Regulation of intestinal c1-and HCO�
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`MSN Exhibit 1018 - Page 1 of 12
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`secretion by uroguanylin
`MSN v. Bausch - IPR2023-00016
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`NAM SOO JOO,1 ROSLYN M. LONDON,1,2 H YUN DJU KIM,1
`LEONARD R. FORTE,
` AND LANE L. CLARKE3
`1Department of Pharmacology, School of Medicine, 3Department of Veterinary Biomedical Sciences,
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`College of Veterinary Medicine, and the 4Dalton Cardiovascular Research Center, University
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`of Missouri and 2The Truman Veterans Affairs Medical Center, Columbia, Missouri 65212
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`1,2
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`,4
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`membrane conductance regulator (CFTR) (3, 7, 16, 21)
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`Joo, Nam Soo, Roslyn M. London, Hyun Dju Kim,
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`and Lane L. Clarke. Regulation of
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`and possibly the inhibition of electroneutral NaCl
`Leonard R. Forte,
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`by uroguanylin. intestinal c1-and HC03 secretion
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`Am. J.
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`absorption (37, 52). Much less is known about the
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`274 (Gastrointest. 37): 0633-0644,
`Physiol.
`Liver Physiol.
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`intestinal actions ofuroguanylin. However, it has been
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`1998.-Uroguanylin is an intestinal peptide hormone that
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`shown that the peptide stimulates intracellular cGMP
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`may regulate epithelial ion transport by activating a receptor
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`production and transepithelial c1-secretion in T84
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`guanylyl cyclase on the luminal surface of the intestine. In
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`cells, a cell line derived from a colonic adenocarcinoma
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`this study, we examined the action of uroguanylin on anion
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`(17, 24, 27). Both uroguanylin and guanylin are abun­
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`transport in different segments of freshly excised mouse
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`dantly expressed in the intestinal epithelium as inac­
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`intestine, using voltage-clamped Ussing chambers. Urogua­
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`tive precursors, or propeptides, that undergo enzymatic
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`nylin induced larger increases in short-circuit current (I,c) in
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`proximal duodenum and cecum compared with jejunum,
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`cleavage to yield the bioactive peptides (11, 29, 43).
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`ileum, and distal colon. The acidification of the lumen of the
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`Uroguanylin and, to a lesser extent, guanylin are
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`proximal duodenum (pH 5.0-5.5) enhanced the stimulatory
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`expressed in other tissues and have been isolated from
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`action of uroguanylin. In physiological Ringer solution, a
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`plasma and urine (11, 25, 27, 28, 40, 44, 46). Recent
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`significant fraction of the I,c stimulated by uroguanylin was
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`studies demonstrate that uroguanylin induces natriure­
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`insensitive to bumetanide and dependent on HC03 in the
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`sis in the perfused rat kidney (M. Fonteles, personal
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`bathing medium. Experiments using pH-stat titration re­
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`communication), suggesting that (intestinal) urogua­
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`vealed that uroguanylin stimulates serosal-to-luminal
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`HC03
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`nylin may also be elaborated into the blood as an
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`secretion (J�_:f;) together with a larger increase in I,c-Both
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`endocrine mediator ofrenal function (14).
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`J�_:f; and I,c were significantly augmented when luminal pH
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`Although uroguanylin and guanylin share nearly
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`was reduced to pH 5.15. Uroguanylin also stimulated the
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`50% identity in their primary amino acid sequences,
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`J�_:f; and I,c across the cecum, but luminal acidity caused a
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`uroguanylin differs from guanylin with regard to intes­
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`generalized decrease in the bioelectric responsiveness to
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`tinal expression and intrinsic biochemical properties
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`agonist stimulation. In cystic fibrosis transmembrane conduc­
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`(26, 27, 34). First, both uroguanylin and guanylin are
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`tance regulator (CFTR) knockout mice, the duodenal I,c
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`found throughout the length of the rat intestinal mu­
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`response to uroguanylin was markedly reduced, but not
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`cosa, but uroguanylin mRNA is most abundant in the
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`eliminated, despite having a similar density of functional
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`proximal small intestine, whereas guanylin mRNA is
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`receptors. It was concluded that uroguanylin is most effective
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`greatest in the distal small intestine and large bowel
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`in acidic regions of the small intestine, where it stimulates
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`(39, 40, 42). In opossums, uroguanylin mRNA is abun­
`both HCO3 and c1-secretion primarily
`via a CFTR-depen­
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`dant in the duodenum but also has high levels of
`dent mechanism.
