In this work, we 1) synthesized rat CCK-58, 2) determined the amounts and forms of rat CCK in whole blood after stimulation of its release by casein, 3) determined the potency of CCK-8 and CCK-58 peptides to displace labeled CCK-8 from CCK(A) and CCK(B) receptors transfected into Chinese hamster ovary (CHO) cells, and 4) examined the biological actions of CCK-8 and rat CCK-58 in an anesthetized rat model. CCK-58 was the only detected endocrine form of CCK in rat blood. Synthetic rat CCK-58 was less potent than CCK-8 for displacing the label from CCK(A) and CCK(B) receptors in transfected CHO cells. However, rat CCK-58 was more potent than CCK-8 for stimulation of pancreatic protein secretion in the anesthetized rat. In addition, CCK-58 but not CCK-8 stimulated fluid secretion in this anesthetized rat model. These data suggest that regions outside the COOH terminus of rat CCK-58 influence the expression of CCK biological activity. The presence of only CCK-58 in the circulation and the fact that its biological activity differs from CCK-8 suggests that CCK-58 deserves scrutiny in other physiological models of CCK activity.

The carboxyl terminal octapeptide of cholecystokinin (CCK-8) has been hypothesized to account for the bioactivity of all the molecular forms of cholecystokinin. However, the physiological relevance of CCK-58 has not been rigorously examined because of the lack of sufficient amounts of the peptide and concerns about inactivation of natural peptides during their purification. Therefore, canine-sulfated CCK-58 was synthesized and conditions determined for its unblocking and purification that preserved the sulfated tyrosine. Synthetic CCK-58 was indistinguishable from natural CCK-58 by amino acid analysis and by mass spectrometry. Synthetic CCK-58 and CCK-8 have different patterns of pancreatic stimulation: both caused a dose-related increase in amylase release, while only CCK-58 stimulated bile-pancreatic output volume. Thus, CCK-58 and CCK-8 are biased agonists at the CCK-A receptor (they have distinct patterns of action mediated by the same receptor). Previous work has demonstrated that the identical carboxyl termini of CCK-8 and CCK-58 have different solution conformations. Taken together, the physiological and structural results support the hypothesis that different carboxyl terminal conformations of CCK-58 and CCK-8 alter the expression of their biological activity.

Introduction Receptor subtypes can be distinguished by different actions of agonists on physiologic responses. In this study, we compared effects of four species variants of secretin (rat, porcine, canine, and human) on pancreatic secretion and gastrin-induced acid secretion in urethane-anesthetized rats. These secretins differ by one to three residues in position 14, 15, or 16 and were used to probe for the presence of different secretin receptor subtypes in the rat. Methodology Pancreatic responses were measured in a two-point parallel line bioassay with porcine secretin (3 and 30 pmol/kg IV bolus) as standard. Inhibition of gastric acid secretion by each secretin (100 pmol/[kg·h]) was quantitated against a threshold dosage of gastrin-17 (200 pmol/[kg·h]), and percent inhibition of incremental acid responses was determined. Results Rat secretin was significantly more potent than other secretins for pancreatic secretion, in the order of rat > porcine > canine > human. The four secretins significantly inhibited gastrin-induced acid secretion by 37% to 49%, with no statistically significant differences among the forms. Conclusions Stimulation of pancreatic secretion was influenced by species variations in secretin structure, but inhibition of gastric acid secretion was not. This finding suggests that secretin receptor subtypes with different recognition patterns mediate these responses.

Differences in the structure of PYY and two important analogs, PYY [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36] and [Pro34]PYY, are evaluated. Y-receptor subtype ligand binding data are used in conjunction with structural data to develop a model for receptor subtype selective agonists. For PYY it is proposed that potent binding to Y1, Y4 and Y5 receptors requires the juxtaposition of the two termini while Y2 binding only requires the C-terminal helix. Further experiments that delineate between primary and tertiary structure contributions for receptor binding and activation are required to support the hypothesis that tertiary structure is stable enough to influence the expression of PYY's bioactivity.

