Association of polymorphisms in myeloperoxidase and catalase genes with precancerous changes in the gastric mucosa of patients at inner-city hospitals in New York.

Gastric carcinogenesis is a multistep process progressing from chronic gastritis, through glandular atrophy (GA), intestinal metaplasia (IM) and dysplasia. We have previously demonstrated that minority patients at New York City hospitals are infected with a relatively virulent strain of H. pylori (Hp) and that Hp infection is associated with an increased incidence of precancerous changes in the gastric mucosa. Nevertheless, precancerous changes are not observed in every Hp-infected individual, suggesting that environmental and genetic factors may also play a role in the formation and appearance of precancerous lesions. In the present study, the association between polymorphisms in the promoter regions of human myeloperoxidase (MPO -463G--> A) and catalase (CAT -262C-->T) genes and the appearance of precancerous changes in the gastric mucosa of our patient population were examined. Patients enrolled in this study were undergoing endoscopy for gastrointestinal complaints. Samples were collected from 126 patients at Kings County Hospital in Brooklyn and St. John's Episcopal Hospital in Queens. One antral biopsy was taken for genotyping, while additional biopsies were taken from the antrum and fundic region for histological analysis and were scored with respect to acute and chronic inflammation, GA, IM and Hp infestation according to the Sydney classification. MPO and CAT genotypes were determined by PCR and RFLP. CAT genotypes did not influence the incidence or severity of precancerous lesions in the fundic or antral regions of the stomach, whereas the MPO -463A allele was associated with an increase in intensity of gastric atrophy in the fundic mucosa. In Hp-infected individuals, the MPO -463G/G genotype was associated with an increase in the incidence of IM in the antrum, whereas the A allele was associated with an increase in IM in the fundic region. These paradoxical findings suggest that different MPO genotypes are associated with the appearance of IM in distinct anatomical regions of the stomach. However, since the majority of gastric cancer (GC) cases in our patient population occurred in the antrum, the MPO -463G/G genotype, which is associated with increased MPO expression and antral IM, may be considered a risk factor for GC.

Helicobacter pylori infection is associated with a high incidence of intestinal metaplasia in the gastric mucosa of patients at inner-city hospitals in New York.

Gastric carcinogenesis is a multistep process progressing from chronic gastritis, through glandular atrophy (GA), intestinal metaplasia (IM) and dysplasia. Infection of the stomach with H. pylori increases the risk of developing gastric cancer. Few studies have examined the degree to which Hp-induced changes occur in specific populations. In the present study, we examined the association between Hp infection and histological changes in the gastric mucosa of patients at two inner-city hospitals in New York. Patients enrolled in this study were undergoing endoscopy for gastrointestinal complaints. One antral biopsy was taken for detecting and genotyping Hp by PCR. Additional biopsies were taken from the antrum and fundic region for histological analysis and were scored with respect to acute and chronic inflammation, GA, IM and Hp infestation according to the Sydney classification. Hp strains infecting these patients were genotyped with respect to the expression of Hp virulence factors including VacA, CagA, and BabA2. Samples were collected from 126 patients at Kings County Hospital in Brooklyn and St. John's Episcopal Hospital in Queens. Hp infection rates were highest in Blacks (41.6%) and Hispanics (29.4%) and lowest in Caucasians (18.8%). Scores for acute and chronic inflammation and IM were higher in Hp-infected individuals in both the antrum and fundic regions, whereas Hp infection did not affect the incidence or intensity of GA. In Hp-infected individuals, the incidence of IM was greater in the antrum (Hp-infected 37.8% vs. non-infected 9.2%, p < 0.05) and fundic region (Hp-infected 15.1% vs. noninfected 1.8%, p < 0.05). Genotyping of the Hp strains infecting these patients revealed that the predominant VacA allele was s1 bm 1 and that the CagA gene was present in 69.8% of Hp-infected samples. Interestingly, the BabA2 gene was detected in only four samples (9.3%). The incidence of IM in the antrum was higher in CagA + samples when compared with CagA- samples (52.2% vs. 15.4%, respectively). Our findings indicate that the virulent Hp strain infecting minority patients treated at inner-city hospitals in New York City is associated with a high incidence of IM and that these patients may be at greater risk for developing gastric cancer than the general population.