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`expression in the large intestine compared with gua­
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`cyclic OMP; bicarbonate transport; chloride transport; cystic
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`nylin (11). Second, the amino acid sequence ofurogua­
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`fibrosis; cystic fibrosis transmembrane conductance regula­
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`nylin has a conserved asparagine residue, instead of
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`tor; guanylyl cyclase; mouse intestine; proximal duodenum
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`the phenylalanine (in opossum) or tyrosine (in other
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`species) found in guanylin, which makes uroguanylin
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`resistant to proteolysis by the pancreatic enzyme chy­
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`motrypsin (25). Third, uroguanylin has two additional
`UROGUANYLIN is an intestinal peptide that is closely
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`related to guanylin, another intestinal peptide that is
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`acidic amino acids that render it a more strongly acidic
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`secreted onto the intestinal epithelial surface and
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`molecule. Interestingly, uroguanylin is more potent at
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`acidic pH, whereas guanylin is more potent at alkaline
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`regulates transepithelial salt and water transport
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`through a receptor-mediated action (19, 26, 27, 34, 50).
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`pH, in stimulating cGMP production and c1-secretion
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`Guanylin was first discovered in attempts to identify an
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`in T84 cell monolayers (24, 27).
`The effects of uroguanylin on anion secretion have
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`endogenous ligand for the apical membrane-bound
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`not been previously examined in the intact mammalian
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`guanylyl cyclase C, which serves as the receptor for
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`Escherichia coli heat-stable enterotoxin (STa) (6, 13,
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`intestinal mucosa. On the basis of its higher expression
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`in duodenum and its pH dependence of action, it was
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`20, 35, 36, 48, 54, 55), the causative agent of traveler's
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`reasoned that uroguanylin may be more effective in
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`diarrhea (12). Guanylin binding to the receptor in­
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`regions of the intestinal tract where the mucosa is
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`creases intracellular cGMP, resulting in activation of
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`exposed to acidic luminal conditions. Therefore, differ­
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`protein kinase G II in the intestinal epithelial cell (18,
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`45). The subsequent intracellular events include stimu­
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`ent segments of the murine intestine were investigated
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`for responsiveness to uroguanylin by measuring the
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`lation of anion secretion via the cystic fibrosis trans-
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`0193-1857/98 $5.00 Copy rig h t © 1998 the American Physiological Society
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`G634
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`PHYSIOLOGICAL FUNCTIONS OF UROGUANYLIN
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`MATERIALS AND METHODS
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`Animals
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`Tissue Preparation
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`2
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`Bioelectric Measurements
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`Bioassay for cGMP Accumulation in Mouse Intestine
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`Transmural I,c (µA/cm 2 tissue surface area) was measured
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`short-circuit current (/sc), an index of anion secretion.
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`with the use ofan automatic voltage clamp device (VCC-600;
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`In the most responsive intestinal segments (proximal
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`Physiologic Instruments, San Diego, CA) that compensates
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`duodenum and cecum), we examined the pH depen­
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`for electrode offset and the fluid resistance between the
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`dence of uroguanylin in stimulating transepithelial
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`potential-measuring electrode bridges. Transepithelial poten­
`ofc1-and HCO3 across the mucosa.
`secretion
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`tial difference (in m V) was measured via a pair of calomel
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`half-cells connected to the serosal and mucosal baths by 4%
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`agar-Ringer (wt/vol) bridges. I,c was applied across the tissue
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`via a pair ofAg/AgCl electrodes that were kept in contact with
`the serosal and mucosal baths through 4% agar-Ringer
`Fem ale C57BL6 mice 8-10 wk old were housed in a
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`bridges. All experiments were carried out under short­
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`standard animal care room with a 12:12-h light-dark cycle.
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`circuited conditions. Total tissue conductance (G1, mS/cm
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`Animals were allowed free access to food and water until the
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`tissue surface area) was calculated by applying Ohm's law to
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`time of study. CFTR knockout and normal littermate mice
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`the current deflection resulting from a 5-m V bipolar pulse
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`(B6.129-Cftr1m/UNC; C57BL/6J-Cftr1m/UNC) were also main­
`across the tissue every 5 min during the course of the
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`tained on standard laboratory chow, but the water contained
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`experiment. In all cases, the serosal side served as ground
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`an osmotic laxative (polyethylene glycol) to reduce intestinal
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`and the I,c was conventionally referred to as negative when
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`malfunction in the cftr(-1 -) mice (5). All experiments involv­
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`current flowed from the lumen to the serosa.
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`ing the animals were approved by the University Institu­
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`After the tissues achieved a stable I,c (-20 min post-TTX),
`tionalAnimal Care and Use Committee.
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`a 20-min period was required to adjust the luminal bath pH.
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`The small intestine and gallbladder preparations were then
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`sequentially exposed to a peptide (uroguanylin, 1.0 µM;
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`guanylin, 1.0 µM; or STa, 0.02 µM) in the luminal bath for 30
`Before each experiment, the mice were fasted for a mini­
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`min and then to bumetanide (0.1 mM) in the serosal bath for
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`mum of 1 hand only water was provided. The mice were killed
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`After pH 10 min to inhibit the Na+-K+-2 c1-cotransporter.
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`(to induce by a brief exposure to a 100% CO2 gas atmosphere
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`adjustment, large intestinal preparations were first treated
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`narcosis), followed immediately by a surgical pneumothorax.
