receptor
`gene-related peptide
`Calcitonin
`antagonist human CGRP-(8-37)
`
`YAMAGUCHI,
`AKINORI
`CHIBA,
`TSUTOMU
`TOMOYUKI
`MORISHITA,
`AKIRA
`NAKAMURA,
`TOSHIHARU
`NODA,
`AND
`MASAAKI
`FUKASE,
`Third Department
`of Internal Medicine, Kobe University
`Kobe 650; and Toyo Jozo Research Laboratory,
`Shixuoka
`
`YAMATANI,
`TOSHIYUKI
`INUI,
`TETSUYA
`FUJITA
`TAKUO
`School of Medicine,
`632-1, Japan
`
`YA-
`TOSHIYUKI
`YAMAGUCHI,
`AKINORI
`TSUTOMU,
`CHIBA,
`TOMOYUKI
`MORISHITA,
`TETSUYA
`AKIRA
`NAKAMURA,
`MATANI,
`Fu-
`TOSHIHARU
`NODA,
`AND
`TAKUO
`MASAAKI
`FUKASE,
`INUI,
`JITA. Calcitonin
`gene-related
`peptide
`receptor antagonist
`human
`Metab. 19):
`CGRP-(8-37).
`Am. J. Physiol.
`256
`(Endocrinol.
`E331-E335,
`1989.-From
`this
`study, we predicted
`that
`the
`human
`calcitonin
`gene-related
`peptide
`(hCGRP)
`fragment
`hCGRP-(8-37)
`would
`be a selective
`antagonist
`for CGRP
`receptors
`but an agonist
`for calcitonin
`(CT)
`receptors.
`In rat
`liver plasma membrane,
`where CGRP
`receptors
`predominate
`and CT appears
`to act through
`these
`receptors,
`hCGRP-(8--
`37) dose dependently
`displaced
`1251- [ Tyr’]
`rat CGRP
`binding.
`However,
`hCGRP-(8-37)
`had no effect on adenylate
`cyclase
`activity
`in liver plasma membrane.
`Furthermore,
`hCGRP-@-
`37) inhibited
`adenylate
`cyclase activation
`induced
`not only by
`hCGRP but also by hCT. On the other hand,
`in LLC-PK1
`cells,
`where calcitonin
`receptors
`are abundant
`and CGRP appears
`to
`act via
`these
`receptors,
`the bindings
`of 1251-[Tyro]rat
`CGRP
`and 1251-hCT were both
`inhibited
`by hCGRP-(8-37).
`In con-
`trast
`to liver membranes,
`interaction
`of hCGRP-(8-37)
`with
`these
`receptors
`led
`to stimulation
`of adenosine
`3’, Y-cyclic
`monophosphate
`(CAMP)
`production
`in LLC-PK1
`cells, and
`moreover,
`this
`fragment
`did not
`inhibit
`the increased
`produc-
`tion of CAMP
`induced
`not only by hCT but also by hCGRP.
`Thus hCGRP-(8-37)
`appears
`to be a useful
`tool
`for determin-
`ing whether
`the action
`of CGRP
`as well as that of CT
`mediated
`via specific CGRP
`receptors
`or CT receptors.
`
`is
`
`LLC-PK1
`
`cells; calcitonin
`
`gene
`
`specific processing of initial mRNA
`TISSUE
`ALTERNATIVE
`from the calcitonin gene generates two dis-
`transcripts
`tinct peptides: calcitonin and calcitonin gene-related
`these two peptides
`(I, 16). Although
`peptide (CGRP)
`have little sequence homology, calcitonin and CGRP
`possess similar structural characteristics,
`i.e., similar
`sizes, a disulfide bridge at the NHz-terminal portion, and
`an amidated carboxy-terminal
`end. On the other hand,
`previous pharmacological studies have demonstrated the
`presence of distinct
`receptors
`for calcitonin and for
`CGRP in various tissues and cells (5, 7, 11, 12, 19, 20).
`0193-1849/89
`$1.50 Copyright
`0 1989
`
`recent receptor cross-linking studies followed
`Moreover,
`by sodium dodecyl sulfate-polyacrylamide gel electropho-
`resis (SDS-PAGE) analysis have revealed the existence
`of specific CGRP
`receptors, which are distinct
`from
`calcitonin receptors (2,13), with differing molecular sizes
`in various tissues (3, 4, 7). Because of the structural
`similarities mentioned above, however, these two pep-
`tides have also been shown to cross-react with each other
`at the levels of their receptors (6, 12, 19, 20). Further-
`more, it has also been reported that specific receptors for
`both calcitonin and CGRP appear to coexist within
`the
`same tissue, such as brain (6). As a result, it is extremely
`difficult
`to elucidate the direct actions of these peptides
`on their respective receptors. Previously, however, we
`have demonstrated that rat liver plasma membrane ex-
`clusively possesses CGRP receptors and that calcitonin
`appears to interact with
`these receptors (20). On the
`other hand, Wohlwend et al. (19) have reported that, in
`LLC-PK1 pig kidney cells, calcitonin
`receptors predom-
`inate and that the stimulation of adenylate cyclase activ-
`ity by CGRP is probably mediated by these receptors.
`Therefore, by using both LLC-PK1 cells and rat liver
`plasma membrane, it should be possible to characterize
`specific CGRP receptors and calcitonin
`receptors sepa-
`rately.
`Recently, we have shown that a human CGRP frag-
`ment, human CGRP-(8-37),
`binds to CGRP receptors
`in rat liver plasma membrane (20).
