of Gila monster
`Actions
`from guinea pig pancreas
`
`venom on dispersed acini
`
`R. T, JENSEN,
`J.-P. RAUFMAN,
`AND
`J. D. GARDNER
`Institute of Arthritis,
`and Digestive
`Diabetes,
`Digestiue Diseases Branch, National
`and Kidney Diseases, National
`Institutes
`of Health, Bethesda, Maryland
`20205
`
`V. E. SUTLIFF,
`
`J. J. PISANO,
`
`RATJFMAN, J.-P.,R. T. JENSEN, V. ELSUTLIFFJ. J. PISANO,
`AND J, D. GARDNER. Actions of Gila monster
`venom on dis-
`persed acini
`from guinea
`pig pancreas.
`Am. J. Physiol.
`242
`(Gastrointest.
`Liver Physiol.
`5): G470-G474,
`1982.--Venom
`from Gila monster
`(family Helodermatidae)
`contains
`a pan-
`creatic
`secretagogue.
`In dispersed
`acini
`from guinea pig pan-
`creas, the venom
`increased
`enzyme secretion
`to the same extent
`as did vasoactive
`intestinal
`peptide,
`secretin,
`or PHI. The
`abilities
`of vasoactive
`intestinal
`peptide
`and Gila monster
`venom
`to stimulate
`enzyme secretion were not altered by boil-
`ing but were abolished
`by incubation
`with
`trypsin
`or chymo-
`trypsin, Like vasoactive
`intestinal
`peptide,
`secretin, and PHI,
`the venom caused a 50- to W-fold
`increase
`in cellular CAMP
`and inhibited
`binding
`of ““I-vasoactive
`intestinal
`peptide
`to its
`membrane
`receptors
`on pancreatic
`acini. The action of venom
`on enzyme secretion was inhibited
`by [Gln!‘]secretin-(5-27),
`a
`vasoactive
`intestinal
`peptide
`receptor
`antagonist,
`but was not
`altered
`by atropine,
`a cholinergic
`receptor
`antagonist,
`or by
`dibutyryl
`cGMP,
`a cholecystokinin
`receptor
`antagonist.
`Gila
`monster venom contained
`no immunoreactive
`vasoactive
`intes-
`tinal peptide by radioimmunoassay,
`These
`results
`indicate
`that
`venom
`from Gila monster
`contains a peptide
`that can stimulate
`pancreatic
`enzyme
`secretion
`by
`interacting
`with
`vasoactive
`intestinal
`peptide
`receptors
`on pancreatic
`acinar
`cells and
`thereby
`activating
`adenylate
`cyclase and
`increasing
`cellular
`CAMP.
`
`pancreatic
`intestinal
`
`acini; enzyme secretion; CAMP;
`peptide;
`secretin
`
`receptors;
`
`vasoactive
`
`there has been much interest in
`OVER THE PAST DECADE,
`bioactive peptides of nonmammalian origin (for review,
`see Ref. 1). Amphibian skin, for example, has proven to
`be a rich source of secretagogues such as caerulein,
`bombesin, and physalaemin, which probably represent
`genetic ancestors of bioactive agents found in mammals
`(cholecystokinin,
`gastrin-releasing peptide, and sub-
`stance P, respectively). Reports of pancreatitis after a
`scorpion bite (16, 20) have led some investigators
`to
`examine scorpion venoms for pancreatic secretagogues
`(18). A recent report (3) has shown that venom from the
`scorpion Tityus serrulatus increases pancreatic enzyme
`secretion indirectly by stimulating neural release of ace-
`tylcholine.
`In the present study, we undertook a systematic ex-
`amination of the effects of venoms from various arthro-
`pods and reptiles on dispersed acini from guinea pig
`pancreas. Venom
`from Gila monster (family Heloder-
`
`matidae) was found to increase pancreatic enzyme secre-
`tion by interacting with vasoactive
`intestinal peptide
`(VIP)
`receptors
`to activate adenylate cyclase and in-
`crease cellular CAMP.
