GASTROENTEROLOGY 1988;94:1014-23
`
`Effect of Cholecystokinin Receptor
`to
`Antagonist on Pancreatic Responses
`Exogenous Gastrin and Cholecystokinin
`and to Meal Stimuli
`
`S. J. KONTUREK,
`M. HLADIJ
`Institute of Physiology, Academy of Medicine, Krakow, Poland
`
`J. TASLER, M. CIESZKOWSKI, K. SZEWCZYK,
`
`and
`
`to
`to food is believed
`response
`Exocrine pancreatic
`result from the interaction
`of neural and hormonal
`factors, but their contribution
`in the net postpran-
`dial secretion
`is unknown. Recent description
`of a
`highly
`specific and potent cholecystokinin
`(CCK)-
`receptor antagonist permitted
`the evaluation
`of the
`physiologic
`role of CCK in postprandial
`pancreatic
`secretion.
`In dogs with chronic pancreatic
`fistula,
`CCK antagonism
`caused
`little alteration
`in sham
`feeding- or urecholine-induced
`pancreatic
`protein
`the pancreatic
`secretion,
`but reduced
`by
`-60%
`protein
`response
`to a gastrointestinal
`meal and
`virtually
`abolished
`the pancreatic
`responses
`to du-
`odenal perfusion with amino acids or oleate and to
`exogenous CCK, but not to secretin or neurotensin.
`The pancreatic
`protein
`responses,
`particularly
`to
`lower dqses of gash-in, were also reduced by CCK-
`receptor antagonist, but no changes
`in the responses
`to secretin or neurotensin were detected. Cholecys-
`tokinin antagonism
`also significantly
`reduced
`the
`pancreatic
`polypeptide
`responses
`to CCK, gastrin,
`and the gastrointestinal
`meal, possibly
`due to re-
`moval of the CCK-mediated
`release of pancreatic
`polypeptide. We conclude
`that CCK plays a crucial
`role in the mediation
`of the gastrointestinal
`phase,
`but not the cephalic phase, of pancreatic
`secretion.
`
`E xocrine pancreatic
`
`to in-
`in response
`secretion
`phases
`from the overlapping
`gested food results
`(1,~).
`of cephalic, gastric, and intestinal
`stimulation
`Although
`it is generally accepted
`that the pancreatic
`secretion
`is controlled
`by
`interacting
`neural
`and
`hormonal mechanisms,
`the controversy
`continues
`over the relative contribution
`of these mechanisms
`in the interdigestive
`and postprandial
`secretion
`(1).
`The old suggestion
`that the reflex vagal-cholinergic
`mechanisms
`play a considerable
`role in the cephalic
`(2-4) of pancreatic
`secre-
`and gastrointestinal
`phases
`
`that
`by recent evidence
`tion has been undermined
`antral gastrin
`intestinal
`cholecystokinin
`(5,6) and
`(CCK) (7,8) may also be important
`physiologic me-
`diators
`in postprandial
`pancreatic
`secretion.
`The
`assessment
`of the hormonal
`contribution
`has been
`possible because of the recent description
`of highly
`specific and potent CCK-receptor antagonists
`(g-12),
`which allow pancreatic
`responses
`to be measured
`in
`the absence of the effect of CCK.
`the role of
`This study was undertaken
`to clarify
`CCK and gastrin
`in the cephalic and gastrointestinal
`phases of pancreatic
`secretion,
`as well as in the
`pancreatic
`responses
`to exogenous
`gut hormones.
`This was accomplished
`by using one of the most
`of CCK action
`and
`potent
`antagonists
`(CR-1409)
`binding
`to the pancreas
`(g-12)
`in conscious
`dogs
`with chronic gastric and pancreatic
`fistulas.
`
`and Methods
`Materials
`Studies
`in vivo were carried out on 6 mongrel dogs,
`weighing 18-20 kg and prepared surgically with esopha-
`geal, gastric, and pancreatic
`fistulas as described previ-
`ously (8,131. The studies reported here started -5 mo after
`surgery. Food was withheld
`for at least 18 h before each
`test. Throughout all tests, except
`those with feeding,
`the
`gastric fistula was left open to allow for draining of gastric
`juice to the outside
`to prevent gastric acid from entering
`the duodenum and releasing endogenous hormones.
`fistula
`Secretions
`from the gastric fistula and pancreatic
`were collected
`continuously
`and divided
`into 15-min
`aliquots. The volume was recorded
`to the nearest 0.1 ml.
`Acid concentration
`in the gastric
`juice and bicarbonate
`and protein concentrations
`in the pancreatic
`juice were
`
`tJds paper: CCK, cholecystokinin;
`in
`used
`Abbreviations
`CCK-8, cholecystokinin-octapeptide; D,,, dose producing half-
`maximal stimulation; PP, pancreatic polypeptide.
`0 1988 by the American Gastroenterological Association
`0016-5085/88/$3.50
`
`
`
`
`MAIA Exhibit 1043
`MAIA V. BRACCO
`IPR PETITION
`
`

`

`April 1988
`
`CCK AND PANCRJZATIC SECRETION
`
`1015
`
`in each sample and presented as 15- or 30-min
`
`measured
`outputs.