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`with amiloride (0.1 mM) in the luminal bath for 20 min to
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`A midline abdominal incision was used to excise the gallblad­
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`inhibit electrogenic Na+ absorption and were then treated
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`der and the following intestinal segments: proximal duode­
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`with peptide addition followed by bumetanide. In studies in
`num (a portion from 2 mm distal to the pylorus to the
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`which the effects of acidic pH on the action of the peptides
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`sphincter of Oddi), mid jejunum, ileum, cecum (a portion 1-2
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`were examined, the proximal duodenum, jejunum, and cecum
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`cm proximal to the cecal apex), and distal colon. The excised
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`were used, and the pH of the luminal bath was decreased to
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`segments were opened along the mesenteric border in ice­
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`pH 5.0-5.5 by addition of 1 N HCl. An equal amount of 1 M
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`cold, oxygenated Krebs-Ringer-bicarbonate (KRB) solution
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`NaCl was added simultaneously to the serosal bath to pre­
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`and pinned mucosal-side down on a pliable silicone surface.
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`vent a trans epithelial c1-diffusion potential. At the end ofan
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`The intestinal sections were stripped of their outer muscle
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`experiment, glucose (10 mM) was added to the luminal bath
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`layers by shallow dissection with a scalpel and fine forceps.
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`of the small intestinal preparations and carbachol (CCh; 0.1
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`mM) was added to the serosal bath of the large intestinal
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`preparations as measures of tissue viability.
`Each intestinal sheet (-1 cm in length) and the microdis­
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`sected gallbladder (with a support of nylon gauze) were
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`mounted in standard Ussing chambers with an exposed
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`surface area of0.25 cm2 for intestinal preparations (or 0.126
`Mucosal epithelium was prepared by scraping the intesti­
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`cm2 for ileum and colon) and 0.049 cm2 for the gallbladder as
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`nal segment from cftr(-1-) and cftr(+I +) mice and washing
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`previously described (5). The tissue sheets were indepen­
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`it gently once in 0.9% NaCl and twice in DMEM containing 20
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`dently bathed on the serosal and mucosal surfaces with 4 ml
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`mM HEPES, pH 7.4. The mucosal suspension (-60 mg wet
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`ofKRB solution containing (in mM) 115 NaCl, 4 K2HPO4, 0.4
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`wt) was placed in 0.2 ml DMEM (pH 7.4) at 4°C. The tissue
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`KH2PO4, 25 N aHCO3, 1.2 MgC12, and 1.2 CaC12, pH 7 .4. To
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`was incubated for 40 min at 37°C with either 1.0 µM
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`facilitate pH adjustment of the medium in some experiments,
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`uroguanylin, 1.0 µM guanylin, or vehicle that was added to
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`a phosphate-free Ringer solution of the following composition
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`the DMEM-HEPES with 1.0 mM 3-isobutyl-1-methylxan­
`was used (in mM): 115 NaCl, 5 KCl, 25 NaHCO3, 1.2 MgSO4,
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`thine. At the end of the 40-min period, perchloric acid was
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`and 1.2 calcium gluconate, pH 7.4. In ion substitution experi­
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`added to a final concentration of3.3%, the cells were centri­
`ments, HCO3 and c1-were replaced with TES and gluconate,
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`fuged, and the resulting supernatants were neutralized with
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`respectively. Glucose (10 mM) was included in the serosal
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`1 N KOH. The supernatants were used to measure cGMP
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`solution (both baths in large intestinal preparations), and 10
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`concentration by radioimmunoassay as described previously
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`mM mannitol was substituted for glucose in the mucosal bath
`(15).
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`to prevent Na +-coupled glucose current stimulation. To mini­
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`mize tissue exposure to endogenously generated prostaglan­
`pH-Stat Titration
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`dins during tissue preparation and mounting, indomethacin
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`Proximal duodenum or cecum was mounted in a standard
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`(1.0 µM) was present in both baths throughout the experi­
`Ussing chamber bathed with 156.2 mM NaCl in the luminal
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`ment. KRB solutions were gassed with 95% 02-5% CO2,
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`1.2 bath and KRB in the serosal bath. In cecal experiments,
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`whereas HCO3-free bathing solutions were gassed with 100%
`mM CaC12 and MgC12 were also added to the luminal bath.
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`recirculation via a gas-lift 02. The solutions were circulated
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`serosal The luminal bath was gassed with 100% 02 and the
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`system and were maintained at 37°C by water-jacketed
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`bath with 95% 02-5% CO2. To decrease the pH of the luminal
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`reservoirs. In all experiments, TTX (0.1 µM) was added to the
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`bath, the luminal saline solution was gassed with 95% 02-5%
`serosal bath at least 20 min before each experiment to
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`flux CO2 and maintained at pH 5.15. The serosal-to-luminal
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`prevent any intrinsic neural influence on ion transport regu­
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`of HCO3 (J�_:f;) was measured by continuously titrat-
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`lation ofthe intestine (47).