`
`MATERIALS
`AND METHODS
`Materials. Male guinea pigs (150-200 g) were obtained
`from the Small Animals Section, Veterinary Resources
`Branch, National
`Institutes of Health, Bethesda, MD.
`N-2 - hydroxyethylpiperazine
`- N’-2-ethanesulfonic
`acid
`(HEPES) and normal rabbit serum were purchased from
`Calbiochem, La Jolla, CA; Gila monster venom (Helo-
`derma horridurn or Heloderma suspecturn), carbamyl-
`choline (carbachol) , atropine sulfate, soybean trypsin
`inhibitor, B-bromo-CAMP
`(B&-CAMP), N’,O’-dibutyryl
`cGMP (Bt,cGMP),
`carbonyl cyanide m-chlorophenylhy-
`drazone
`(CCCP),
`trypsin, and a-chymotrypsin
`from
`Sigma Chemical St. Louis, MO; basal medium (Eagle)
`times concentrated)
`from Grand Island
`amino acids (100
`Biological, Grand
`Island, NY; Drabkin’s reagent from
`Fisher Scientific, Pittsburgh, PA; essential vitamin mix-
`ture (100 times concentrated)
`from Microbiological As-
`sociates, Bethesda, MD; glutamine from Research Plus
`Laboratories, Denville, NJ; bovine plasma albumin (frac-
`tion V)
`from Armour Pharmaceutical, Phoenix, AZ;
`Phadebas amylase test from Pharmacia Diagnostics, Pis-
`cataway, NJ; purified collagenase (Clostridium histoly-
`ticum
`type CLSPA)
`from Worthington Biochemical,
`Freehold, NJ; [“Hlaminobutyric
`acid, 45Ca, CAMP Y-O-
`succinyl[ ““Iliodotyrosine methyl ester, CAMP antiserum
`(preconjugated to a second antibody), and Aquasol from
`New England Nuclear, Boston, MA; and synthetic VIP
`and synthetic bombesin from Peninsula Laboratories,
`Belmont, CA. Natural porcine VIP, natural porcine se-
`cretin, and natural porcine PHI1 were gifts from Dr.
`Viktor Mutt, Gastrointestinal Hormone Research Unit,
`Karolinska
`Institute, Stockholm, Sweden. Synthetic C-
`terminal octapeptide of cholecystokinin
`(CCK-8) was a
`gift from Dr. Miguel Ondetti, Squibb Institute
`for Medi-
`cal Research, Princeton, NJ. A23187 was a gift from Dr.
`Robert Hamill, Eli Lilly, Indianapolis, IN. [Gln”]secretin-
`(5-27) was a gift
`from Dr. Miklos Bodanszky, Case
`Western Reserve University, Cleveland, OH.
`
`’ PHI
`histidine
`
`(P) having amino-terminal
`for the peptide
`is the acronym
`(H) and carboxyl-terminal
`isoleucine
`(I) amide.
`
`SANOFI-AVENTIS Exhibit 1019 - Page G470
`
`IPR for Patent No. 8,951,962
`
`

`
`GILA
`
`MONSTER
`
`VENOM
`
`so-
`incubation
`the standard
`stated otherwise,
`Unless
`lution contained 24.5 mM HEPES
`(pH 7.4), 98 mM NaCl,
`6 mM KCl, 2.5 mM NaHgP04,
`5 mM Na pyruvate,
`5 mM
`Na fumarate,
`5 mM Na glutamate,
`11.5 mM glucose, O-5
`mM CaClz, 1.0 mM MgClz, 2 mM glutamine,
`5 mM
`theophylline,
`1% (wt/vol)
`albumin, 0.01% (wt/vol)
`tryp-
`sin inhibitor,
`1% (vol/vol)
`essential amino acid mixture,
`and 1% (vol/vol)
`essential
`vitamin mixture.
`The pH of
`the incubation
`solution was 7.4, and all incubations were
`performed with 100% oxygen as the gas phase.
`Tissue preparation.
`A guinea pig was killed by a blow
`to the head. The pancreas was
`removed and trimmed
`of
`fat and mesentery.