`In tests
`in each animal.
`tests were performed
`Several
`with basal secretion,
`the CCK-receptor
`antagonist
`DL-J-
`(3,4-dichloro-benzoyl-amino)-5(di-n-pentyl-amino)-5-oxo-
`pentanoic acid (gift of Dr. L. Rovati from Rotta Research
`Lab., Milano,
`Italy) was infused
`intravenously
`in graded
`doses (0.03-2.0 pmol/kg h), each dose being given for 60
`min and then doubled during separate
`tests. This com-
`pound
`is named CR-1409
`(lo), but it is also known from
`other reports as compound 53 (9), proglumide analogue 10
`(ll), or compound B (12).
`two types of ex-
`secretion,
`In tests with meal-induced
`sham
`feeding and ordinary
`periments were performed:
`feeding. For the sham feeding procedure,
`the esophagus
`was totally obstructed distal to the esophageal
`fistula so
`that the possibility
`of entry of food particles
`into
`the
`stomach was completely
`excluded
`(13). Each dog was
`offered 500 g of cooked homogenized ground beef for 15
`min; the ingested meal fell from the esophagus back into
`the feeding pan and was repeatedly
`reconsumed. Gastric
`and pancreatic
`collections were made for 60 min before,
`during, and 90 min after the sham feeding. CR-1409
`[l.O
`pmol/kg h) was given for 30 min before, during, and after
`sham feeding. In tests with the gastrointestinal phase, 500
`g of cooked beef liver homogenized with 100 ml of water
`was
`introduced
`from
`the plastic bag directly
`into
`the
`stomach
`through
`the esophageal
`fistula, without animals
`being allowed
`to see or smell the meal. The gastric fistula
`was kept closed and pancreatic
`secretion was measured
`during the next 3 h. CR-1409
`(0.5 or 1.0 pmol/kg h) was
`infused
`intravenously
`30 min before and throughout
`the
`postprandial period.
`a solution of 100
`In tests with duodenal acidification,
`mM HCl was instilled
`into the duodenum
`through
`the
`intestinal
`limb of the pancreatic cannulas at a constant rate
`(8 mmol/h) to evoke near-maximal pancreatic bicarbonate
`secretion.
`In tests with duodenal perfusion with oleate,
`oleic acid (Sigma Chemical Co., St. Louis, MO.) was pre-
`pared as the sodium soap by adding the desired amount of
`fatty acid to an aqueous
`solution of NaOH and stirring
`vigorously
`for as long as 60 min to obtain proper disper-
`sion (8). The final pH was 9.4 and the final concentration
`of oleate used was 100 mM. L-Isomers of tryptophan and
`phenylalanine
`(Calbiochem,
`San Diego, Calif.) were used
`in solutions
`containing
`100 mM of each, adjusted
`to pH
`6.0. All solutions were prepared on the day of the experi-
`ment and were made isotonic
`(300 2 10 mosmol/kg) by the
`addition of NaCl as needed. Solutions were infused at a
`constant
`rate of 80 ml/h. CR-1409
`(1.0 pmol/kg h) was
`infused
`in the middle hour of duodenal
`instillation of HCl,
`oleate, or amino acid mixture.
`In control
`tests, the duode-
`nal HCl, oleate, or amino acid mixture was administered
`alone for the duration of the experiment.
`synthetic
`In tests with exogenous hormonal stimulation,
`CCK-octapeptide
`(CCK-8)
`(Squibb
`Institute
`for Medical
`Research, Princeton, N.J.) was infused intravenously
`either
`h) doubled
`every
`in graded doses
`(25-800
`pmol/kg
`30-min period or in a constant dose (100 pmol/kg h) given
`for a 5-h period. CR-1409 was administered
`either
`in a
`constant dose (0.5, 1.0, or 2.0 pmol/kg
`h) given through-
`
`out the period of infusion of graded doses of CCK or in
`gradually
`increasing doses (0.03-2.0
`h) admin-
`pmol/kg
`istered during a constant administration
`of CCK-8. Gastrin
`heptadecapeptide
`(a gift of Professor R. A. Gregory,
`Liverpool, U.K.) was administered
`intravenously
`either in
`h) doubled every 30-min
`graded doses (31-2000 pmol/kg
`period or in a constant dose (250 pmol/kg
`. h) given for 5.5
`h. CR-1409 was added to an intravenous
`infusion of gastrin
`h) or in
`either in a constant dose (1.0, 2.0, or 4.0 pmol/kg
`gradually increasing doses (0.03-4.0 pmolikg
`h), changed
`every 30 min.
`In tests with graded doses of CCK and
`gastrin without and with CR-1409, actual maximal pancre-
`atic protein and gastric acid responses were determined
`in
`each dog and then combined
`from 6 dogs to calculate
`the
`mean maximum
`and the mean dose of CCK or gastrin
`producing half-maximal
`stimulation
`(D5,,). In tests with
`h) and neurotensin
`(50 pmol/kg h),
`secretin
`(82 pmolikg
`each of these hormones was infused
`intravenously
`in a
`constant dose
`throughout
`the experiment
`to achieve a
`secretory
`rate similar
`to that obtained with duodenal
`perfusion of HCl or oleate, respectively. When the pancre-
`atic secretion
`reached a well-sustained
`plateau, CR-1409
`was added
`to
`the
`infusion
`in a constant
`dose
`(1.0
`pmol/kg h). Secretin
`and neurotensin wete purchased
`from Peninsula Laboratories, Belmont, Calif.
