Robert D. Raffaniello

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.

Histamine-2 Receptor Antagonists Do Not Alter Serum Ethanol Levels in Fed, Nonalcoholic Men

Histamine-2 (H-2) receptor antagonists, used primarily for the treatment of acid-peptic diseases, rank among the safest and most frequently used drugs in clinical medicine, and some of these agents are now poised to go over the counter [1]. As a consequence of similar efficacy, there has been little basis to choose among the four drugs currently available in the United States. Recently, however, data have been presented to suggest that several of these agents may be dangerous. We refer to studies, primarily by Lieber and colleagues, indicating that therapeutic doses of cimetidine, ranitidine, and nizatidine, but not famotidine, elevate blood ethanol levels in nonalcoholic men following ingestion of moderate amounts of alcohol (approximately one or two glasses of wine or cans of beer) [2-4]. According to these studies, in men taking these agents, peak ethanol levels and the area-under-the-curve value may be elevated as much as twofold compared to levels observed in those taking famotidine or no H-2 blocker [2-4]. Liebers group believes that these effects may result in blood ethanol levels that exceed the legal limit set for automobile driving in many countries in addition to unexpected impairment of attention and motor coordination after modest ingestion of alcohol [4]. Because H-2 blockers are prescribed so frequently and social drinking is common, such effects would impact greatly on the safety of these agents and the wisdom of allowing them to go over the counter. Attempts to confirm the results of Liebers group have met with conflicting results. Some investigators have found that cimetidine, but not ranitidine, interferes with alcohol metabolism [5]. Others have found that ranitidine, but not cimetidine, interferes with alcohol metabolism [6]. Some have found no effect with cimetidine or ranitidine [7-10]. Inadequate sample size and designs in which participants did not serve as their own controls may have contributed to discrepant results. Other possible explanations for differing results include the influence of smoking, previous alcohol consumption, and the effect of racial variation on the activity of gastric alcohol dehydrogenase [11]. Our study was designed to examine the potential interaction of H-2 receptor antagonists with alcohol using a randomized crossover design in which each of 25 participants would be treated with a therapeutic regimen of the four drugs and serve as his own control. We chose to study nonalcoholic men, a group reported to be particularly sensitive to the effects of H-2 blockers on alcohol metabolism [4]. Particular attention was paid to reproducing the standard meal, the rate of alcohol consumption, sample collection, and methods of alcohol measurement for each treatment arm. Methods Our study was done at the State University of New York- Health Science Center at Brooklyn between January and April 1992. Twenty-five healthy men were recruited by posting an advertisement at the medical center. Men with alcoholism were excluded by extensive interviewing by two trained observers and by completion of the CAGE questionnaire [12]. All participants had physical examinations, and their blood counts, serum electrolytes, blood urea nitrogen, creatinine, liver enzymes, and urinalysis were determined. Study Design This was a prospective, observer-blinded, randomized, five-arm crossover study. At entry, the participants were randomized to receive 7 days of treatment with each of the following: cimetidine (Tagamet; SmithKline Beecham, Philadelphia, Pennsylvania) 400 mg twice daily; famotidine (Pepcid; Merck Sharp and Dohme, West Point, Pennsylvania) 20 mg twice daily; nizatidine (Axid; Eli Lilly and Company, Indianapolis, Indiana) 150 mg twice daily; ranitidine (Zantac; Glaxo Pharmaceuticals, Research Triangle Park, North Carolina) 150 mg twice daily; and no treatment. Participants were contacted daily to remind them to take their drugs, and tablet counts were done at the end of each treatment week to assess compliance. Participants abstained from alcohol and any other agents, such as aspirin [13], that might affect ethanol metabolism during the treatment week. At 1800 hours in the evening of the last treatment day, the participants ingested the remaining tablet of their assigned drug followed by a 15-minute meal, consisting of a turkey sandwich, potato salad, a roll with two pats of margarine, a cookie, and 170 mL of water. An indwelling catheter was inserted in the arm of each participant. One hour after starting the meal, the participants ingested 0.3 g/kg body weight ethanol (92.42% ethyl alcohol, Grain Processing Corporation, Muscatine, Iowa) in 500 mL of orange juice over 8 minutes. Blood samples and exhaled breath for alcohol determination were taken 0, 10, 20, 30, 45, 60, 90, 120, 150, and 180 min after ingestion of ethanol began. During the 4-hour study period further food and fluids were withheld and smoking was not permitted. Each treatment period was separated by a 7-day washout period. Participants received each study drug in randomized sequence and the procedure described above repeated until all had received each of the study drugs and a control period. At the end of the study, a complete physical examination; repeated evaluation of blood counts, serum electrolytes, blood urea nitrogen, creatinine, and