`Dispersed
`acini were
`then prepared
`using purified
`collagenase
`and mild shearing
`forces as
`described previously
`(15).
`from pancreatic
`Amylase
`release. Release of amylase
`acini was measured as described previously
`(6, 15). Acini
`from
`the pancreas
`of one animal were suspended
`in 150
`ml of standard
`incubation
`solution.
`Incubations
`con-
`tained 1. ml of cell suspension
`and were
`for 30 min at
`37°C. Amylase
`release was calculated
`as the percentage
`of the amylase activity
`in the acini at the beginning
`of
`the
`incubation
`that was
`released
`into
`the extracellular
`medium during
`the incubation.
`by radioim-
`CAMP. Cellular CAMP was determined
`and Brooker
`munoassay
`using
`the procedure
`of Harper
`the pancreas
`(B), as described
`previously
`(7). Acini
`from
`of one animal were
`suspended
`in 50 ml of standard
`incubation
`solution, and then a portion of the suspension
`was diluted with 8 vol of standard
`incubation
`solution.
`Incubations
`contained 0.5 ml of cell suspension
`and were
`for 30 min at 37°C. Basal values
`for cellular CAMP were
`determined
`using
`the undiluted
`cell suspension;
`values
`from acini
`incubated
`with Gila monster
`venom, VIP,
`PHI, or secretin were determined
`using
`the diluted
`cell
`suspension.
`In acini incubated with Gila monster
`venom,
`VIP, PHI, or secretin, CAMP became maximal by 20 min
`and remained
`constant
`for
`the subsequent
`40 min
`(not
`shown) q
`Binding of “‘I- VIP.
`was pre-
`(830 Ci/mmol)
`““I-VIP
`of Hunter
`pared using a modification
`of the procedure
`and Greenwood
`(10) published
`previously
`(2). Binding of
`“:‘I-VIP
`to dispersed pancreatic
`acini was measured using
`the procedure
`also published previously
`(2, 7). Acini
`from
`the pancreases
`of two animals were suspended
`in 25 ml
`of standard
`incubation
`solution.
`Incubations
`contained
`1.0 ml of cell suspension and 50 pM ‘“‘I-VIP and were for
`30 min at 37°C. At 37°C binding of ““I-VIP
`became
`maximal by 10 min and remained constant for the sub-
`sequent 30 min (2, 7). Nonsaturable binding of ““I-VIP
`was measured as the amount of radioactivity associated
`with the acini when the incubation contained 50 pM ““I-
`VIP plus 1 PM VIP (2, 7). All values in the present paper
`are for saturable binding, i.e., binding measured with ““I-
`VIP alone (total binding) minus binding measured with
`1 PM VIP
`(nonsaturable binding).
`In all experiments,
`nonsaturable binding was less than 25% of the total
`binding.
`OutfZux of 4q5Ca. Outflux of 4’r)Ca from pancreatic acini
`was measured using the procedure described previously
`(14). Acini
`from the pancreas of one animal were sus-
`pended in 15 ml of standard
`incubation solution and
`
`G471
`
`labeled with 4r’Ca during a 60-min incubation at 37°C.
`The acini were washed with standard incubation solution
`to remove extracellular radioactivity and resuspended in
`40 ml of standard incubation solution. Samples (1.0 ml)
`were incubated
`for 5 min at 37”C, and outflux of 45Ca
`was measured as the percentage of radioactivity
`in the
`acini at the beginning of the 5-min incubation
`that was
`released into the extracellular medium during the incu-
`bation.
`Immunoreactive VIP was
`radioimmunoassay.
`V.P
`kindly determined by Dr. T. O’Dorisio, Department of
`Internal Medicine, Ohio State University School of Med-
`icine, Columbus, OH, This assay can detect VIP at con-
`centrations as low as 5 pg/ml and does not cross-react
`with up to 10 rig/ml of calcitonin, CCK, secretin, gastric
`inhibitory
`peptide, glucagon, gastrin, or bovine pan-
`creatic polypeptide
`(13).