`In all tests
`involving administration
`of CCK, gastkin,
`secretin,
`or neurotensin,
`a solution
`of 0.5% albumin
`(Sigma) was used to dissolve
`these peptides
`to pre&nt
`their degradation
`and adsorption
`into the plastic
`tubes
`during intravenous
`infusion.
`feeding, and in
`In tests with sham feeding and ordinary
`tests with
`infusion of exogenous gastrin, blood samples
`were taken from the peripheral vein at 15-30-min
`intervals
`for radioimmunoassay
`of plasma gastrin and pancreatic
`polypeptide
`(PP). Blood samples were collected
`in chilled
`tubes with 10 U of heparin and 400 KIU of aprotonin
`(Trasylol, Bayer Farma, Copenhagen, Denmark) per milli-
`liter and centrifuged, and the plasma was frozen within 15
`min of sampling. The plasma gastrin level was determined
`using gastrin antiserum 4562 (a gift of Prof. J. F. Rehfeld,
`Aarhus, Denmark) and plasma PP was assayed using PP
`antiserum
`(a gift of Dr. R. E. Chance, Eli-Lilly,
`Indianapo-
`lis, Ind.) as presented previously
`(13).
`Results are expressed as mean ? SEM. In tests compar-
`ing various nutrients
`and hormones with and without
`CR-1409,
`the increments
`in pancreatic
`secretory outputs
`and plasma hormone concentrations were calculated and
`averaged to provide the incremental
`secretory outputs and
`incremental plasma hormone
`levels for the experimental
`period. The significance of the differences between means
`was evaluated using Student’s
`t-test
`for paired values.
`Differences were considered
`significant
`if p < 0.05.
`
`Results
`Effects of CR-1409
`Secretion
`secretion
`acid
`gastric
`dogs, basal
`In
`fasted
`fistula was negligible, whereas
`the
`the gastric
`from
`pancreatic
`secretion
`showed
`some
`fluctuations
`in
`
`on Basal Pancreatic
`
`
`
`
`

`

`1016 KONTUtiK ET AL.
`
`GASTROENTEROLOGY Vol. 94. No. 4
`
`to
`dogs. Protein outputs varied from -20
`individual
`240 mg/l5 min, averaging -80 k 15 mg/15 min
`during
`the 2-h basal collection
`period
`(Table 1).
`HC03 output also fluctuated
`from -30 + 5 to 76 + 12
`pmol/l5 min, averaging
`-32
`? 8 pm01115 min.
`Intravenous
`infusion of CR-1409 in doses of O-25-2.0
`. h tended
`to reduce mean basal protein
`pmol/kg
`secretion, but this was significant only at the highest
`dose of CCK antagonist. No changes
`in basal gastrin
`or PP levels were observed
`after
`intravenous
`infu-
`sion of CR-1409 (Table 1).
`
`Effects of CR-1409 on Pancreatic Responses
`to Exogenous Hormones
`in a dose-related
`Infusion of CCK-8 resulted
`that was reflected
`increase
`in pancreatic
`secretion
`mainly
`in the protein outputs
`(Figure l), whereas
`the
`volunie flow and &OS outputs showed only a small
`increase
`(data not shown).
`in tests
`outputs
`protein
`The maximal
`observed
`. h and
`with CCK-8 occurred at a dose of 400 pmol/kg
`averaged 2350 ? 250 mg/30 min. The dose of CCK-8
`required
`for half-maximal
`protein output averaged
`86 + 12 pmol/kg
`h. When CR-1409 was added at 0.5
`. h to the CCK infusion,
`the respec-
`and 1.0 pmollkg
`tive maximal protein
`responses
`to CCK-8 averaged
`780 2 168 and 460 + 96 mg/30 min. The respective
`D5,, values
`in these tests were 67 t 18 and 134 + 28
`pmol/kg
`h. At the highest dose of CR-1409
`(2.0
`. h) the pancreatic
`protein
`responses
`to all
`pmollkg
`doses of CCK-8 were almost completely
`suppress&d.
`Thus,
`in the presence of CR-1409 the maximal pan-
`creatic protein
`responses
`to CCK were significantly
`reduced
`in a dose-dependent
`fashion, whereas
`the
`DSo was not changed significantly.
`by the in-
`Plasma gastrin
`levels were unchanged
`travenous
`infusibn
`of CCK-8 with or without
`the
`addiiion
`of CR-1409. Plasma PP levels showed
`a
`
`Table 1. Pancreatic HC03 and Protein Outputs and
`Plasma Gastrin and Pancreatic Polypeptide
`Concentrations
`in Fasted Dogs Without and
`With intravenous Administration of CR-1409
`in Gradually Increasing Doses
`HCOB
`Protein
`(~molll5
`(mg/l5
`min)
`min)
`
`Gastrin
`(PM)
`
`CCK 8 (pmollkg-h1
`
`to graded
`in response
`in pancreatic outputs
`Figure 1. Increments
`h). Mean + SEM of
`doses of CCK-8 (25-800 pmol/kg
`indicate significant
`six tests on 6 dogs. Single asterisks
`decrease below the control value obtained with CCK-8
`alone. Double asterisks
`indicate
`significant
`decrease
`below the values obtained with CCK-8 plus CR-1409 at
`h. Triple asterisks
`indicate
`a dose of 0.5 pmol/kg
`significant
`decrease below
`the values
`obtained
`with
`CR-1409 at a dose of 1.0 pmolikg
`h.