`MSN Exhibit 1018 - Page 2 of 12
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`PHYSIOLOGICAL FUNCTIONS OF UROGUANYLIN
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`G635
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`Receptor Autoradiography
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`RESULTS
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`Materials
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`mM. TTX was dissolved in 0.2% acetic acid at a stock
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`ing the luminal solution to pH 7.4 (or pH 5.15) with 5 mM
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`concentration of 0.1 mM. Indomethacin (s.c., 10 mM), bu­
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`HCl, using either a computer-aided titrimeter (model 455/
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`metanide (s.c., 0.1 M), methazolamide (s.c., 1.0 M), DIDS (s.c.,
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`465; Fisher) or manual addition of titrant. The volume of
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`0.3 M), and amiloride (s.c., 0.1 M) were dissolved in DMSO.
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`added acid was used to calculate the HC03 flux, taking into
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`account the exposed surface area of tissue (0.25 cm2) and
`Data Analysis
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`time. Typically, J�_:f; stabilized within 30 min after the
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`tissue was mounted and a basal flux period was initiated.
`Data are means ±: SE. Student's t-test for paired or
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`After 30 min, uroguanylin (1.0 µM) was added to the luminal
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`unpaired data or an ANO VA protected least-significant differ­
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`ent test was used for comparisons of means among different
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`bath, and when the J�_:f; stabilized (-20 min), a second
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`intestinal segments and different treatment groups. In all
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`30-min flux period was initiated.
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`cases, P < 0.05 was accepted as a statistically significant
`difference.
`Dissected intestinal segments (proximal duodenum, mid je­
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`junum, ileum, cecum, and distal colon) from control and
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`CFTR knockout mice were quickly frozen with dry ice and
`Segmental Responses to Uroguanylin in the
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`stored at -80°C. The frozen tissue samples were sectioned to
`Mouse Intestine
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`a 12-µm thickness in a cryostat device (2800 Frigocut N,
`Figure IA shows the pattern of /sc responses to
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`Reichert-Jung; Leica Instrument, Germany) maintained at
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`sequential treatment with specific agents on proximal
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`-20°C. Two sections were placed on opposite ends of a
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`gelatin-coated slide, one for total binding (TB) and one for
`duodenum. After the tissue was mounted, the addition
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`nonspecific binding (NSB), and then dried and stored at
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`ofTTX (0.1 µM, serosal bath) resulted in a decrease in
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`-80°C until used. Iodinated STa was used as the radioligand
`the baseline lsc to a stable value within 20 min.
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`for this assay and was prepared by a modification of the
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`Uroguanylin (1.0 µM, luminal bath) caused a rapid
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`method described by Krause and co-workers (35). The use of
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`increase in lsc, which was sustained
`for a 40-min period.
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`STa was necessary because iodination of guanylin (Tyr-9)
`Subsequent addition of STa (1.0 µM, luminal bath)
`
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`may decrease the biological activity of this ligand (35) and
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`elicited a further increase in lsc· Bumetanide
`(0.1 mM,
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`uroguanylin has no tyrosine residue available for labeling.
`serosal
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`bath) treatment resulted in a decrease in the lsc
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`For the assay, each section on the slide was incubated with 30
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`but to a level that was elevated relative to the lsc before
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`µl of DMEM containing 0.5% BSA to minimize background
`
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`the uroguanylin/STa treatment. The inhibitory effect of
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`labeling (pH 5.5 at 37°C for 20 min). Total binding of
`125I-labeled
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`STa was assessed by incubating 30 µl DMEM
`on lsc typically reached steady state by 10
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`bumetanide
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`containing 1,000 cpm/µl 125I-STa on one section, and nonspe­
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`min posttreatment, i.e., the percentage ofbumetanide
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`cific binding was assessed by incubating 30 µl DMEM contain­
`at 5 min was equal to 98 :::'::: 1.1% of the lsc at
`inhibition
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`ing both 125I-STa and unlabeled STa (1.0 µM), uroguanylin (10
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`10 min postbumetanide (n = 15) in proximal duode­
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`µM), or guanylin (10 µM) on the other section. After a 20-min
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`num. Glucose (10 mM, luminal bath) addition intended
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`incubation at 37°C, slides were washed five times with
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`to stimulate Na+-coupled glucose transport caused a
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`ice-cold phosphate-buffered saline solution and then air­
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`the in /sc· In contrast to apical treatment,
`rapid increase
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`dried. To verify labeling in the different intestinal segments,
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`addition of either uroguanylin or STa to the serosal
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`the slides were arranged in cassettes and exposed to Kodak
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`bath solution had no effect on the lsc (Fig. lB).