We examined the role of CCK-A receptors in acid inhibition by intestinal nutrients. Gastric acid and plasma CCK and gastrin levels were measured in rats with gastric and duodenal fistulas during intragastric 8% peptone and duodenal perfusion with saline, complete liquid diet (CLD; 20% carbohydrate, 6% fat, and 5% protein), and the individual components of CLD. Acid output was significantly inhibited (50-60%) by CLD, lipid, and dextrose. Plasma CCK was significantly increased by CLD (from 2.6 +/- 0.3 to 4.8 +/- 0.5 pM) and lipid (4.6 +/- 0.5 pM). CCK levels 50-fold higher (218 +/- 33 pM) were required to achieve similar acid inhibition by exogenous CCK-8 (10 nmol x kg(-1) x h(-1) iv). Intestinal soybean trypsin inhibitor elevated CCK (10.9 +/- 2.5 pM) without inhibiting acid secretion. The CCK-A antagonist MK-329 (1 mg/kg iv) reversed acid inhibition caused by CLD, lipid, and dextrose. Peptone-stimulated gastrin (21.7 +/- 1.9 pM) was significantly inhibited by CLD (14.5 +/- 3.6 pM), lipid (12.3 +/- 2.2 pM), and dextrose (11.9 +/- 1.5 pM). Lipid and carbohydrate inhibit acid secretion by activating CCK-A receptors but not by altering plasma CCK concentrations.

Only one secretin receptor has been cloned and its properties characterized in native and transfected cells. To test the hypothesis that stimulatory and inhibitory effects of secretin are mediated by different secretin receptor subtypes, pancreatic and gastric secretory responses to secretin and secretin-Gly were determined in rats. Pancreatic fluid secretion was increased equipotently by secretin and secretin-Gly, but secretin was markedly more potent for inhibition of basal and gastrin-induced acid secretion. In Chinese hamster ovary cells stably transfected with the rat secretin receptor, secretin and secretin-Gly equipotently displaced (125)I-labeled secretin (IC(50) values 5.3 +/- 0.5 and 6.4 +/- 0.6 nM, respectively). Secretin, but not secretin-Gly, caused release of somatostatin from rat gastric mucosal D cells. Thus the equipotent actions of secretin and secretin-Gly on pancreatic secretion appear to result from equal binding and activation of the pancreatic secretin receptor. Conversely, secretin more potently inhibited gastric acid secretion in vivo, and only secretin released somatostatin from D cells in vitro. These results support the existence of a secretin receptor subtype mediating inhibition of gastric acid secretion that is distinct from the previously characterized pancreatic secretin receptor.

We synthesized PYY-(1-36) (nonselective between Y(1) and Y(2) receptor subtype agonists), [Pro(34)]PYY (selective for Y(1)), and PYY-(3-36) (selective for Y(2)) to determine whether solution conformation plays a role in receptor subtype selectivity. The three peptides exhibited the expected specificities in displacing labeled PYY-(1-36) from cells transfected with Y(1) receptors (dissociation constants = 0.42, 0.21, and 1,050 nM, respectively) and from cells transfected with Y(2) receptors (dissociation constants = 0.03, 710, and 0.11 nM, respectively) for PYY-(1-36), [Pro(34)]PYY, and PYY-(3-36). Sedimentation equilibrium analyses revealed that the three PYY analogs were 80-90% monomer at the concentrations used for the subsequent circular dichroism (CD) and (1)H-nuclear magnetic resonance (NMR) studies. CD analysis measured helicities for PYY-(1-36), [Pro(34)]PYY, and PYY-(3-36) of 42%, 31%, and 24%, suggesting distinct differences in secondary structure. The backbone (1)H-NMR resonances of the three peptides further substantiated marked conformational differences. These patterns support the hypothesis that Y(1) and Y(2) receptor subtype binding affinities depend on the secondary and tertiary solution state structures of PYY and its analogs.

Peptide YY (PYY) belongs to a family of peptides including neuropeptide Y (NPY) and pancreatic peptide (PP) that regulate numerous functions through both central and peripheral receptors. The solution structure of these peptides is hypothesized to be critically important in receptor selectivity and activation, based on prior demonstration of a stable tertiary conformation of PP called the "PP-fold". Circular dichroism (CD) spectra show a pH-dependent structural transition in the pH range 3-4. Thus we describe the tertiary structure of porcine PYY in water at pH 5.5, 25 degrees C, and 150 mM NaCl, as determined from 2D (1)H NMR data recorded at 500 MHz. A constraint set consisting of 396 interproton distances from NOE data was used as input for distance geometry, simulated annealing, and restrained energy minimization calculations in X-PLOR. The RMSDs of the 20 X-PLOR-generated structures were 0.71 +/- 0.14 and 1.16 +/- 0.17 A, respectively, for backbone and heavy atom overlays of residues 1-34. The resulting structure consists of two C-terminal helical segments from residues 17 to 22 and 25 to 33 separated by a kink at residues 23, 24, and 25, a turn centered around residues 12-14, and the N-terminus folded near residues 30 and 31. The well-defined portions of the PYY structure reported here bear a marked similarity to the structure of PP. Our findings strongly support the importance of the stable folded structure of this family of peptides for binding and activation of Y receptor subtypes.