`
`rise with increasing doses of CCK-8.
`dose-dependent
`Treatment with CR-1409 did not affect basal PP
`levels, but reduced
`significantly
`the PP responses
`to
`all doses of CCK-8, including
`that producing
`the
`maximal PP response. At the higher dose of CR-1409
`. h), CCK-8 failed
`to produce
`any sig-
`(2.0 pmollkg
`nificant alterations
`in plasma
`levels of PP [Table 2).
`. h)
`In tests with a constant-dose
`(100 pmol/kg
`infusion of CCK-8 producing
`-50% maximal stimu-
`lation of pancreatic
`protein output,
`the addition of
`CR-1409
`in gradually
`increasing
`doses
`(0.03-2.0
`pmol/kg
`. h) resulted
`in a significant
`decrease
`in
`protein
`secretion
`at a dose of 0.03 pmol/kg
`. h and
`almost complete
`suppression
`of the protein
`response
`to CCK at doses of 1.0 and 2.0 pmol/kg
`. h (Figure 2).
`In tests with gastrin heptadecapeptide,
`graded
`doses of hormone
`infused
`intravenously
`resulted
`in
`a dose-dependent
`increase
`in both gastric acid and
`pancreatic
`protein outputs,
`reaching
`the maximal
`observed outputs at a dose of 500 pmol/kg
`h. These
`observed acid and protein maximums
`averaged 7940
`? 1090 pmol/30 min and 1410 t 230 mg/30 min,
`respectively. Addition of CR-1409 to the intravenous
`infusion
`reduced
`both gastric acid and pancreatic
`protein
`responses
`to gastrin
`in a dose-dependent
`fashion. Gastric acid responses
`to lower doses of
`. h) were significantly
`re-
`gastrin
`(31-125 pmol/kg
`duced by CR-1409 at a dose of 1.0 pmol/kg
`h and
`completely
`suppressed
`at doses of 2.0 and 4.0
`pmol/kg
`. h. With higher doses of gastrin,
`such as
`500 and 2000 pmol/kg
`h, the acid output was not
`
`PP (PM)
`14 + 2
`
`32 ” 6
`
`80 + 15
`
`21 + 4
`
`Basal
`CR-1409
`0.25 pmol/kg . h
`94 2 18
`34 * 6
`16 + 2
`29 + 5
`. h
`0.5 pmol/kg
`42 k 9
`15 t 2
`30 ? 4
`70 2 12
`1.0 pmol/kg
`. h
`35 + 7
`17 2 4
`26 +- 3
`62 2 14
`2.0 qol/kg
`. h
`26? 7
`11 -t 2
`42 2 10" 24 2 6
`PP, pancreatic polypeptide. Values shown are mean + SEM of six
`tests in 6 dogs. ’ Significant (p < 0.05) decrease below the basal
`value.
`
`
`
`
`

`

`April 1988
`
`CCK AND PANCREATIC SECRETION
`
`1017
`
`Table 2. Plasma Gastrin and Pancreatic Polypeptide
`Levels in Tests With
`Increasing
`Doses of
`Cholecystokinin-Octapeptide
`Without
`and
`With Addition
`of CR-1409 Throughout
`the
`Experiment
`
`Basal
`
`CCK-8
`25 pmol/kg . h
`50 pmol/kg
`. h
`100 pmol/kg
`. h
`200 pmol/kg
`. h
`400 pmol/kg
`. h
`800 pmolikg
`h
`
`Gastrin (PM)
`
`22 + 4
`
`26 t 2
`28 + 6
`25 k 5
`30 2 3
`31 2 4
`32 2 6
`
`CR-1409 (1.0 pmolikg
`25 pmolikg . h
`50 pmolikg . h
`100 pmol/kg
`h
`200 pmolikg
`. h
`400 pmolikg
`. h
`800 pmol/kg . h
`
`. h) + CCK-8
`26 + 5
`30 r 4
`32 + 5
`34 t 7
`30 ” 2
`31 2 5
`
`CR-1409 (2.0 Fmol/kg
`25 pmol/kg . h
`50 pmol/kg
`. h
`100 pmol/kg
`. h
`200 pmol/kg
`. h
`400 pmolikg
`. h
`800 pmol/kg . h
`
`. h) + CCK-8
`23 2 3
`24 * 5
`26 + 5
`28 k 4
`27 2 7
`30 f 9
`
`PP (PM)
`12 z!I 3
`
`18 + 4
`24 k 2a
`32 k 4”
`56 k 8”
`79 ‘- 14”
`92 +- 17”
`
`11 It 2
`10 ? lb
`12 r 2b
`27 ” 4b
`33 2 Sb
`39 2 12b
`
`12 + 5
`14 r 4
`12 + 3b
`16 t 5b
`12 2 6c
`14 ” 4”
`
`CCK-8, cholecystokinin-octapeptide;
`PP, pancreatic polypeptide.