`
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`X-OMAT AR (XAR 5) film overnight at -80°C, and then the
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`to uroguanylin Cumulative data of the lsc responses
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`film was developed. Slides were then transferred in a dark
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`room, coated with Kodak NTB-2 emulsion solution, and dried
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`(1.0 µM) by different intestinal segments and the
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`overnight. The emulsion-coated slides were sealed in light­
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`gallbladder are shown in Fig. 2. Uroguanylin elicited
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`tight boxes and stored at 4°C for 2-3 wk until they were
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`had no an lsc response in all intestinal segments but
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`developed. After development, fixation, and covers lipping, the
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`stimulatory effect on the gallbladder preparations.
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`radiolabeled TB and NSB sections were examined under
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`Proximal duodenum and cecum had the greatest mean
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`bright-and dark-field microscopy.
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`(1.0 µM) and were not lsc responses to uroguanylin
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`
`
`significantly different from each other. However, the lsc
`response in the proximal duodenum was significantly
`Opossum uroguanylin (QEDCELCINVACTGC) and E. coli
`
`
`
`greater than the responses in the other small intestinal
`
`
`ST (CCELCCNPACTGC), STal3, were synthesized by the
`
`segments (jejunum and ileum). Likewise, the cecum
`
`
`solid-phase method, using an Applied Biosystems peptide
`
`
`than did the distal colon. The had a greater lsc response
`
`
`
`synthesizer, as previously described (25). Opossum urogua­
`
`
`concentration of uroguanylin (1.0 µM) used in these
`
`
`nylin differs from murine uroguanylin (TDECELCINVAC­
`
`
`experiments was not a maximally stimulatory dose,
`
`TGC) only in the sequence of the first three amino acids.
`
`since subsequent addition of STa (1.0 µM) elicited
`
`
`Purified rat guanylin (PNTCEICAYAACTGC) was generously
`
`in the lsc in all intestinal segments
`further increases
`
`
`
`provided by Dr. Mark Currie (Searle Research and Develop­
`
`ment, St. Louis, MO). The iodination of E. coli ST (NSSNYC­
`
`that were tested (Msc, in µA/cm2): 95.5 :::'::: 10.3 in
`
`CELCCNP ACTGCY) was performed using a lactoperoxidase
`proximal duodenum (n = 7), 112.0 :::'::: 18.5 in jejunum
`
`
`method as previously described (15, 16). Membrane-perme­
`(n = 9), 64.5 :::'::: 10.5 in ileum (n = 9), 77.5 :::'::: 15.8 in
`
`
`
`able 8-bromo-cAMP (8-BrcAMP) and 8-bromo-cGMP (8-
`cecum (n = 9), and 58.4 :::'::: 13.5 in distal colon (n = 7).
`
`
`
`BrcGMP) were obtained from Research Biochemical Interna­
`
`
`tional (Natick, MA). All other chemicals were purchased from
`Proximal Duodenum
`either Sigma Chemical (St. Louis, MO) or Fisher Scientific
`To
`
`
`
`(Springfield, IL). Uroguanylin, guanylin, and E.coli STa were
`
`
`Effect of lum inal acidic pH on uroguanylin action.
`
`
`
`
`dissolved in deionized water at a stock concentration (s.c.) of 1
`
`examine the effect of luminal acidity on uroguanylin
`MSN Exhibit 1018 - Page 3 of 12
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`
`
`
`
`
`

`

`TTX Uroguanylin
`STa
`-250
`
`Burnet Glc
`
`!
`
`-225
`
`-200
`
`-175
`
`e .150
`< -125
`::i.
`� -100
`.!!l
`
`-75
`
`-50
`
`-25
`
`20 40
`
`60 80
`
`100 120
`Time (min)
`
`B
`
`TTX
`
`-250
`
`Uroguanylin STa
`
`Burnet Glc
`
`-225
`
`-200
`
`!
`! !
`!
`!
`
`baseline in about 5 min (Fig. 3A, pH adjustment
`
`
`
`period). A recent study suggests that the acid-induced
`
`
`current may represent stimulation of electrogenic,
`
`
`CF TR-dependent HCO� secretion in the murine duode­
`
`
`
`num (30). Subsequent uroguanylin treatment at the
`
`
`
`
`acidic pH produced an approximately twofold greater
`
`in lsc than was observed at pH 7.4
`(P < 0.05) increase
`lsc elicited by
`
`(Fig. 3, A and B). Again, the increased
`
`
`
`uroguanylin was only partially inhibited by bumetanide
`
`treatment: 18.7 :::'::: 5.7% at pH 7.4 and 27.0 :::'::: 8.0% at pH
`
`
`5.0-5.5 (Fig. 3B). At both pH conditions, uroguanylin
`G1, but the changes were
`
`
`
`treatment slightly increased
`
`
`not statistically different from each other: 2.0 :::'::: 0.4
`2
`2
`
`
`
`mS/cmat pH 7.4 and 3.1 :::'::: 0.3 mS/cmat acidic pH.