liver enzymes; and urinalysis were performed. The protocol was approved by the Institutional Review Board at the State University of New York-Health Science Center at Brooklyn on 20 November 1991. Written informed consent was obtained from all participants before entry into the study. Alcohol Determination Immediately after completing the ingestion of alcohol, each participant rinsed his mouth and pharynx of residual alcohol by vigorous gargling and expectoration (20 times with water). Breath alcohol was assayed with a Lion Alcolmeter S-D2 (MPD Inc., Owensboro, Kentucky) using the methods recommended by the manufacturer, including the use of a fresh mouthpiece for each determination. Alcolmeters were calibrated using an artificial breath alcohol standard (NALCO, MPD Inc.) before each study session. Venous blood was drawn into a vacuum tube at the times indicated above and placed in an ice-water bath (4 C). Immediately after the study session, the tubes were centrifuged at 2100 rpm for 10 min at 4 C. The serum was aliquoted into 0.5-mL microfuge tubes and stored at 70C. To determine serum ethanol, 100 L of sample or standard was placed in a 14-mL vial with 100 L of 2 M ammonium sulfate [14]. The vials were immediately capped with silicone septae and aluminum crimp seals. Ethanol in the samples was determined using an HS-40 automatic headspace sampler (Perkin-Elmer, Norwalk, Connecticut) connected to a Perkin-Elmer Autosystem gas chromatography system containing a 2-meter 80/100 mesh Carbopak C column and flame ionization detector. A calibration curve was constructed (Perkin-Elmer 1020S integrator) using known standards that were assayed along with each set of test samples. Alcohol measurements by breath analysis and gas chromatography were done by persons who had no knowledge of the randomization sequence. Statistical Analysis The area-under-the-curve value for serum ethanol levels determined by gas chromatography was calculated by the trapezoidal method (SigmaPlot, Jandel Scientific, Corte Madera, California) using a Macintosh IIsi computer (Apple Computer, Cupertino, California) and expressed as mmol/L x h. Peak serum alcohol concentration and area-under-the-curve value were compared for each treatment. The data were evaluated using analysis of variance for repeated measures with the Tukey test and paired t-test as appropriate (Systat version 5.0, Evanston, Illinois) [15-17]. Differences in values for serum ethanol or area-under-the-curve between treatment groups were considered significant if P was less than 0.05. The sample size was chosen to provide 90% power, = 0.05, to detect a 20% difference between groups [15-17]. Results Of the 25 participants entered, 2 were withdrawn before starting the protocol; 1 because it was discovered that he did not meet the inclusion criteria and the other because of an unrelated hospitalization. The remaining 23 participants completed the protocol without incident. The mean age of these 23 men was 25.6 years (range, 21 to 35 years); mean weight was 74.3 kg (range, 52.3 to 99.1 kg); and mean height 175 cm (range, 165 to 183 cm). Sixteen participants were white; 6 were Asian (4 Chinese, 2 Korean); and 1 was an American Indian. In terms of alcohol consumption, no one gave an affirmative response to any item in the CAGE questionnaire [12]; 1 participant reported drinking 1 can of beer/d; 7 participants reported drinking 1 can of beer/wk; and 15 participants reported that they did not drink alcohol at all. One participant smoked more than 10 cigarettes/d. Figure 1 shows the mean serum ethanol concentration curves after no treatment, cimetidine, famotidine, nizatidine, and ranitidine. The curves were superimposable and, when these data were analyzed by analysis of variance for repeated measures, no significant difference (P > 0.2) was apparent between treatment arms. Table 1 shows the peak serum ethanol concentrations, area-under-the-curve observed with each treatment arm, and P values (paired t-tests) when each H-2 blocker was compared to no drug. As indicated, there was no significant difference in peak ethanol levels or area-under-the-curve after treatment with cimetidine, famotidine, nizatidine, ranitidine, or no treatment. Table 1. Effects of No Treatment Compared with H-2 Receptor Antagonists on Serum Ethanol Levels after Ingestion of Ethanol (0.3 g/kg Body Weight)* Figure 1. Effect of H-2 blocker treatment on serum ethanol concentration. Figure 2 shows histograms for the frequency of within-person differences (treatment minus no treatment) of peak serum ethanol concentration after treatment with each H-2 blocker. Ninety-five percent confi

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 μM), 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 μM calcium, and PMA plus calcium, respectively. A PKC inhibitor (1 μM 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 μM 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. J. Cell. Biochem. 64:514–523.

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.

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 μM 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.

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 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 Na2CO3, 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. J. Cell. Physiol. 185:339–347, 2000.

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. J. Cell. Biochem. 72:540–548, 1999.

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.