`Values shown are mean t SEM of six tests in 6 dogs. ’ Significant
`(p < 0.05)
`increase
`above basal value. b Significant
`(p < 0.05)
`decrease
`below
`the value
`obtained
`with
`the
`respective
`dose of
`CCK-8 alone. ’ Significant
`(p < 0.05) decrease
`below
`the value
`obtained
`with
`the
`respective
`dose of CCK-8 + CR-1409
`at 1.0
`. h.
`pmolikg
`
`(1.0
`of CR-1409
`dose
`lower
`the
`by
`affected
`by
`significantly
`reduced
`it was
`. h), but
`pmollkg
`. h),
`higher doses of CR-1409 (2.0 and 4.0 pmol/kg
`by
`the maximal
`acid
`response
`being diminished
`-50% by these doses of CR-1409. The DSO for gastrin
`
`CCKB ~lOOomollko-hl
`
`._,
`
`1
`
`1
`
`5
`
`13
`4
`15min PERIODS
`
`17
`
`21
`
`Figure
`
`2
`
`h) of
`pmolikg
`(0.03-2.0
`doses
`of graded
`Effects
`to a constant
`protein
`responses
`on pancreatic
`CR-1409
`? SEM of six
`dose of CCK-8
`(100 pmol/kg
`h). Mean
`tests on 6 dogs. Asterisks
`indicate
`significant
`decrease
`below
`the values
`obtained with CCK-8 alone.
`
`GASTGiN(pmollkghl
`
`Figure
`
`3.
`
`out-
`protein
`in gastric H+ and pancreatic
`Increments
`puts
`in response
`to graded
`doses of gastrin
`(31-2000
`pmolikg h) without
`and with CR-1409
`(1.0, 2.0, and
`4.0 pmol/kg
`h). Mean 2 SEM of six tests on 6 dogs.
`Asterisks
`indicate
`significant
`decrease
`below
`the con-
`trol values obtained with CCK-8 alone.
`
`. h, and the addition
`alone averaged 92 ~fr 12 pmol/kg
`of CR-1409 at doses of 1.0, 2.0, and 4.0 pmol/kg
`h
`increased
`the DSO value
`to 132 + 18, 294 ? 35, and
`246 2 42 pmol/kg
`. h, respectively
`(Figure 3).
`Pancreatic protein
`responses
`to low doses (31-125
`. h) of gastrin were reduced by low doses of
`pmol/kg
`CR-1409 and abolished
`by high doses of CR-1409.
`The maximal protein
`response
`to gastrin, which was
`not affected by CR-1409 at a dose of 1.0 pmol/kg
`. h,
`was reduced by 67% and 74% by CR-1409 at doses of
`2.0 and 4.0 pmollkg
`. h, respectively.
`The DSO for
`. h and the
`gastrin alone averaged 172 ? 26 pmol/kg
`addition
`of CR-1409 at doses of 1.0, 2.0, and 4.0
`pmol/kg
`h increased
`the DSO to respective values of
`380 + 32, 254 ? 27, and 480 + 57 pmol/kg
`. h.
`Gastrin infused
`intravenously
`in a constant dose of
`250 pmol/kg
`. h produced
`a pancreatic
`protein
`re-
`sponse similar
`to that occurring during
`the adminis-
`tration of CCK at a dose of 100 pmol/kg
`h in these
`animals. CR-1409 added
`to the intravenous
`infusion
`in graded doses
`resulted
`in a dose-dependent
`de-
`crease
`in both gastric acid and pancreatic
`protein
`secretion. At
`the highest
`dose of CR-1409
`(4.0
`. h) the gastric acid and pancreatic
`protein
`pmol/kg
`responses
`to gastrin were reduced by -60%
`(Figure
`4). After withdrawal
`of the
`infusion
`of CR-1409,
`there was an
`immediate
`increase
`in gastric and
`pancreatic
`secretion
`toward
`the control
`level. Plasma
`gastrin and PP levels
`in tests with intravenous
`infu-
`sion of gastrin heptadecapeptide
`are shown
`in Table
`3. Gastrin concentrations
`showed a gradual
`increase
`
`
`
`
`

`

`1018 KONTUREK ET AL.
`
`GASTROENTEROLOGY Vol. 94. No. 4
`
`GASTRIN IZ5O~molko-hl
`
`Table 4.
`
`in Pancreatic HC03 and Protein
`Increments
`Outputs
`in Response
`to Secretin and
`Neurotensin
`in Tests Without and With the
`Addition
`of CR-1409
`
`HCOB
`(pmol/30 min)
`
`Protein
`(mgi30 min)
`
`Secretin”
`Secretin” + CR-140Sb
`Neurotensin”
`Neurotensin” + CR-140Sb
`
`3668 2 720
`3248 ? 460
`828 2 104
`750 + 92
`
`210 2 34
`287 + 52
`772 + 82
`848 + 66
`
`t SEM of six tests in 6 dogs. a 82 pmoli
`Values shown are mean
`kg. h. b 1.0 pmol/kg . h. ’ 50 pmol/kg
`h.