Posttranslational processing of preprosecretin generates several COOH-terminally extended forms of secretin and α-carboxyl amidated secretin. We used synthetic canine secretin analogs with COOH-terminal -amide, -Gly, or -Gly-Lys-Arg to examine the effects of COOH-terminal extensions of secretin on bioactivity and detection in RIA. Synthetic products were purified by reverse-phase and ion-exchange HPLC and characterized by reverse-phase isocratic HPLC and amino acid, sequence, and mass spectral analyses. Secretin and secretin-Gly were noted to coelute during reverse-phase HPLC. In RIA using eight different antisera raised against amidated secretin, COOH-terminally extended secretins had little or no cross-reactivity. Bioactivity was assessed by measuring pancreatic responses in anesthetized rats. Amidated canine and porcine secretins were equipotent. Secretin-Gly and secretin-Gly-Lys-Arg had potencies of 81 ± 9% ( P > 0.05) and 176 ± 13% ( P < 0.01), respectively, compared with amidated secretin, and the response to secretin-Gly-Lys-Arg lasted significantly longer. These data demonstrate that 1) amidated secretin and secretin-Gly are not separable under some chromatographic conditions, 2) current RIA may not detect bioactive COOH-terminally extended forms of secretin in tissue extracts or blood, and 3) the secretin receptor mediating stimulation of pancreatic secretion recognizes both amidated and COOH-terminally extended secretins.

Due to the prevalence of degenerative neuronal disease in aging, there is considerable interest in elucidating pathways involved in neuronal injury and death.Neuronal sphingolipids, particularly ceramide, may protect against apoptotic death in some neuronal systems.We previously documented a significant decrease in neuronal ceramide concentration in aging (Hall et al., Soc.Neurosci.23:533, 1997).Here, we present evidence that treatment with the cell-permeable ceramide analog, C 2, decreases apoptotic cell death in aged neurons induced by both NGF withdrawal, and by the combination of NGF withdrawal and treatment with TNFct.Dorsal root ganglion neurons from young (Y: 4-6 ran), middle-aged (M: 10-14 ran), and old (O: 22-28 mo) Fisher 344 x BN F1 hybrid rats were dissociated and plated on culture dishes.All experiments were run in duplicate.Neurons were cultured in serum-free medium containing NGF (100 ng/ml) x 24 hours, then treated for 24 hours with 1) medium without NGF (NGF-), 2) NGF-medium + 20 pM cellpermeable C2-ceramide, N-acetyl-sphingosine (NGF-/C2), 3) NGF-medium + 20 pM inactive analog dihydro-C 2 (N-acetyldihydro-sphingosine) (NGF-/dh-C2), or 4) medium containing NGF (NGF+).At 48 hours neurons were preserved in methanol, and apoptosis rates calculated by counting the number of apoptotic cells/200 neurons/dish using the TUNEL staining method with fluorescence microscopy.Apoptosis rates (%) were low in NGF+ neurons (Y: 0.35±0.2;n=2,M: 0.45;n=1, O: 0.54+_0.02;n=2).NGF withdrawal was associated with significantly greater rates of apoptosis in aged animals (3.12+0.9;n=2,p<0.01)compared to youthful animals (1.4 -+ 0.2;n=2), Addition of C 2 ceramide decreased % apoptosis in all groups (NGF-/C2: Y: 0.8 • 0.05;n=2, M: 0.3;n=l; O: 1.51 +__ 0.25; n=2) to levels comparable to NGF+ treatment.The effect appeared to be specific for C 2, as addition of the analog dh-C 2 did not reduce apoptosis in Y (1.35-+ 0.08; n=2) and M (1.9%;n=1) neurons, and actually significantly increased apoptosis further in aged neurons (4.22 -+ 0.66;n=2; p<0.005).Addition of TNFa (20 laM) at the time of NGF withdrawal significantly elevated % apoptosis in neurons from aged animals compared to NGF withdrawal alone, with a similar beneficial effect of C 2 observed (NGF+: 5.5 ± 2.5; NGF-: 12.0 ± 1.5;p<0.01;NGF-/C2: 6.75-+0.25;NGF-/dh-C2:11.25 +-2.75;p<0.01;n=2).We conclude that, compared to younger animals, neurons from aged animals are more sensitive to induction of apoptosis by NGF withdrawal.Treatment with C 2 significantly decreased apoptosis induced by NGF withdrawal alone, and NGF withdrawal+TNF~t, suggesting that our previous finding that aging is associated with decreased neuronal cerarnide has functional significance.