H. pylori infection and genotyping in patients undergoing upper endoscopy at inner city hospitals.

Kings County Hospital (KCH), and St. John's Episcopal Hospital (SJH) are inner-city hospitals in New York City serving predominantly minority populations. Staten Island University Hospital (SIUH) serves a predominantly middle-class Caucasian population. We examined H. pylori (HP) infection in patients undergoing upper endoscopy at these hospitals. Two gastric biopsies were obtained from each patient. One biopsy was examined by histology or the rapid urease test for the presence of HP. The other was subjected to analysis by PCR to detect HP DNA and to identify putative HP virulence factors. Of 200 subjects, 54% were African-American, 10% were Hispanic, and 36% were Caucasian. HP infection rates in African-American, Hispanic, and Caucasian patients were 43%, 20%, and 11%, respectively. Many of the African-American patients are recent immigrants from the Caribbean Islands. In these patients, an inverse relationship was observed between HP infection and the number of years living in the United States. Higher levels of HP infection were observed in patients with duodenitis and peptic ulcer disease. With respect to HP virulence factors, the vacA s1b and m1 alleles, as well as the iceA2 allele were the predominant alleles expressed in HP-positive samples obtained from African-Americans. The cagA gene was detected in 81% of HP-positive samples. However, CagA positivity was not related to any specific gastrointestinal disorder. Our findings indicate that among several ethnic groups served by three hospitals, African-American patients have the highest rate of HP infection. Moreover, in AfricanAmerican patients undergoing endoscopy: (1) HP infection was inversely related to the number of years the patients have been living in the USA; (2) HP infection rates were higher in patients diagnosed with duodenitis and peptic ulcer disease versus other disorders; (3) expression of the CagA gene was not associated with any specific gastroduodenal disorder; and (4) there was little allelic heterogeneity with respect to VacA and IceA subtypes. These findings suggest that inner-city African-Americans are more likely to be infected with HP and suffer from more serious gastroduodenal disorders than other ethnic groups.

Expression and localization of rab escort protein isoforms in parotid acinar cells from rat.

Rab proteins are geranylgeranylated on their carboxyl terminal cysteine motifs by geranylgeranyltransferase II (GGTase). Rab escort protein (REP) is required to present Rab proteins to GGTase. REP may remain bound to newly isoprenylated Rab proteins and present them to their target membrane. Other studies have shown that Rab proteins cycle between the membrane and cytosolic compartments and that cytosolic Rab proteins are complexed with rab-GDI. In the present study, we examined the expression and localization of REP isoforms in parotid acinar cells. Although both REP isoforms, REP-1 and REP-2, were detected in parotid cytosol, REP-2 was the predominant isoform. Subcellular fractionation revealed that approximately 42% of cellular REP-2 is membrane-associated. REP-2 was partially removed from parotid membranes with 1 M NaCl or Na(2)CO(3), indicating that REP-2 is a peripheral membrane protein. Membrane-associated REP-2 did not colocalize with Rab3D on secretory granule membranes. However, density gradient centrifugation revealed that membrane-associated REP-2 and Rab3D colocalize on low- and high-density membrane fractions in parotid acinar cells. Isoproterenol, an agent which induces amylase release from parotid glands, caused a shift in both REP-2 and Rab3D to less dense membrane fractions. When acinar cell cytosol was fractionated by gel filtration chromatography, Rab3D eluted exclusively with REP, not rab-GDI. In contrast, Rab1B and Rab5 eluted with both REP and Rab-GDI. Colocalization of Rab3D and REP-2 on acinar cell membranes suggests that REP-2 plays a role in delivering Rab3D to parotid membranes and may regulate guanine nucleotide binding to membrane-associated Rab3D. In addition, unlike other Rab proteins, cytosolic Rab3D appears to associate exclusively with REP, not rab-GDI in parotid acinar cells.

Expression and localization of Rab3D in rat parotid gland.