`
`1
`
`5
`
`17 21
`13
`9
`15min PERIODS
`h) of
`pmol/kg
`Figure 4. Effects of graded
`doses
`(0.03-4.0
`CR-1409 on gastric acid and pancreatic
`protein
`re-
`sponses
`to a constant dose of gastrin (250 pmol/kg
`h).
`Mean -t SEM of six tests on 6 dogs. Asterisks
`indicate
`significant decrease below the control values obtained
`with gastrin alone.
`
`Table 3. Plasma Gastrin and Pancreatic Polypeptide
`Concentrations During Intravenous Infusion of
`Graded Doses of Gastrin in Tests Without and
`With the Addition
`of CR-1409
`
`Basal
`Gastrin
`31 pmol/kg
`125 pmol/kg
`500 pmol/kg
`2000 pmol/kg
`
`. h
`. h
`. h
`. h
`
`Gastrin
`(PM)
`26 + 3
`
`45 & 5O
`91 r lla
`231 ? 330
`685 rfr 74a
`
`CR-1409 (1.0 pmol/kg
`31 pmol/kg
`. h
`. h
`125 pmol/kg
`. h
`500 pmol/kg
`2000 pmol/kg
`. h
`
`CR-1409 (2.0 pmol/kg
`31 pmol/kg . h
`125 pmol/kg
`. h
`500 pmollkg
`. h
`2000 pmol/kg
`. h
`
`. h) + gastrin
`39 ? 4O
`112 5 24”
`552 * 48’
`852 2 140”
`. h) + gastrin
`38 t 4
`110 * 21
`358 f 87
`992 * 127
`
`CR-1409 (4.0 pmol/kg
`31 pmol/kg
`. h
`125 pmol/kg
`. h
`500 pmol/kg ’ h
`2000 pmol/kg . h
`
`. h) + gastrin
`31 +- 12
`121 2 26
`424 2 118
`792 f 180
`
`12 + 2
`18 2 2a
`23 2 5'
`27 k 4"
`
`10 + 1
`11 + 2
`14 2 2b
`20 f 4b
`
`12 2 3
`10 z!z 2
`14 f 2b
`18 f gb
`
`9+3
`18 + 5
`21 + 3
`20 + 2b
`
`the infusion of graded doses of gastrin, and
`during
`these
`increments were not significantly
`different
`in
`tests without and with CR-1409. Plasma PP levels,
`which showed a small but significant
`increase dur-
`ing infusion of higher doses of gastrin, were signifi-
`cantly reduced by the addition of CR-1409.
`(50
`Secretin
`(82 pmol/kg
`. h) and neurotensin
`h) infusion produced
`a potent stimulation
`pmoi/kg
`of pancreatic HC03 secretion.
`The
`increment
`in
`protein output was negligible
`in tests with secretin
`and somewhat
`higher
`in
`tests with neurotensin.
`CR-1409 given intravenously
`did not affect HCO, or
`protein
`responses
`to secretin or neurotensin
`(Table
`4).
`in graded doses
`intravenously
`infused
`Urecholine
`resulted
`in a dose-dependent
`increase
`in pancreatic
`protein secretion,
`reaching a peak that amounted
`to
`attained with CCK-8. There
`-42%
`of the maximum
`was also a small
`increase
`in gastric acid secretion,
`but
`it was not dose-dependent,
`and
`these
`results
`have not been included. CR-1409 added
`to the intra-
`venous
`infusion
`(1.0 pmol/kg
`. h) did not affect sig-
`nificantly
`the gastric acid or pancreatic
`protein
`se-
`cretion
`(Figure 5).
`
`1.0
`012 025 a5
`M6
`URECHOLINE lumollkg-h)
`
`PP, pancreatic polypeptide. Values shown are mean 2 SEM of six
`tests in 6 dogs. ’ Significant
`(p < 0.05) increase above basal value.
`b Significant
`(p < 0.05) decrease below the values obtained with
`respective dose of gastrin alone.
`
`to
`in response
`in pancreatic protein outputs
`Figure 5. Increments
`graded doses of urecholine
`(0.061.0 pmolikg
`h) with-
`out and with CR-1409 (1.0 pmol/kg
`h). Mean t SEM of
`six tests on 6 dogs.
`
`
`
`
`

`

`April 1988
`
`CCK AND PANCREATIC SECRETION
`
`1019
`
`SHAM-FEEDING
`Y
`
`1
`
`7
`5
`3
`15 min PERIODS
`
`9
`
`11
`
`Figure 6.
`
`under basal
`secretion
`protein
`Gastric H+ and pancreatic
`conditions
`and
`after
`IS-min
`sham
`feeding
`in
`tests
`(1.0 pmol/kg h). Mean 2
`and with CR-1409
`without
`SEM of six tests on 6 dogs. Asterisks
`indicate
`signifi-
`cant decrease
`versus
`results obtained
`in tests with sham
`feeding
`alone.