`
`
`
`
`The effect ofluminal acidity on uroguanylin bioactivity
`
`
`
`in jejunal tissue was also examined. The reduction of
`
`
`
`intraluminal pH to 5.0-5.5 again increased basal lsc
`
`
`2), and subsequent treatment with
`(19.6 :::'::: 4.8 µA/cm
`
`
`1.0 µM uroguanylin at acidic pH produced a signifi­
`
`in lsc than was observed at pH 7.4
`
`
`cantly larger increase
`2, P < 0.05, n = 4). In a
`(66 :::'::: 4.2 vs. 44.6 :::'::: 5.8 µA/cm
`
`
`
`
`dose-response study, uroguanylin was more active (P <
`
`
`
`0.05) in acidic luminal conditions than at pH 7.4 within
`
`the tested range of concentrations (Fig. 4). It was not
`
`
`possible to test higher concentrations of uroguanylin
`
`
`
`because the supply of the peptide was inadequate for
`the experiment.
`Effect of lum inal acidity on the actions of 8-BrcGMP
`
`
`
`
`and 8-BrcAMP. To test whether the effect of luminal
`
`
`
`
`acidity on uroguanylin action resulted from an effect of
`
`
`
`low extracellular pH on the bioelectric properties or
`
`
`
`
`intracellular signaling pathways of the epithelial cells,
`
`
`
`cy­the /sc responses elicited by membrane-permeable
`
`
`clic nucleotides, 8-BrcGMP and 8-BrcAMP, were exam­
`
`
`ined at acidic and physiological pH. In the proximal
`
`G636
`
`A
`
`
`
`PHYSIOLOGICAL FUNCTIONS OF UROGUANYLIN
`
`-175
`
`"'e .150
`< -125
`_::!,
`(..) -100
`.!!l
`.75
`
`-50
`
`-25
`
`20 40
`
`60 80
`
`100 120
`
`Time (min)
`
`60
`
`a
`
`50
`
`"' 40
`
`u
`
`Fig. 1. Time course of short-circuit current (/8,) response
`
`
`to sequen­
`30
`_::!,
`
`
`
`tial treatments in mouse intestine. Data are representative of 11
`u
`
`
`
`separate experiments with proximal duodenum bathed in standard
`<l 20
`
`
`Krebs-Ringer-bicarbonate (KRB). A: uroguanylin (1.0 µM), Esch­
`
`
`
`
`(Glc, enterotoxin (STal3; 1.0 µM), and glucose erichia coli heat-stable
`
`
`
`10 mM) were applied to the luminal bath. B: uroguanylin (1.0 µM)
`
`and STa (1.0 µM) were added to serosal bath. TTX (0.1 µM) and
`
`
`
`bumetanide (Burnet; 0.1 mM)were added to serosal bath.
`
`10
`
`0
`
`bioactivity in the native intestine, the pH of the luminal
`
`
`
`
`
`bath was reduced to 5.0-5.5 for 20 min before the tissue
`Fig. 2. Segmental responses to luminal uroguanylin in mouse intesti­
`
`
`
`
`
`
`
`was treated with the peptide agonists (pH adjustment
`
`
`
`nal preparations. All tissues were bathed with standard KRB solu­
`
`
`period). The proximal duodenum was chosen because
`
`
`tion. lilsc was calculated as baseline ls, minus maximal ls, response to
`
`
`
`this segment elicited pronounced increases in urogua­
`
`
`
`
`uroguanylin. Large intestinal epithelia (cecum and distal colon) were
`
`lsc (Fig. 2) and because this part of the
`nylin-induced
`
`
`pretreated with amiloride (0.1 mM, luminal bath) 20 min before
`
`
`
`uroguanylin addition. Values are means ± SE obtained from proxi­
`
`
`
`small intestine has an acidic intraluminal environment
`
`mal duodenum (n = 11), midjejunum (n = 9), ileum (n = 9), cecum
`
`
`during digestion (1). The intraluminal pH reduction
`
`
`(n = 12), distal colon (n = 5), and gallbladder (n = 4). Means without
`
`
`caused a rapid and reproducible increase in the lsc of
`
`
`a letter in common are significantly different; one-way ANOVA
`
`2, reaching a new steady-state
`13.4 :::'::: 2.7 µA/cm
`/sc
`
`
`
`protected least-significant difference test, P < 0.05.
`
`Proximal Mid Ileum Cecum Distal Gallbladder
`Duodenum Jejunum
`Colon
`
`MSN Exhibit 1018 - Page 4 of 12
`MSN v. Bausch - IPR2023-00016
`
`
`
`Downloaded fromjoumals.physiology.org/journal/ajpgi (098.109.055.010) on January 13, 2021.