Rab3 proteins (isoforms A, B, C and D) are low molecular weight GTP-binding proteins proposed to be involved in regulated exocytosis. In the present study, Rab3 protein expression and localization was examined in rat parotid gland by reverse transcription (rt) PCR, Western blotting and immunocytochemistry. An approximately 200 bp PCR product was obtained from parotid RNA by rtPCR and this fragment was cloned and sequenced. Nucleotide and deduced amino acid sequences obtained from five clones were identical to rab3D. Membrane and cytosolic fractions prepared from parotid acini were immunoblotted with antisera specific for each of the four Rab3 isoforms. A 28 kDa protein was detected with Rab3D-specific antisera in both fractions with staining being more intense in the membrane fraction. No other Rab3 isoforms were detected by immunoblotting, a result consistent with those obtained by rtPCR. Rab3D was enriched in zymogen granule membranes and Triton X-114 extraction revealed that this isoform is predominantly lipid-modified in parotid. Localization of Rab3D was done on frozen sections of parotid gland by immunofluorescence microscopy. Staining was observed primarily in the acinar cells and was adjacent to the acinar lumen. Incubation of dispersed acini with isoproterenol and substance P stimulated amylase secretion 4- and 2-fold above basal, respectively. Isoproterenol, but not substance P, induced redistribution of Rab3D from the cytosol to the membrane fraction in dispersed parotid acini. Consistent with these findings, isoproterenol injections into fasted rats also resulted in increased membrane-associated Rab3D in the parotid acini. These results indicate that Rab3D is: (1) the major Rab3 isoform expressed in rat parotid gland; (2) localized to zymogen granule membranes; and (3) involved with regulated enzyme secretion in acinar cells.

Cytosolic RAB3D is associated with RAB escort protein (REP), not RAB-GDP dissociation inhibitor (GDI), in dispersed chief cells from guinea pig stomach.

Rab3D, a low-molecular-weight GTP-binding protein believed to be involved with regulated exocytosis, is associated with secretory granules in gastric chief cells. Although Rab3D is predominantly membrane associated, a significant fraction is cytosolic. Rab proteins are geranylgeranylated on their C-terminal cysteine motifs by geranylgeranyltransferase (GGTase). Rab escort protein (REP) is required to present Rab proteins to GGTase and may accompany newly modified Rab proteins to their target membrane. In most tissues, cytosolic Rab proteins are complexed with rab-GDP dissociation inhibitor (rab-GDI). In the present study, we examined the interactions of Rab3D with cytosolic proteins in dispersed chief cells. Two REP isoforms and at least two GDI isoforms are present in chief cell and brain cytosol. When chief cell cytosol was fractionated by gel filtration chromatography, Rab3D eluted with REP at >150 kDa, whereas rab-GDI eluted as a separate 65-kDa peak, suggesting that Rab3D exists as a complex with REP, but not with rab-GDI. In addition, a small fraction of Rab3D eluted as a monomer at 29 kDa. As has been demonstrated previously, in brain cytosol, Rab3 proteins co-elute with rab-GDI at approx. 90 kDa, suggesting that Rab3 proteins undergo active cycling between membrane and cytosolic compartments in this tissue. In vitro experiments revealed that Rab3D remains associated with REP after geranylgeranylation. Our findings suggest that, in gastric chief cells, Rab3D remains associated with REP after geranylgeranylation until it is presented to its target membrane.

Protein kinase C isoform expression and function in transformed and non-transformed pancreatic acinar cell lines.

Members of the protein kinase C (PKC) family of multifunctional serine/threonine phosphorylating enzymes are believed to play a role in regulating cellular differentiation and proliferation in many cell types. In the present study, we examined the expression of PKC isoforms in non-transformed (BMRPA.430) and transformed (TUC3) rat pancreatic acinar cell lines and compared this to PKC expression in freshly dispersed acini from rat pancreas. BMRPA.430 cells maintain characteristics of normal acini and are not tumorigenic, whereas TUC3 cells do not express tight junctions or polygonal morphology and are tumorigenic. As reported previously, PKC alpha, delta, epsilon, and zeta are expressed in freshly prepared acini. Likewise, these isoforms were detected in both the BMRPA.430 and TUC3 cell lines. In addition, PKC theta, a novel isoform, was detected in all three cell types at low levels. We used two PKC inhibitors to examine the role of PKC in acinar cell proliferation. CGP 41 251, a selective PKC inhibitor, and Go 6976, an agent which specifically inhibits calcium-dependent PKC isoforms, inhibited cell proliferation of both cell lines. Translocation of PKC alpha to the membrane was not observed in either cell line. Hence, our data indicate that ras-induced transformation does not alter PKC isoform expression in pancreatic acinar cells and that activation of PKC alpha is involved with acinar cell growth.