`
`on Pancreatic Responses
`of CR-1409
`Effects
`to Sham Feeding, Gastrointestinal Meal, and
`Duodenal Perfusion with HCI, Oleate, or
`Amino Acids
`
`a 15-min
`carried out during
`feeding
`Sham
`resulted
`in almost
`immediate
`increase
`in
`period
`gastric acid secretion,
`reaching peaks amounting
`to
`-95% of gastrin maximum
`and then slowly declin-
`ing toward
`the basal values
`(Figure 6). CR-1409
`(1.0
`h) given before, during,
`and after sham
`pmol/kg
`feeding
`reduced
`only
`the
`initial 30-min peak acid
`response
`(by --200X,), without affecting
`the remaining
`acid secretory
`rate. Pancreatic
`protein
`response also
`peaked
`immediately
`after sham
`feeding,
`reaching
`of the CCK maximum;
`it then declined within
`-27%
`1 h to the basal level. CR-1409
`caused a small and
`insignificant
`decline
`in protein output only during
`the
`initial period of sham
`feeding. Plasma gastrin
`and PP concentrations
`were significantly
`elevated
`51, but
`the addition
`of
`after sham
`feeding
`(Table
`did not alter these hormonal
`responses.
`CR-1409
`in
`The gastrointestinal meal evoked an increase
`pancreatic
`secretion of both HC03 and protein
`(Fig-
`ure i’), reaching
`respective
`peaks
`in the second and
`third 15min period after feeding. Both parameters of
`pancreatic
`secretion
`tended
`to decline during the 3-h
`(0.5 pmol/kg
`. h) given
`postprandial
`period. CR-1409
`before, during, and after feeding resulted
`in a marked
`reduction
`in protein output
`ranging
`from
`in
`-70%
`the first postprandial
`hour to 40%
`in the third hour.
`The HC03
`response was significantly
`reduced
`by
`
`
`
`
`

`

`1020 KONTURRK ET AL.
`
`GASTROENTEROLOGY
`
`Vol. 94, No. 4
`
`in the second and third postpran-
`CR-1409 by -30%
`dial hour only. A larger dose
`(1.0 pmollkg
`h) of
`CR-1409 did not cause any stronger
`inhibition
`in the
`postprandial HC03 or protein secretion.
`a marked
`Plasma gastrin concentration
`showed
`rise after feeding, and CR-1409 did not affect these
`levels significantly
`(Table 5). Pancreatic polypeptide
`also showed
`a marked
`increment
`that was well
`sustained
`throughout
`the postprandial
`period. CR-
`1409 reduced
`the PP increment by -20%
`throughout
`the postprandial
`period.
`of 100 mM HCl or 100 mM
`Duodenal
`instillation
`sodium
`oleate produced
`pancreatic
`secretion
`of
`HC03
`similar
`to
`that obtained with
`exogenous
`secretin
`and neurotensin,
`respectively
`(Figure 8).
`Protein outputs with duodenal HCl or oleate were
`significantly
`higher
`than
`in respective
`tests with
`doses of secretin and neurotensin
`producing
`equal
`rates of HC03
`secretion.
`These outputs
`reached
`-21% and 68% of the maximum obtained with CCK.
`. h) infused before and during
`CR-1409 (1.0 pmol/kg
`duodenal
`perfusion with HCl or oleate caused
`a
`significant decrease
`in protein output. HC03 output
`was reduced
`in tests with oleate, but not with duo-
`denal acid. Duodenal
`instillation
`of phenylalanine
`and
`tryptophan
`increased
`the pancreatic
`protein
`outputs
`(but not HC03 secretion)
`to -31%
`of the
`maximum
`induced
`by CCK, and
`this was almost
`completely
`abolished by CR-1409 (1.0 pmol/kg
`h).
`
`MEATFEEDING
`
`1
`
`3
`
`7
`5
`15mln PERIODS
`
`9
`
`11
`
`13
`
`Figure
`
`7.
`
`to a
`in response
`outputs
`HCO, and protein
`Pancreatic
`and with CR-1409
`gastrointestinal
`meal
`in tests without
`? SEM of six tests on 6 dogs.
`(1.0 pmol/kg
`h). Mean
`Asterisks
`indicate
`significant
`decrease
`below
`the con-
`trol values obtained with meal alone
`
`Cl
`
`WOHCI WOOLEATE WO PHE+TRP
`18mmoVh) i8mmollhl
`l8mmolfhl
`
`Figure
`
`to
`in response
`secretion
`HCO, and protein
`8. Pancreatic
`of HCl, oleate. or
`(DUO) perfusion
`(8 mmolih)
`duodenal
`amino
`acids
`(PHE + TRP)
`in tests without
`and with
`CR-1409 (hatched
`columns). Mean + SEM of six tests
`on 6 dogs. Asterisks
`indicate
`significant
`decrease
`below
`the values obtained
`in control
`tests.
`
`Discussion
`
`that CCK plays
`This study provides evidence
`of pancreatic
`an important
`role
`in the stimulation
`protein
`secretion
`in response
`to a gastrointestinal
`meal and to intestinal
`perfusion with amino acids
`and oleate, but not
`to basal or vagal-cholinergic
`stimulation.