`
`RESULTS
`
`increase in
`Gila monster venom caused a significant
`amylase secretion, and theophylline
`increased the effi-
`cacy of the venom but not its potency
`(Fig. 1). Gila
`monster venom also caused a significant increase in cel-
`lular CAMP, and this increase was augmented by theoph-
`ylline
`(Table 1). These effects of theophylline on the
`actions of Gila monster venom are similar to those ob-
`tained previously with VIP and secretin (12). Because
`theophylline augmented
`the actions of Gila monster
`venom on amylase secretion and CAMP, all subsequent
`experiments were performed with
`incubation solution
`containing 5 mM theophylline.
`The increase in amylase secretion caused by a maxi-
`mally effective concentration of Gila monster venom was
`
`’
`I
`’
`’
`1
`Ll
`-2
`2
`1
`0
`-1
`-3
`GILA MONSTER VENOM
`(log
`pg/ml)
`on dose-response
`theophylline
`of
`1. Effect
`FIG.
`release
`from
`on amylase
`Gila monster
`venom
`agents
`indicated
`acini. Acini were
`incubated
`with
`each experiment,
`each
`value was determined
`given
`are means
`from
`3 separate
`experiments.
`I SD.
`
`of
`
`for action
`curve
`pancreatic
`dispersed
`In
`for 30 min at 37°C.
`in duplicate,
`and
`results
`bars
`represent
`Vertical
`
`SANOFI-AVENTIS Exhibit 1019 - Page G471
`
`IPR for Patent No. 8,951,962
`
`

`
`G472
`
`concen-
`effective
`the same as that caused by a maximally
`tration
`of VIP, PHI,
`or secretin
`(Fig. 2). Significant
`stimulation
`of amylase secretion
`occurred with 10 rig/ml
`Gila monster
`venom, and stimulation
`was half maximal
`with 100 rig/ml and maximal with 750 rig/ml
`(Fig. 2). As
`has been reported
`previously
`for VIP, PHI, and secretin
`the rate of amylase secretion stimulated by Gila
`(15),
`monster venom remained constant for at least 60 min
`(not shown).
`Boiling (100°C for 30 min) did not alter the ability of
`VIP or Gila monster venom to stimulate amylase secre-
`tion, whereas incubating these secretagogues with trypsin
`or chymotrypsin
`followed by boiling abolished their abil-
`ities to stimulate enzyme secretion (Table 2).
`Because some venoms contain cytolytic
`substances
`(19), we examined the possibility
`that
`the release of
`amylase caused by Gila monster reflects lysis of acinar
`cells instead of secretion. Incubating acini with 0.1 mM
`CCCP, an uncoupler of oxidative phosphorylation
`(9),
`abolished the increase in amylase release caused by Gila
`monster venom, as well as that caused by carbachol or
`VIP (not shown). When pancreatic acini were preloaded
`with a nonmetabolizable amino acid, [“Hlaminobutyric
`acid, and then washed to remove extracellular
`radioac-
`tivity, Gila monster venom failed to accelerate the release
`of cellular radioactivity
`during a 60-min incubation at
`37OC (not shown). Gila monster venom also failed to
`
`1. Effect of Gila monster uenum alone
`TABLE
`or in combination with theophylline on CAMP
`in dispersed pancreatic acini
`
`Additions
`
`CAMP
`
`(experimental/control)
`
`Alone
`
`+Theo,
`5mM
`
`None
`venom,
`Gila monster
`In each
`experiment,
`results
`given
`are means
`
`2.5 pg/ml
`each
`value
`k SD
`from
`
`1.0
`3.9
`t 3.4
`t 9.5
`62.7
`t 5.r
`28.7
`in duplicate,
`was determined
`and
`3 separate
`experiments.
`
`RAUFMAN
`
`ET AL.
`
`cause release of hemoglobin from intact human erythro-
`cytes during a 30-min incubation at 37OC (not shown).