Regulation of calcium-induced exocytosis from gastric chief cells by protein phosphatase-2B (calcineurin).

The molecular mechanisms whereby calcium stimulates secretion are uncertain. In the present study, we used streptolysin O (SLO)-permeabilized chief cells from guinea pig stomach to investigate whether protein phosphatase-2B (calcineurin), a calcium/calmodulin-dependent, serine/threonine phosphatase plays a role in mediating calcium-induced pepsinogen secretion. Preincubation of cells with alpha-naphthylphosphate, a non-specific phosphatase inhibitor, decreased calcium-induced secretion. Likewise, specific inhibitors of protein phosphatase-2B (cyclosporin-A and FK-506) caused a dose-dependent reduction in calcium-induced pepsinogen secretion. Moreover, in intact cells, cyclosporin-A and FK-506 inhibited pepsinogen secretion caused by cholecystokinin, carbamylcholine and A23187, agonists known to increase chief cell cytosolic calcium. Okadaic acid, an inhibitor of protein phosphatase-1 and -2A, had no effect on secretion caused by these agonists. Chief cell calcium-dependent phosphatase activity, measured using radiolabeled casein as substrate, was reduced selectively by inhibitors of protein phosphatase-2B. Endogenous substrates for calcium/calmodulin-dependent phosphatase activity were identified by analyzing chief cell lysates using 2-dimensional gel electrophoresis. Increasing the cytosolic calcium concentration resulted in dephosphorylation of a 55-kDa, acidic cytoskeletal protein. FK-506 inhibited dephosphorylation of this protein. Thus, in permeabilized chief cells, specific inhibitors of protein phosphatase-2B inhibit calcium-induced pepsinogen secretion, calcium/calmodulin-dependent phosphatase activity and calcium-induced dephosphorylation of a 55-kDa, acidic cytoskeletal protein. These results support the hypothesis that protein phosphatase-2B (calcineurin) plays an important role in mediating calcium-induced exocytosis.

Expression and phosphorylation of a MARCKS-like protein in gastric chief cells: further evidence for modulation of pepsinogen secretion by interaction of Ca2+/calmodulin with protein kinase C.

In gastric chief cells, agents that activate protein kinase C (PKC) stimulate pepsinogen secretion and phosphorylation of an acidic 72-kDa protein. The isoelectric point and molecular mass of this protein are similar to those for a common PKC substrate; the MARCKS (for Myristoylated Alanine-Rich C Kinase Substrate) protein. We examined expression and phosphorylation of the MARCKS-like protein in a nearly homogeneous suspension of chief cells from guinea pig stomach. Western blotting of fractions from chief cell lysates with a specific MARCKS antibody resulted in staining of a myristoylated 72-kDA protein (pp72), associated predominantly with the membrane fraction. Using permeabilized chief cells, we examined the effect of PKC activation (with the phorbol ester PMA), in the presence of basal (100 nM) or elevated cellular calcium (1 microM), on pepsinogen secretion and phosphorylation of the 72-KDa MARCKS-like protein. Secretion was increased 2.3-, 2.6-, and 4.5-fold by incubation with 100 nM PMA, 1 microM calcium, and PMA plus calcium, respectively. A PKC inhibitor (1 microM CGP 41 251) abolished PMA-induced secretion, but did not alter calcium-induced secretion. This indicates that calcium-induced secretion is independent of PKC activation. Chief cell proteins were labeled with 32P-orthophosphate and phosphorylation of pp72 was detected by autoradiography of 2-dimensional polyacrylamide gels. In the presence of basal calcium, PMA (100 nM) caused a > two-fold increase in phosphorylation of pp72. Without PMA, calcium did not alter phosphorylation of pp72. However, 1 microM calcium caused an approx. 50% attenuation of PMA-induced phosphorylation of pp72. Experiments with a MARCKS "phosphorylation/calmodulin binding domain peptide" indicated that calcium/calmodulin inhibits phosphorylation of pp72 by binding to the phosphorylation/calmodulin binding domain and not by inhibiting PKC activity. These observations support the hypothesis that, in gastric chief cells, interplay between calcium/calmodulin binding and phosphorylation of a common domain on the 72-kDa MARCKS-like protein plays a role in modulating pepsinogen secretion.