`
`
`
`
`
`
`
`

`

`PHYSIOLOGICAL FUNCTIONS OF UROGUANYLIN
`
`G637
`
`A
`
`··•--pH 5.0~5.5
`-o-pH 7.4
`
`
`TTX
`-200
`
`pH adjustment Uroguanylin Burnet Glc
`
`120
`-o- pH 5.0~5.5
`-pH7.4
`100
`
`*
`
`-180
`
`-160
`
`i
`
`-8
`
`-7
`
`-6
`
`-5
`
`
`
`Log [Uroguanylin, M]
`
`--8
`
`0
`
`(J
`<i: 60
`-::!,
`(J
`
`40
`
`20
`
`0
`-9
`
`-140
`e -120
`<j'. -100
`-::!,
`(J -80
`J!l.
`-60
`
`-40
`
`-20
`
`0
`0
`
`B
`
`125
`
`100
`
`75
`
`(J
`<i:
`-::!,
`(J
`50
`J!l.
`<I
`
`25
`
`0
`
`20
`
`40
`
`60
`
`80
`
`Time (min)
`
`Fig. 4. Effect ofluminal bath pH on noncumulative concentration-ls,
`
`
`
`
`
`
`response curve for uroguanylin in intact mouse proximal duodenum.
`100
`
`
`Luminal bath pH was reduced to 5.0-5.5 by addition of 1.0 N HCI,
`
`
`
`as and basolateral pH was maintained at pH 7.4. M s, was calculated
`
`baseline ls, minus maximal ls, response
`
`to uroguanylin. Values are
`
`
`means ± SE obtained from 4-9 different mice. * Significantly differ­
`
`
`ent from pH 7.4 group (unpaired < 0.01).
`t-test,P
`
`i::::::J Uroguanylin (1.0 µM)
`
`- +Burnet (0.1 mM)
`
`8-BrcAMP was significantly inhibited (P < 0.05) by
`
`
`
`
`bumetanide (0.1 mM) treatment: 59.3 :::'::: 1.8% at pH 7.4
`
`and 65.7 :::'::: 3.4% at pH 5.0-5.5, or 61.5 :::'::: 5.9% at pH 7.4
`
`
`and 90.8 :::'::: 4.2% at pH 5.0-5.5, respectively.
`
`
`Effect of lum inal acidity on guanylin and S Ta ac­
`
`
`
`tions. To investigate whether the pH dependence was
`
`
`
`
`specific for uroguanylin action, we examined the effects
`
`of acidic pH on the secretagogue actions of guanylin
`
`and STa (Fig. 6). Whereas uroguanylin is more effective
`
`conditions in stimulating the lsc under acidic luminal
`
`
`120
`
`100
`
`c::::J pH 7.4
`-pH5.0~5.5
`
`pH7.4
`
`pH 5.0~5.5
`
`.-.
`Fig. 3. Effect of pH on l
`in mouse proximal s, response to uroguanylin
`
`
`80
`
`
`
`to agents in time course of lduodenum. A: representative s, response
`
`
`
`
`proximal duodenum superfused with Ringer solution at either pH
`
`5.0�5.5 or pH 7.4 in luminal bath. Luminal pH was reduced to pH
`60
`-::!,
`
`
`
`
`5.0-5.5 by addition of 1.0 N HCI (pH adjustment), and serosal pH was
`
`
`
`
`maintained at 7.4. TTX (0.1 µM), uroguanylin (1.0 µM), and glucose
`<I
`
`
`(10 mM) were added to luminal bath, and bumetanide (0.1 mM) was
`
`
`
`added to serosal bath. B: cumulative data showing maximal change
`
`
`
`treatment and after in ls, (Ms,) from baseline during uroguanylin
`
`
`
`
`sequential addition ofbumetanide (+Burnet). Results are means ±
`
`SE from intact proximal duodena of 8 (pH 7.4 group) and 9 (acid­
`
`
`
`
`treated group) different mice. *Significantly different from urogua­
`
`
`
`
`
`nylin treatment (paired < 0.05). tSignificantly different from
`t-test,P
`
`
`pH 7.4 group (unpaired < 0.05).
`t-test,P
`
`(J
`
`40
`
`20
`
`*
`
`0
`8-Br-cAMP +Burnet
`8-Br-cGMP +Burnet
`
`Proximal Duodenum
`
`
`to membrane­Fig. 5. Effect of luminal bath pH on ls, response
`
`
`permeable cyclic nucleotides in mouse proximal duodenum. 8-
`duodenum, membrane-permeable 8-BrcGMP (20 µM)
`
`
`
`BrcGMP (20 µM) or 8-BrcAMP (20 µM) was added to both luminal
`
`stimulated the /sc more than did an equimolar
`concen­
`
`
`and serosal baths 20 min after pH adjustment. Bumetanide (0.1 mM)
`
`tration of 8-BrcAMP (P < 0.001) (Fig. 5). However, the
`
`was added to serosal bath 30 min later. M s, was calculated
`as
`
`/sc response elicited by 8-BrcGMP at pH 7.4 was similar
`
`
`
`during cyclic nucleotide baseline ls, minus maximal ls, response
`
`
`
`
`treatment and after subsequent treatment with bumetanide. Data
`
`
`
`
`to that observed under acidic luminal conditions. In
`
`
`are means± SE; proximal duodena were obtained from 4-6 different
`
`
`contrast, 8-BrcAMP (20 µM) was significantly less
`
`
`
`
`
`mice in each cyclic nucleotide treatment group. * Significantly differ­
`
`
`effective under acidic conditions than at pH 7.4 (P <
`
`
`
`ent from 8-BrcGMP-treated group (unpaired t-test; P < 0.001).
