SMA）, collagen type Ⅰ , and ERK were determined by Western blot. Results： SAB and curcumin inhibited the proliferation and activation of rat＇s HSC-T6 in dose-dependent fashion and significantly reduced the expression level of a-SMA （ P〈0.01 ）. Curcumin significantly reduced the expression of collagen type Ⅰ （P〈0.05）. Both SAB and curcumin showed insignificant effect on the ERK expression level, but they could significantly reduce the level of phosphorylated-ERK expression, showing significant difference as compared with that in the control group （ P〈0.01 and P〈0.05 respectively）. Conclusion： SAB and curcumin could significantly inhibit the proliferation, activation of HSC, and the production of type Ⅰ collagen in HSC, the mechanism may be associated with their inhibition on ERK phosphorylation.
基金Supported by Natural Science Foundation of Anhui Province,No.1408085MH148Natural Science Fund of Education Bureau of Anhui Province,No.kj2010b242+1 种基金Natural Science Fund of Wannan Medical College,No.wk2012zf02the key science and technology project of Wuhu City,No.health-2-4
文摘AIM:To explore the role and mechanisms of extracellular signal-regulated protein kinase-mitogen-activated protein kinase(ERK-MAPK) signaling in pentagastrinregulated growth of large intestinal carcinoma.METHODS:HT-29 cells were incubated in different media and divided into the control group,pentagastrin group,proglumide group,and pentagastrin + proglumide group.No reagent was added to the control group,and other groups were incubated with reagent at different concentrations.Changes in proliferation of HT-29 cells were detected by MTT assay,and the optimal concentrations of pentagastrin and proglumide were determined.The changes in proliferation index(PI) and apoptosis rate(AR) of HT-29 cells were detected by Annexin V-fluorescein isothiocyanate flow cytometry.mRNA expression of pentagastrin receptor/cholecystokinin-B receptor(CCK-BR),ERK1/2 and K-ras were detected by reverse transcriptase polymerase chain reaction.The protein and phosphorylation level of ERK1/2 and K-ras were detected by western blotting.All data were analyzed by analysis of variance and SNK-q test.RESULTS:The proliferation of HT-29 cells was stimulated by pentagastrin at a concentration of 6.25-100 mg/L,and the optimal concentration of pentagastrin was 25.0 mg/L(F = 31.36,P < 0.05).Proglumide had no obvious effect on the proliferation of HT-29 cells,while it significantly inhibited the proliferation of HT-29 cells stimulated by pentagastrin when the concentration of proglumide was 8.0-128.0 mg/L,and the optimal concentration was 32.0 mg/L(F = 24.31,P < 0.05).The PI of the pentagastrin(25.0 mg/L) group was 37.5% ± 5.2%,which was significantly higher than 27.7% ± 5.0% of the control group and 27.3% ± 5.8% of the pentagastrin(25.0 mg/L) + proglumide(32.0 mg/L) group(Q = 4.56-4.75,P < 0.05).The AR of the pentagastrin(25.0 mg/L) group was 1.9% ± 0.4%,which was significantly lower than 2.5% ± 0.4% of the control group and 2.4% ± 0.3% of the pentagastrin(25.0 mg/L) + proglumide(32.0 mg/L) group(Q = 4.23-4.06,P < 0.05).mRNA expression of CCK-BR was detected in HT-29 cells.The phosphorylation levels of ERK1/2 protein and phosphorylated K-ras protein of the pentagastrin group were 0.43% ± 0.04% and 0.45% ± 0.06%,which were significantly higher than 0.32% ± 0.02% and 0.31% ± 0.05% of the control group(Q = 7.78-4.95,P < 0.05),and 0.36% ± 0.01% and 0.35% ± 0.04% of the pentagastrin + proglumide group(Q = 5.72-4.08,P < 0.05).There were no significant differences in the mRNA and protein expression of ERK1/2 and K-ras among the control,pentagastrin,proglumide and pentagastrin + proglumide groups(F = 0.52,0.72,0.78,0.28;P > 0.05).CONCLUSION:Gastrin stimulates proliferation of HT-29 cells and inhibits apoptosis by upregulating phosphorylation of ERK and K-ras through the Ras-Raf-MEK1/2-ERK1/2 pathway,and this is restrained by proglumide.