Actions and expression of RAB-GDP dissociation inhibitor in dispersed chief cells from guinea pig stomach.

Rab proteins are a family of ras-like proteins that are involved in intracellular membrane trafficking. Rab-GDP dissociation inhibitor prevents dissociation of GDP from Rab proteins and extracts Rab proteins from cell membranes in vitro. In the present study, we examined the effects of recombinant rab-GDI on Rab proteins in gastric chief cells. Rab-GDI extracted GTP-binding proteins, including Rab3 and Rab5, from chief cell membranes in a dose-dependent manner. Maximal concentrations of rab-GDI (> or = 50 micrograms/ml) removed approx. 40% of membrane-associated Rab3. Moreover, preincubation of permeabilized chief cells with rab-GDI resulted in a 35% decrease in GTP gamma S(100 microM)-induced pepsinogen secretion, suggesting that membrane-associated Rab proteins are involved in the final stages of secretion. Immunostaining of chief cell cytosolic and membrane fractions with GDI-specific antisera revealed bands at 56 and 48 kDa, respectively, indicating that chief cells express two isoforms of rab-GDI. In gastric chief cells, regulation of Rab proteins by rab-GDI plays an important role in mediating exocytosis.

Calcineurin mediates calcium-induced potentiation of adenylyl cyclase activity in dispersed chief cells from guinea pig stomach. Further evidence for cross-talk between signal transduction pathways that regulate pepsinogen secretion.

In cholera toxin-treated gastric chief cells, incubation with a cholinergic agonist (carbamylcholine), a regulatory peptide (cholecystokinin), or a calcium ionophore (A23187) causes a dose- and time-dependent potentiation of cAMP levels. Because this augmented response is calcium/calmodulin-dependent, we hypothesized that it was mediated by calcineurin (protein phosphatase 2B). To test this hypothesis, we examined the actions of calcineurin inhibitors on secretagogue-induced potentiation of cAMP levels in guinea pig chief cells. Preincubation of cells with 0.1 microM FK-506 completely prevented carbachol-induced augmentation of cAMP levels and pepsinogen secretion from cholera toxin-treated cells. Cyclosporin-A, another calcineurin inhibitor, also prevented the augmented cAMP response. FK-506 and cyclosporin inhibited augmentation of cAMP levels following treatment with cholecystokinin(26-33) and A23187, but not the smaller increase in cAMP following treatment with a phorbol ester that activates protein kinase C. Hence, the actions of calcineurin inhibitors were limited to secretagogues that increase cellular calcium. Rapamycin, an agent that competes with FK-506 for the immunophilin, FK binding protein 12, does not inhibit calcineurin. In the present study, preincubation with rapamycin did not prevent carbachol-induced augmentation of cAMP levels in cholera toxin-treated chief cells. However, a molar excess of rapamycin reversed the inhibitory actions of FK-506. These experiments provide further evidence that the actions of FK-506 on cholera toxin-treated gastric chief cells are caused by its inhibitory actions on calcineurin. FK-506 also inhibited potentiation of cAMP levels when carbachol was added to cells that were preincubated with forskolin, an agent that directly activates adenylyl cyclase. We conclude that, in gastric chief cells, calcineurin mediates cross-talk between the calcium/calmodulin and adenylyl cyclase signaling pathways.

Expression of Rab3D in dispersed chief cells from guinea pig stomach.