`
`
`0.05). The increased lsc elicited
`
`by either 8-BrcGMP or
`
`
`
`tSignificantly different from pH 7.4 (unpaired < 0.05).
`t-test;P
`
`
`
`Downloaded fromjoumals.physiology.org/journal/ajpgi (098.109.055.010) on January 13, 2021.
`
`MSN Exhibit 1018 - Page 5 of 12
`MSN v. Bausch - IPR2023-00016
`
`

`

`G638
`
`*
`
`100
`
`80
`
`N
`
`E 60
`u
`
`..:!,
`u 40
`!!!.
`<I
`
`20
`
`
`
`PHYSIOLOGICAL FUNCTIONS OF UROGUANYLIN
`
`c::::JpH 7.4
`
`-pHS.0~5.5
`
`*
`
`50
`
`N
`
`E 40 �
`
`..:!,
`u 30
`!!!.
`
`Ill
`
`20
`
`E
`10
`
`Ill 0
`
`*
`
`i::=J Uroguanylin (1.0 µM)
`
`
`-+Bumetanide (0.1 mM)
`
`0
`
`
`
`
`(1.0 µM) Uroguanylin (1.0 µM) Guanylin
`
`STa(20 nM}
`
`Cl-free
`
`-10 HC03-free
`Ringer Solution
`
`Proximal Duodenum
`
`to Fig. 7. Effect of anion-substituted Ringer solutions on ls, response
`
`
`
`
`
`
`to uroguanylin, Fig. 6. Effect of luminal bath pH on ls, response
`
`
`uroguanylin in intact mouse proximal duodenum. Sodium bicarbon­
`
`
`
`
`guanylin, and STa in intact mouse proximal duodenum. Each peptide
`
`
`
`ate was replaced with TES. Gluconate was substituted for c1-in
`
`
`was added to luminal bath 40 min after TTX treatment (20 min after
`c1--free and HCO3 /Cl--free solutions.
`
`Methazolamide (1.0 mM) was
`
`
`
`
`as baseline pH adjustment period). M s, was calculated ls, minus
`
`
`
`added to both luminal and serosal bath solutions 20 min before
`
`
`
`Results are means ± SE; proximal maximal ls, response to a peptide.
`
`
`luminal uroguanylin application in HCO3-free Ringer solutions.
`
`
`duodena were obtained from 6-9 different mice for each peptide
`
`
`Bumetanide (0.1 mM) was added to serosal bath 30 min after
`
`
`
`
`tested. *Significantly different from pH 7.4 group (unpaired t-test,
`
`
`as pretreatment uroguanylin. Change from basal ls, was calculated
`P < 0.05).
`ls, minus maximal ls, recorded
`
`after uroguanylin or 10 min after
`
`
`
`bumetanide treatment. Values are means ± SE; proximal duodena
`
`
`
`(1.0 µM) was to guanylin (P < 0.01), the lsc response
`
`
`
`were obtained from 6-9 different mice in each treatment group.
`
`
`
`significantly reduced at acidic pH compared with pH
`
`
`* Significantly different from uroguanylin-treated group (paired t­
`
`the lsc responses to STa (20
`
`7.4 (P = 0.05). In contrast,
`test, P < 0.01).
`nM) did not appear to be pH dependent. The l
`clamp conditions were used to measure the effect of
`
`
`
`
`
`responses induced by guanylin or STa were incom­
`on J�__:f3. The values of lsc in Table 1 are
`uroguanylin
`
`
`
`
`
`pletely inhibited by serosal bumetanide treatment (25-
`
`
`given in microequivalents per square centimeter per
`
`
`35% at pH 7.4; 50% at acidic pH)(data not shown). Gt in
`
`hour to facilitate the comparison with J�__:f3. As shown
`
`
`
`STa-treated or guanylin-treated groups under acidic
`
`
`
`
`intraluminal conditions was increased by 3-4 mS/cm2
`
`in Table 1, uroguanylin (1.0 µM) at pH 7.4 stimulated
`whereas at pH 7.4, �Gt in this period was ~0.5-1.0
`the J�__:f3 and the /sc· At luminal
`pH 5.15, the basal
`mS/cm2
`mean J�__:f3 and lsc were larger than the basal param­
`
`
`by urogua­Stimulation of Cl-and HC0-3 secretion
`
`
`
`eters measured at pH 7.4 (P < 0.05, unpaired t-test). In
`
`
`As stated, nylin. a major fraction of the lsc stimulated
`
`
`
`
`part, t