`Bt,cGMP,
`a CCK receptor antagonist,
`inhibited
`the
`increase in amylase secretion caused by CCK-8 but not
`that caused by Gila monster venom, and atropine, a
`muscarinic cholinergic antagonist, inhibited the increase
`in amylase secretion caused by carbachol but not that
`caused by Gila monster venom (Table 3).
`Gila monster venom caused a 65-fold increase in cel-
`lular CAMP, and the increase caused by a maximally
`effective concentration of Gila monster venom was not
`significantly different
`from that caused by a maximally
`effective concentration of secretin, VIP, or PHI (Fig. 3).
`An increase in cellular CAMP could be detected with 10
`rig/ml of Gila monster venom, and maximal stimulation
`occurred with 2.5 pg/ml (Fig. 3). The configuration of the
`dose-response curve for the action of Gila monster venom
`on cellular CAMP
`resembled that
`for PHI
`in that a
`relatively small increase (5-fold) occurred with concen-
`trations of Gila monster venom up to 100 rig/ml, and
`concentrations above 100 rig/ml caused an additional 60-
`fold increase in CAMP (Fig. 3) l
`Like other pancreatic secretagogues that increase cel-
`lular CAMP (4), Gila monster venom did not alter outflux
`of 4”Ca from pancreatic acini and did not alter the in-
`
`2, Effect of boiling, trypsin, and chymotrypsin
`TABLE
`OIZ ability of VIP and Gila monster venom
`to stimulate amylase secretion from pancreatic acini
`
`Secretagogue
`
`Alone
`
`for
`100°C
`30 min
`
`+
`Trypsin
`for
`100°C
`30 min
`
`Chymo-
`+
`trypsin
`for
`100°C
`30 min
`
`12.0 k 3.1
`12.6 k 2.1
`
`3.1 t 0.4
`3.3
`t 0.7
`
`t 0.6
`3.6
`3.3 +- 0.5
`
`venom,
`
`t 2.8
`12.1
`12.1 k 2.2
`
`0.3 nM
`VIP,
`Gila monster
`0.8
`lug/ml
`and
`in duplicate,
`was determined
`value
`each
`experiment,
`In each
`Stock
`experiments.
`-t- SD
`from
`3 separate
`results
`given
`are means
`solutions
`of vasoactive
`intestinal
`peptide
`(VIP,
`300 nM)
`or Gila monster
`venom
`(100 pg/ml)
`were divided
`into 4 equal
`portions.
`One portion
`was
`incubated
`for 150 min at 37°C. A second
`portion
`was
`incubated
`for 120
`min
`at 37°C
`and
`then
`for 30 min
`at 100°C.
`The
`remaining
`portions
`were
`incubated
`for 120 min with
`trypsin
`(192
`rig/ml)
`or chymotrypsin
`(19
`rig/ml)
`and
`then
`for
`30 min
`at 100°C.
`Samples
`(10 ~1) of each
`portion
`were
`then
`assayed
`for
`their
`ability
`to stimulate
`amylase
`secre-
`tion
`from
`pancreatic
`acini
`during
`a 30-min
`incubation
`at 37°C.
`The
`value
`of amylase
`secretion
`from
`acini
`incubated
`with
`no additions
`was
`3.3 t, 0.5.
`
`.eti n
`
`CONCENTRATION
`(log pgiml)
`(log M)
`secretin,
`PHI,
`peptide
`(VIP),
`intestinal
`of vasoactive
`venom
`on amylase
`release
`from
`dispersed
`pancreatic
`In
`incubated
`with
`agents
`indicated
`for 30 min at 37°C.
`each
`value was determined
`in duplicate,
`and
`results
`from
`4 separate
`experiments.
`Verticd
`bars
`represent
`
`2. Effect
`FIG.
`and Gila monster
`acini. Acini were
`each experiment,
`given
`are means
`1 SD.