Rab3 proteins are low molecular weight GTP-binding proteins that are expressed in neurons and other secretory cells. These proteins are localized to secretory vesicles and may play a role in regulated exocytosis. Presently, four highly homologous Rab3 isoforms (A, B, C, D) have been identified. We examined the expression of Rab3 isoforms in dispersed chief cells from guinea pig stomach. Immunoblotting with a specific monoclonal Rab3 antibody detected a 27-kDa protein in chief cell cytosolic and membrane fractions, but staining was more intense in membrane fractions. Using the Rab3 antibody, immunohistochemical staining was detected in chief cells but not in parietal or mucous cells. To determine which Rab3 isoform(s) is (are) expressed, chief cell cDNA was obtained by reverse transcription and subjected to PCR using degenerate primers that are specific for rab3 isoforms. The resulting PCR products were cloned and sequenced. The nucleotide sequences obtained were 89% homologous to the nucleotide sequence of mouse rab3D. The deduced amino acid sequence was identical to that of mouse Rab3D (amino acids 16-83). Moreover, Rab3D was the only isoform detected in chief cells by these methods. To identify rab3 transcripts, the guinea pig rab3D fragment obtained by reverse transcription PCR cloning was used as a probe for Northern blotting. The 4.0- and 2.3-kb transcripts identified in chief cells with the rab3 probe were the same size as those detected by others in mouse adipocytes using a rab3D-specific probe. These results indicate that Rab3D is expressed in gastric chief cells.

Actions of Rab3 effector domain peptides in chief cells from guinea pig stomach.

Rab3 proteins are low molecular weight guanine nucleotide-binding proteins that belong to the Ras superfamily and are believed to play a role in the final steps of exocytosis. To examine potential interactions of these proteins with signaling pathways that mediate pepsinogen secretion from gastric chief cells, we synthesized peptides corresponding to the effector domain of Rab3. In the absence of added calcium [calcium concentration ([Ca2+]) < 1 nM], a maximal concentration (15 microM) of the Rab3 effector domain peptide or Rab3AL peptide, containing alanine and leucine substitutions, stimulated the release of 62 and 66%, respectively, of total pepsinogen from streptolysin O-permeabilized chief cells. A Rab2AL peptide, corresponding to the Rab2 effector domain, and modified (scrambled and truncated) Rab3AL peptides did not alter secretion from permeabilized cells. An additive secretory response was observed when 5 microM Rab3AL peptide was combined with increasing calcium ([Ca2+] < 1 nM to 3 microM). In contrast, adding up to 3 mM adenosine 3',5'-cyclic monophosphate (cAMP) had no effect on Rab3AL peptide-induced secretion, and Rab3AL peptide did not alter endogenous cAMP production. The addition of a nonhydrolyzable GTP analogue [0.01 to 100 microM guanosine 5'-O-(3-thiotriphosphate)] potentiated the secretory response to Rab3AL peptide. This potentiated response indicates that other GTP-binding proteins are involved in calcium-independent secretion. Preincubation of cells with streptolysin O (10-30 min), to allow egress of cytosolic constituents, enhanced the response to Rab3AL peptide, suggesting that the target(s) for this peptide is (are) anchored to chief cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

GTP gamma S-induced pepsinogen secretion from gastric chief cells does not require carboxyl methylation or translocation of low molecular weight (LMW) GTP-binding proteins.

We identified at least four LMW GTP-binding proteins in membrane and cytosolic fractions from dispersed gastric chief cells. Extraction of membrane-bound GTP-binding proteins with various agents revealed that these proteins are intimately associated with chief cell membranes. Upon extraction with Triton X-114, the majority of GTP-binding proteins partitioned into the detergent phase, indicative of their hydrophobic nature. Although carboxyl methylation of LMW GTP-binding proteins has been shown to regulate their localization and function, inhibitors of carboxyl methylation had no effect on GTP gamma S-induced pepsinogen secretion. Moreover, pre-permeabilization of chief cells for up to 20 min did not alter GTP gamma S-induced secretion, suggesting that the guanine nucleotide analogue interacts with membrane-bound GTP-binding proteins to elicit a secretory response.

Protein kinase C expression and translocation in dispersed chief cells from guinea-pig stomach.