`(14~5) used in
`studies with proglumide
`Previous
`dogs
`a massive dose (300 mg/kg . h) in conscious
`showed
`that it was also an effective
`inhibitor of the
`pancreatic
`responses
`to CCK-8 and
`to duodenal
`perfusion
`with
`amino
`acids
`and
`fat, but not
`bethanechol
`(16). The action of proglumide was
`specific for CCK, but its widespread
`usefulness was
`limited because of its very
`low potency. Recently,
`Makovec et al. (9,lO) synthesized
`a series of proglu-
`mide derivatives by systematically
`altering
`the struc-
`ture of the dialkyl amide and by additions
`to the
`benzoyl moiety. Some of them were found to be 1000
`times more potent
`than proglumide
`in inhibiting
`CCK-induced
`contractions
`of
`isolated
`guinea pig
`gallbladder muscle strips and inhibiting
`the binding
`of lz51-CCK to rat pancreatic
`acini
`(9,lO). Further
`studies confirmed
`that for some of these proglumide
`analogues
`there was a significant
`correlation
`be-
`tween
`their ability
`to inhibit CCK-stimulated
`amy-
`lase release and gallbladder
`contractions
`(I I).
`In this study we used
`the proglumide
`derivative
`named CR-1409 (lo), compound
`53 (9), proglumide
`analogue 10 (ll), or compound B (12), which was
`found in in vitro studies
`to be one of the most potent
`CCK-receptor
`antagonists, with anti-CCK
`activity
`>lOOO times higher
`than
`its chemical
`prototype
`(9-12). This analogue was found
`to cause a parallel
`rightward
`shift in the amylase dose-response
`curves
`
`
`
`
`

`

`April 1988
`
`CCK AND PANCREATIC SECRETION
`
`1021
`
`in
`increase
`also the
`to CCK-8 (11,12). It inhibited
`that inter-
`amylase
`release caused by secretagogues
`and gas-
`act with CCK receptors,
`such as caerulein
`to other
`trin, but did not alter the amylase
`response
`secretagogues
`that do not interact with CCK recep-
`tors, such as carbachol or secretin
`(ll,l2).
`CR-1409
`was employed
`in this study
`for the first
`time
`in
`conscious
`dogs to determine
`the role of CCK in the
`pancreatic
`responses
`to a variety of exogenous
`and
`endogenous
`stimulants.
`The analysis of the dose-
`response
`functions
`of CCK-8 without
`and with
`CR-1409 in conscious
`dogs showed
`that the antago-
`nism of CCK results
`in the dose-dependent
`suppres-
`sion of the action of CCK-8 on pancreatic
`protein
`secretion. Partial
`reduction
`by CR-1409 of the pro-
`tein response
`to CCK-8 was achieved at a dose of 0.5
`pmol/kg
`h, and complete
`suppression
`of this re-
`sponse was obtained at a dose of 2.0 pmolikg
`h. As
`CR-1409 reduced
`the maximal protein
`response
`to
`CCK in a dose-dependent
`fashion but did not reduce
`it is evident
`that
`in vivo, unlike
`in vitro
`the I&,
`(11,121, CR-1409
`functions
`as a noncompetitive
`CCK-receptor
`antagonist. The reason for the discrep-
`ancy
`in the inhibitory
`action of CR-1409 on CCK-
`stimulated
`pancreatic
`secretion
`in vivo and in vitro
`is not apparent, but it is certain
`that the pancreatic
`response
`to CCK-8 at a dose (100 pmolikg
`h) pro-
`ducing a similar
`rate of protein output
`to that ob-
`served after a meat meal can be completely
`pre-
`vented or blocked by CR-1409. By comparison with
`proglumide, which was used by Stubbs and Stabile
`(16) in an experimental
`protocol
`similar
`to ours,
`CR-1409
`is roughly 1200 times more potent an in-
`hibitor of CCK-induced pancreatic protein secretion.
`This remains
`in good agreement with in vitro studies
`on dispersed pancreatic
`acini from guinea pigs, rats,
`and mice showing
`that our CCK antagonist was 4-5
`times more potent
`(11) than asperleucin
`(17), a
`nonpeptide
`receptor
`antagonist,
`and about 1300
`times more potent
`than proglumide
`(11).
`The crucial question
`raised by the use of CCK
`antagonists
`is whether
`these agents also
`interfere
`with
`the effects of gastrin on gastric acid and pan-
`creatic secretion. Gastrin and CCK share a common
`COOH-terminal
`pentapeptide
`amide;
`therefore, CCK
`agonists
`and antagonists
`should
`affect
`in similar
`fashion
`the secretory
`activity of both oxyntic
`cells
`and acinar cells. Indeed,
`in vitro studies showed
`that
`CCK and gastrin are equipotent
`stimulants
`of acid
`production
`in canine oxyntic cells (18) and exhibit
`similar efficacy
`in the stimulation
`of amylase release
`from canine pancreatic
`acini (19). However,
`in vivo
`studies demonstrated
`that CCK behaves as a partial
`agonist of gastric acid secretion
`(20), and gastrin
`behaves
`as a partial agonist of pancreatic
`protein
`secretion
`(21). According
`to our results, gastrin stim-
`
`in conscious dogs
`ulates pancreatic protein secretion
`to -60% of the maximum
`caused by CCK, and the
`addition
`of a CCK antagonist
`causes only partial
`inhibition
`of gastrin-induced