`
`3. Effect of atropine and Bt,cGMP
`TABLE
`on increase in amylase secretion caused
`by Gila monster venom, carbachol, or CCK-8
`
`Secretagogue
`
`None
`Gila monster
`Carbachol,
`CCK-8,
`
`venom,
`30 FM
`0.3 nM
`
`2.5 pg/ml
`
`Amylase
`
`Release,
`
`Xtotal
`
`Alone
`
`+Atropine,
`1PM
`
`+Bt,cGMP,
`1mM
`
`4.3
`-t 1.5
`-+ 4.4
`22.2
`28.9 k 6.5
`30.6
`-i 5.7
`
`4.7
`1.1
`t
`t 4.9
`21.9
`4.6 k 1.3
`29.6 k 5.7
`
`6.1 -t- 2.1
`21.1
`* 3.9
`28.3 k 2.3
`6.3 k 2.3
`
`In each experiment,
`results
`given
`are means
`acini were
`incubated
`&cGMP,
`dibutyryl
`cystokinin.
`
`and
`in duplicate,
`was determined
`value
`each
`3 separate
`experiments.
`Dispersed
`from
`k SD
`with
`the agents
`indicated
`for 30 min
`at 37OC.
`cGMP;
`CCK-8,
`C-terminal
`octapeptide
`of chole-
`
`SANOFI-AVENTIS Exhibit 1019 - Page G472
`
`IPR for Patent No. 8,951,962
`
`

`
`GILA
`
`MONSTER
`
`VENOM
`
`(not
`
`in outflux
`
`caused by CCK-8
`
`or carbachol
`
`crease
`shown).
`to
`venom
`of Gila monster
`the ability
`To examine
`on pancreatic
`acini, we meas-
`interact with VIP receptors
`ured
`the ability of the venom
`to inhibit binding of lZ51-
`VIP. Gila monster
`venom,
`like VIP, PHI, and secretin,
`inhibited binding of ‘251-VIP to dispersed pancreatic
`acini
`(Fig. 4) l Significant,
`inhibition
`of binding occurred with
`75 rig/ml Gila monster
`venom, and bindine: was abolished
`
`G473
`
`by 75 lug/ml (Fig. 4). Although Gila monster venom was
`able to inhibit binding of 12”1-VIP, the venom at a con-
`centration of 1. mg/ml contained no immunoreactive VIP
`by radioimmunoassay on three separate determinations.
`In each determination,
`the venom was tested undiluted
`and at dilutions from 15 to 120,000.
`fragments
`Previously, we have shown that C-terminal
`of secretin, such as secretin-(5-27),
`can antagonize the
`interaction of secretin and VIP with their receptors on
`pancreatic acini and by so doing inhibit
`the effects of
`these secretagogues on CAMP and amylase secretion (5,
`7). In the present study, [ Glng]secretin- (5-27)
`inhibited
`the stimulation of amylase secretion caused by Gila mon-
`ster venom, and the potency with which the secretin
`fragment inhibited the action of Gila monster venom was
`the same as that with which the fragment inhibited
`the
`stimulation of amylase secretion caused by VIP or secre-
`tin (Fig. 5).
`
`IP
`
`-5
`
`-11
`
`3
`2
`1
`0
`-1
`-2
`-3
`-6
`-7
`-8
`-9
`-10
`GILA MONSTER VENOM
`CONCENTRATION
`(log pg/ml)
`(log M)
`PHI,
`(VIP),
`intestinal
`peptide
`vasoactive
`of secretin,
`FIG. 3. Effect
`acini. Acini
`venom
`on CAMP
`in dispersed
`pancreatic
`and Gila monster
`with
`agents
`indicated
`for
`30 min
`at 37°C.
`In each
`were
`incubated
`experiment,
`each value was determined
`in duplicate,
`and
`results
`given
`are means
`from
`6 separate
`experiments.
`VerticaZ
`bars
`represent
`1 SD.
`In some
`instances,
`vertical
`bars bave
`been
`omitted
`to enhance
`visual
`clarity.
`
`-7
`
`-6
`
`-5
`
`-7
`
`-5
`
`-6
`
`-5
`
`-7
`-6
`(log MI
`SECRETIN,-,,
`[Gin’]
`re-
`in amylase
`on
`increase
`[Gln”]secretin-(5-27)
`of
`5. Effect
`FIG.