The protein kinase C (PKC) family of enzymes is comprised of at least nine isoforms that vary with respect to co-factor dependence, cellular distribution and substrate specificity. Using specific antibodies for alpha, beta, gamma, delta, epsilon, zeta and eta PKC isoforms, and Western blot analysis, we found that alpha and zeta PKC are expressed in gastric chief cells. We then used these methods to examine the effects of carbamylcholine, a cholinergic agonist that increases cellular calcium and diacylglycerol concentrations, and PMA, a phorbol ester that activates PKC, on the subcellular distribution of these isoforms. Carbamylcholine and PMA caused an increase in membrane-associated alpha PKC, but did not alter the subcellular distribution of zeta PKC. Comparison of the dose-response curves for carbamylcholine-induced pepsinogen secretion and alpha PKC membrane-association indicates that PKC translocation is not required for carbamylcholine-induced secretion. Nevertheless, maximal carbachol-induced secretion occurs at concentrations that also cause translocation of the alpha isoform. Whereas treatment of chief cells with PMA (300 nM) for 4 h down-regulated levels of alpha PKC by 61%, there was no change in the levels of zeta PKC. Separation of the two PKC isoforms in chief cell lysates by DEAE-column chromatography revealed that kinase activity in fractions containing the alpha isoform was increased more than 3-fold by calcium and lipids. In contrast, kinase activity in fractions containing the zeta isoform was not altered. In gastric chief cells, translocation and activation of alpha PKC occurs in response to agonist-induced increases in calcium and diacylglycerol. Zeta PKC may be involved in the regulation of basal pepsinogen secretion.

Carbachol activates protein kinase C in dispersed gastric chief cells.

We used an 'in situ' kinase assay to examine agonist-induced protein kinase C. (PKC) activation in dispersed chief cells from guinea-pig stomach. Phorbol 12-myristate 13-acetate (PMA), a phorbol ester, and carbamoylcholine, a cholinergic agent, caused a 4- and 3-fold increase in pepsinogen secretion from dispersed chief cells respectively. Whereas PMA caused a rapid 3-fold increase in peptide kinase activity, carbachol caused a 15% increase in activity that was inhibited by the PKC inhibitor, CGP 41,251. Concentrations of carbamoylcholine and a Ca2+ ionophore that were sub-maximal for stimulation of pepsinogen secretion did not cause PKC activation. These results indicate that, in the absence of PKC activation, other mechanisms, most likely involving changes in cellular Ca2+, are sufficient to stimulate pepsinogen secretion. Nevertheless, carbamoylcholine stimulated maximal secretion of pepsinogen only at concentrations that also resulted in activation of PKC. Moreover, these data indicate that relatively small increases in PKC activity (5-10%) can stimulate pepsinogen secretion from dispersed chief cells.

Histamine-2 receptor antagonists do not alter serum ethanol levels in fed, nonalcoholic men.

OBJECTIVE: To determine whether the four histamine-2 receptor antagonists currently available for the treatment of acid-peptic disorders in the United States alter serum ethanol levels after moderate alcohol consumption. DESIGN: Prospective, randomized crossover design comparing the effects of histamine-2 receptor antagonists and no treatment on serum ethanol levels. Each participant served as his own control. PARTICIPANTS: Twenty-five healthy nonalcoholic men (21 to 35 years old); two participants were withdrawn before starting the study. SETTING: University medical center. INTERVENTION: Cimetidine (400 mg twice daily), famotidine (20 mg twice daily), nizatidine (150 mg twice daily), ranitidine (150 mg twice daily), and no treatment for 7 days. After the last dose of medication, participants ate a standard meal; 1 hour later they drank ethanol (0.3 g/kg body weight in 500 mL of orange juice) over 8 minutes. MEASUREMENTS: Simultaneous measurements of breath and serum (headspace gas chromatography) ethanol were made before and 10, 20, 30, 45, 60, 90, 120, 150, and 180 minutes after ingestion of ethanol. RESULTS: Peak ethanol levels did not differ (mmol/L; mean +/- SE) after cimetidine (3.0 +/- 0.3), famotidine (2.9 +/- 0.3), nizatidine (2.9 +/- 0.3), ranitidine (3.1 +/- 0.4), and no treatment (2.9 +/- 0.4). Similarly, there was no difference in the area under the curve (mmol/L.h; mean +/- SE) after cimetidine (4.3 +/- 0.5), famotidine (3.8 +/- 0.4), nizatidine (4.2 +/- 0.5), ranitidine (3.9 +/- 0.4), and no treatment (4.0 +/- 0.5). CONCLUSIONS: In healthy nonalcoholic men, the histamine-2 receptor antagonists currently available in the United States do not alter serum ethanol levels following moderate alcohol consumption after an evening meal.

Guanine nucleotides activate multiple signaling pathways in permeabilized gastric chief cells. Evidence for GTP gamma S-induced calcium-independent pepsinogen secretion.