H. pylori infection is difficult to eradicate.Some of the best antibiotic regimens to treat H. pylori infection to this point have included a protonpump inhibitor and two antibiotics taken for 2 weeks.There is limited data available from the United States on the effectiveness of ranitidine bismuth citrate (RBC) plus two antibiotics to treat H. pylori.Therefore, the following study was undertaken to evaluate RBC with two antibiotics, which have been used successfully in combination, to treat H. pylori.Methods: Adults with and without abdominal symptoms, who had never received H. pylori eradication therapy, were tested for the presence of H. pylori infection either by in-office rapid serology assays or histology.Positive subjects were administered the 13C-UBT (Meretek Diag.).Subjects, who also had a positive UBT were then treated with RBC 400 mg bid, clarithromycin 500 mg bid and metronidazole 500 mg bid for 10 days.Four to six weeks after completing antibiotics all subjects were asked to return for a second UBT to assess treatment success.Results: So far 40 subjects have completed the antibiotic regimen and have returned for their second UBT.Thirty-one subjects were negative for H. pylori by UBT and 9 were positive, resulting in a 77.5% (95% C.I.: 90.4%-64.6%)eradication rate.Five subjects stopped their medication due to side effects (12.5%).When analysis is performed on the 35 subjects who took >-80% of their medication (per-protocol), the eradication rate is 88.6% (95% C.I.: 99%-78%) with this regimen.Conclusion: The combination of RBC with clarithromycin and metronidazole successfully treats H~ pylori infection after only 10 days of therapy.The per-protocol eradication rate from this study is similar to that seen with FDA approved regimens.The number of patients who stopped their medication due to side effects is similar to rates seen with other bismuth containing regimens.In conclusion, RBC plus clarithromycin and metronidazole should be considered as a first line treatment regimen for H. pylori infection, and may only need to be taken for a period of 10 days as opposed to 14 days for FDA approved regimens.

The annual incidence of significant GI events (bleeding, obstruction, perforation) in patients on NSAIDs is 2-4%; morbidity and mortality can be reduced with coadministration of misoprostol or PPIs.In the absence of head to head medical or economic outcome trials, the purpose of this study is to evaluate and compare the cost considerations of three prophylactic options: fixed-dose formulations of diclofenac 50 mg/misoprostol 200 meg PO BID/TID (D50/M200) or diclofenac 75 mg/misoprostol 200 meg PO BID (D75/M200) vs. NSAID (diclofenac 75 mg PO BID) plus omeprazole 20 mg PO QD prophylaxis.Methods: Using DATA 3.0.14® software (TreeAge Inc.Williamstown, MA), a decision-analysis model was constructed to conceptualize and quantify six-month economic implications of these three therapeutic options.Patients, not stratified according to risk of NSAID complications, were equally assigned to each arm.Six-month hospitalization rates, and ulcer and outcomes probabilities were derived from clinical literature and expert opinion.Costs were assigned to successful prophylaxis and to treatment failures with intervention and maintenance; drug costs represent 1996 A WP costs.Six-month per-patient costs were then calculated.Results: Six-month per patient cost of therapy with D50/M200 BID/TID was $1,017 (ulcer incidence 4.5%; Verdickt V, et al Scand J Rheumatol 1992; 21:85-91), D75/M200 BID was $942 (ulcer incidence, 4.8%; Bocanegra TS et al Rheumatol Eur 1996; 25 (suppl 1):46.), and PPI costs were $1,410 (ulcer incidence, 14.6%; Yeomans ND et al, 9th EULAR Symposium, 1996), or $1,382 (ulcer incidence, 6%).Net potential savings comparing the combined use of D50/M200 and D75/M200 (assuming equal use of the two strengths) vs. the lowest cost PPI regimen was $402 over 6 months.The model's robustness was verified by Monte Carlo analysis of 10,000 patients; this analysis reported costs for the PPI arm at $1,414 (ulceration incidence, 14.6%) and $1,386 (ulceration incidence, 6.0%), and cost for the DIM arm at $975.As ulceration and healing rates were similar in the D/M and PPI arms, the majority of patients in either arm remained asymptomatic, and therefore did not require intervention.As a result, the cost differences were primarily due to the costs of medication (DIM50 -$2.52/day;D/M75 -$2.10 per day; D-$1.98 per day; PPI-$3.63 per day).In sensitivity analyses, reducing the cost of diclofenac to $1.00 per day (generic) revealed that the break-evan PPI cost would be either $1.79 per day (ulceration incidence 14.6%) or 1.97 per day (ulceration incidence 6%).Conclusions: Based on similar gastroduodenal injury rates, this model demonstrates that, in patients requiring NSAIDs, the diclofenac/misoprostol formulations are more cost-effective than PPI prophylaxis therapy.