`secretin,
`or Gila
`peptide
`(VIP),
`caused
`by vasoactive
`intestinal
`lease
`with
`agents
`indicated
`for 30 min
`monster
`venom.
`Acini were
`incubated
`at 37°C
`Results
`are expressed
`as percentage
`of
`increase
`in amylase
`release
`caused
`by VIP
`(12.6 k 1.9), secretin
`(15.9 k 2.0), or Gila monster
`venom
`(13.2
`* 1.0) alone.
`In each experiment,
`each
`value was deter-
`mined
`in duplicate,
`and
`results
`given
`are means
`from
`3 separate
`experiments.
`VerticaZ
`bars
`represent
`1 SD.
`
`E 80
`
`CONCENTRATION
`(log pg/ml)
`(log M)
`secretin,
`(VIP),
`PHI,
`peptide
`intestinal
`of vasoactive
`pancreatic
`to dispersed
`‘““I-VIP
`venom
`on binding
`of
`In
`for 30 min at 37°C.
`indicated
`incubated
`with
`agents
`each
`value was determined
`in duplicate,
`and
`results
`from
`6 separate
`experiments.
`VerticaZ
`bars
`represent
`
`4. Effect
`FIG.
`and Gila monster
`acini. Acini were
`each experiment,
`given
`are means
`1 SD.
`
`4. Effect of various secretagogues alone
`TABLE
`or in combination with Gila monster venom
`on amylase release from dispersed pancreatic acini
`
`Secretagogue
`
`Amylase
`
`Release
`
`T&total
`
`Alone
`
`+ Gila monster
`2.5 pg/ml
`
`venom,
`
`8.4 t 0.1
`2.4 X!I 1.6
`None
`8.2 k 0.3
`8.2
`t 0.3
`10 nM
`VIP,
`8.0 k 0.4
`8.2 k 0.5
`Secretin,
`0.3 PM
`8.6 t 1.1
`7.9 k 0.9
`8Br-CAMP,
`I mM
`(18.2)
`22.0 AI 4.1
`12.0 * 1.6
`Bombesin,
`10 nM
`25.5
`t 5.3 (18.0)
`11.8
`t 1.6
`Carbachol,
`30 PM
`25.9 & 5*1 (20.1)
`13.9
`t 1.2
`CCK-8,
`0.3 nM
`16.3 k 3.3 (13.8)
`7.6 t 1.9
`A23187,
`10 j-&M
`and
`in duplicate,
`was determined
`value
`each
`In each
`experiment,
`3 separate
`experiments.
`Numbers
`from
`t SD
`results
`given
`are means
`calculated
`by adding
`the
`increase
`values
`in parentheses
`are additive
`amylase
`release
`caused
`by Gila monster
`venom
`alone
`to
`that
`obtained
`with
`the secretagogue
`alone, Dispersed
`acini were
`incubated
`with
`the
`agents
`indicated
`for 10 min at 37°C. VIP,
`vasoactive
`intestinal
`peptide;
`8Br-CAMP,
`8-bromo-CAMP;
`CCK-8,
`C-terminal
`octapeptide
`of chole-
`cystokinin.
`
`in
`
`SANOFI-AVENTIS Exhibit 1019 - Page G473
`
`IPR for Patent No. 8,951,962
`
`

`
`G474
`
`caused
`secretion
`in amylase
`increase
`the
`To examine
`secretagogues,
`we
`plus other
`venom
`by Gila monster
`with
`other
`secreta-
`in combination
`tested
`the venom
`gogues
`that
`increase
`cellular CAMP and with
`secreta-
`gogues
`that cause mobilization
`of cellular
`calcium, The
`increase
`in amylase
`secretion
`caused by a maximally
`effective
`concentration
`of Gila monster
`venom plus an-
`other secretagogue
`that
`increases or mimics
`the action of
`CAMP
`(VIP,
`secretin,
`or B&-CAMP)
`was
`the same as
`that caused by Gila monster
`venom alone
`(Table 4). In
`contrast,
`the increase
`in amylase
`release caused by Gila
`monster
`venom plus a secretagogue
`that mobilizes
`cel-
`lular calcium
`(bombesin,
`carbachol, A23187, or CCK-8)
`was significantly
`greater
`than
`the sum of the
`increase
`caused by each secretagogue
`acting alone (Table 4).