Nonhydrolyzable guanine nucleotide analogues were used to evaluate the role of guanine nucleotide binding (G) proteins in regulating pepsinogen secretion from streptolysin O-permeabilized chief cells from guinea pig stomach. In the presence of 100 nM calcium, 100 microM guanosine 5'-(beta,gamma-imido)triphosphate or guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) caused a 2- to 4-fold increase in pepsinogen secretion. GTP gamma S stimulated secretion in the absence of calcium (up to 10 mM EGTA). With or without added calcium, GTP analogues caused a 2- to 3-fold increase in cAMP, whereas guanosine 5'-O-2-(thio)diphosphate and calcium alone had no effect on cAMP levels. GTP analogue-induced activation of phospholipase C was evidenced by a calcium-independent increase in cytidine diphospho-1,2-diacylglycerol levels (50% above basal). Phorbol ester- and GTP gamma S-stimulated phosphorylation of a 72-kDa acidic protein was abolished by an inhibitor of protein kinase C (CGP 41251). However, GTP gamma S-induced pepsinogen secretion was only partially inhibited by adding CGP 41251 or a protein kinase C inhibitor peptide. These results indicate that guanine nucleotides activate major signaling pathways in gastric chief cells. Nevertheless, GTP gamma S can induce pepsinogen secretion independently of changes in calcium, cAMP, or activation of protein kinase C.

Pepsinogen secretion from streptolysin O-permeabilized chief cells from guinea pig stomach.

To evaluate directly the actions of cellular mediators on pepsinogen secretion, a nearly homogeneous population of dispersed chief cells was permeabilized using the bacterial toxin streptolysin O (30 IU/ml for 10 min). This resulted in the release of > 60% of cellular lactate dehydrogenase activity, whereas cellular pepsinogen levels were not altered. Examination of permeabilized cells using light and electron microscopy revealed preservation of cellular polarity and organelles. Pepsinogen secretion from permeabilized chief cells could be stimulated with increasing concentrations of calcium (300 nM to 10 microM), adenosine 3',5'-cyclic monophosphate (cAMP; 1 microM to 1 mM), phorbol 12-myristate 13-acetate (10 nM to 1 microM), or by addition of carbamylcholine (0.1 mM) to the incubation solution. In the absence of calcium [0.4 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], activators of protein kinase C stimulated a two- to threefold increase in pepsinogen secretion, and potentiation of secretion was observed when these agents were combined with 0.3 and 1.0 microM calcium. In contrast to the effects of activators of protein kinase C, cAMP-induced pepsinogen secretion was observed only with calcium concentrations > or = 100 nM. Combinations of increasing concentrations of cAMP and calcium resulted in an additive secretory response. This study indicates the utility of streptolysin O-permeabilized chief cells for the study of signal transduction mechanisms that mediate pepsinogen secretion.

Distinct mechanisms of zinc uptake at the apical and basolateral membranes of caco-2 cells.

Zinc uptake mechanisms at the apical and basolateral membrane borders of caco-2 cells were examined. This human-derived cell line possesses many morphological and functional characteristics of absorptive small intestinal cells. By day 14, confluent and well-differentiated monolayers were formed when the cells were grown on porous polycarbonate filters. Labelled zinc was placed on the apical or basal side of the monolayer and its uptake by the cells, as well as its transport across the monolayer, were measured. Zinc uptake by the cells from the apical side was found to be a saturable process (Kt = 41 microM; Vmax = 0.3 nmols/cm2/10 min) with a diffusional term at higher concentrations (1.0 sec/cm). Apical uptake was not affected by metabolic inhibitors or potential zinc ligands. Zinc uptake from the basolateral side was concentration dependent (Kd = 1.3 sec/cm) and was partially inhibited (30%) by ouabain and vanadate, suggesting that the (Na-K)-ATPase on the basolateral membrane is involved in the serosal uptake of zinc by the cell. Transport of zinc across the monolayers from the apical or basolateral compartment was concentration dependent and was not affected by metabolic inhibitors. Zinc transport from the basolateral side was greater than 2-fold greater than apical transport. Hence, separate mechanisms can be distinguished with respect to zinc uptake at the apical and basolateral membranes of caco-2 cells.