`
`DISCUSSION
`
`from Gila
`that venom
`indicate
`findings
`The present
`pancreatic
`monster
`contains
`a substance
`that
`increases
`enzyme
`secretion
`and
`that
`this action
`is mediated
`by
`CAMP. Gila monster
`venom
`increases CAMP as well as
`enzyme
`secretion,
`and both of these actions
`are aug-
`mented by theophylline.
`The venom also potentiates
`the
`increase
`in enzyme
`secretion
`caused by secretagogues
`that mobilize
`cellular
`calcium
`but does not alter
`the
`increase
`in enzyme
`secretion
`caused by other
`secreta-
`gogues that
`increase cellular CAMP. Gila monster
`venom
`does not
`increase 4”Ca outflux
`or alter
`the
`increase
`in
`outflux
`caused by other secretagogues
`and does not cause
`lysis or altered permeability
`of the acinar cell.
`In previous
`studies, we have shown
`that pancreatic
`acinar
`cells possess
`two
`classes of receptors,
`each of
`
`REFERENCES
`
`RAUFMAN
`
`ET AL.
`
`(2, 5, 7, 11,
`interact with VIP, PHI, and secretin
`which
`17). One class is “VIP-preferring”
`and has a high affinity
`for VIP and lower affinities
`for PHI and secretin
`(2, 5, 7,
`11, 17). The other class
`is “secretin-preferring”
`and has
`a high affinity
`for secretin
`and lower affinities
`for PHI
`and VIP
`(2, 5, 7, 11, 17). The
`increase
`in CAMP
`caused
`by occupation
`of the VIP-preferring
`receptors mediates
`the
`increase
`in enzyme
`secretion
`caused by VIP, PHI,
`and secretin
`(2, 5, 7, 11, 17). Occupation
`of the secretin-
`preferring
`receptors
`by secretin, PHI, or VIP causes a
`substantial
`increase
`in CAMP; however,
`what
`cellular
`function,
`if any,
`is altered by
`this
`increase
`in CAMP
`is
`not known
`(2, 11, 17). Our present
`results
`indicate
`that
`Gila monster
`venom,
`like VIP, PHI, and secretin,
`inter-
`acts with VIP-preferring
`receptors
`as well as secretin-
`preferring
`receptors;
`however,
`the apparent
`affinity
`of
`the substance
`in
`the venom
`for
`these
`two
`classes of
`receptors
`cannot be determined
`until
`the substance
`is
`isolated and its molecular weight
`is determined.
`Although we do not know
`the structure
`of the secre-
`tagogue
`in Gila monster
`venom, we
`find
`that
`the secre-
`tagogue
`is resistant
`to boiling and inactivated
`by trypsin
`or chymotrypsin,
`does not cross-react
`with VIP-specific
`antiserum,
`and has a higher affinity
`for VIP-preferring
`receptors
`(see Fig. 2) than
`for secretin-preferring
`recep-
`tors
`(see Fig. 3). These
`findings
`suggest
`that
`the secre-
`tagogue
`in Gila monster
`venom
`is a peptide
`that
`is
`structurally
`related
`to but distinct
`from VIP.
`
`O’Shaughnessy
`thank Mary
`We
`Address
`for
`reprint
`requests:
`Health,
`Building
`10, Room
`9D-15,
`
`for preparing
`J.-P. Raufman,
`Bethesda,
`
`MD
`
`the manuscript,.
`National
`Institutes
`20205.
`
`of
`
`Received
`
`8 October
`
`3981; accepted
`
`in final
`
`form
`
`I6 December
`
`1981.
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`in Trinidad.
`
`SANOFI-AVENTIS Exhibit 1019 - Page G474
`
`IPR for Patent No. 8,951,962