`In this study, by
`examining
`the effects on not only CGRP receptors in
`liver plasma membrane but also calcitonin receptors on
`LLC-PK1 cells, we found that
`this fragment, human
`CGRP-(8-37),
`is a specific antagonist for CGRP recep-
`tors but not for calcitonin receptors, raising the possibil-
`ity of being able to clarify whether
`the CGRP or the
`calcitonin receptor is responsible for the action of either
`of these two peptides under various conditions using this
`CGRP antagonist.
`
`MATERIALS
`
`AND
`
`METHODS
`
`Peptides and radioligands. Synthetic
`and human calcitonin were purchased
`the American
`Physiological
`Society
`
`0
`]rat CGRP
`[TY
`from
`Peninsula
`E331
`
`Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (079.177.020.145) on November 6, 2018.
`
`Copyright © 1989 American Physiological Society. All rights reserved.
`
`EX2004
`Eli Lilly & Co. v. Teva Pharms. Int'l GMBH
`IPR2018-01423
`
`1
`
`

`

`E332
`
`A SPECIFIC
`
`CGRP
`
`ANTAGONIST
`
`F .-
`7
`m
`E
`
`E’
`.-
`X
`
`z-
`
`x
`
`100
`
`60
`
`40
`
`20
`
`0
`
`11
`Peptide
`
`9
`10
`Concentration
`
`8
`(-log
`
`7
`M)
`
`5
`
`calcitonin
`-rat
`of 1251- [Tyr’]
`inhibition
`1. Dose-response
`FIG.
`membranes
`rat
`liver
`plasma
`to
`binding
`peptide
`(CGRP)
`related
`(m), and human
`calcitonin
`CGRP-(8-37)
`CGRP
`(o),
`human
`human
`incubated
`for 60 min
`at 24°C
`liver
`plasma
`membranes
`were
`(0). Rat
`with
`25 pM
`1251-[Tyro]rat
`CGRP
`plus peptides
`as indicated.
`Total
`and
`were 11.4 + 1.8 and 10.3 & 1.4% of total
`specific
`bindings
`radioactivity
`added,
`respectively.
`Data
`are expressed
`as percentages
`of maximal
`of
`specific
`binding
`in absence
`of unlabeled
`peptides.
`Values
`are means
`6 separate
`experiments
`differing
`by ~5%.
`Each
`experiment
`was done
`in duplicate.
`
`gene-
`by
`
`CA). Human CGRP and human CGRP-(8-
`(Belmont,
`synthesized
`using an automatic
`solid-phase
`37) were
`(430-A Peptide Synthesizer,
`Applied Biosys-
`synthesizer
`terns.
`lz51- [ Tyr’]rat
`CGRP
`(2,000 Ci/mmol)
`was prepared
`as previously
`described
`(l4), and
`‘?-human
`calcitonin
`(2,000 Ci/mmol)
`was supplied by Amersham
`(Bucking-
`hamshire, UK). Human
`calcitonin was radioiodinated
`by
`the chloramine-T
`method, and the methionine-oxidized
`form of 1251-human calcitonin
`was
`removed by reverse-
`phase high-performance
`liquid chromatography
`(HPLC).
`An adenosine 3’,5’-cyclic
`monophosphate
`(CAMP)
`assay
`kit was obtained
`from Yamasa Shoyu
`(Choshi,
`Japan)
`(17)
`Preparation
`of rat liver plasma membranes, membrane
`receptor binding study, and membrane adenylate cyclase
`assay. Rat liver plasma membranes were prepared from
`adult male Sprague-Dawley rats by the method of Pilkis
`et al. (15) with slight modification, as described previ-
`ously (20). The
`fraction collected at the 42.5-4&Z%
`sucrose interface was removed and washed twice with 10
`mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic
`acid (HEPES) buffer (pH 7.4) containing 1 mg/ml baci-
`tracin. The
`final pellet was resuspended in 10 mM
`HEPES buffer (pH 7.4) containing 120 mM NaCl, 4.7
`mM KCl, 5 mM MgC12, 1 mM ethylene glycol-bis(P-
`aminoethyl ether)-N,N,N’,N’-tetraacetic
`acid (EGTA),
`1 mg/ml bacitracin, and 5 mg/ml bovine serum albumin
`(BSA). One-half milliliter of this membrane suspension
`(200 pg of tissue protein) containing 1251- [Tyr’lrat CGRP
`(25 PM) was incubated at 24°C for 60 min with or without
`various unlabeled peptides. Bound and free hormone
`were separated by centrifugation and radioactivity bound
`to the pellets was counted by an autogamma counter.
`Nonspecific binding was assessed as the fraction of label
`that remained bound in the presence of 10m6 M [Tyr’lrat
`CGRP.
`in the membranes was
`The adenylate cyclase activity
`determined by following
`the synthesis of CAMP
`from
`nonradioactive ATP, as described by Katada and Ui (8).
`Membranes (10 pg of protein) were incubated for 10 min
`at 37°C in 100 ~1 of tris(hydroxymethyl)aminomethane
`(Tris) buffer (25 mM, pH 7.6) containing 1 mM EGTA,
`1 mg/ml of BSA, 100 mM NaCl, 100 PM GTP, 1 mM
`ATP, 5 mM MgC12, 1 mM 3-isobutyl-1-methylxanthine
`(IBMX),
`5 mM phosphocreatine, 50 U/ml of creatine
`phosphokinase, and 1 mM aprotinin with or without
`various stimulants. The reactions were terminated by
`adding 0.33 M ZnS04(40 ~1) and 0.5 M NaC03 (40 pl),
`and after being left to stand for 1 h at 4”C, samples were
`centrifuged and CAMP
`levels in the supernatant were
`measured by a sensitive radioimmunoassay (RIA)
`(17).
`The adenylate cyclase activity was expressed as pico-
`moles of CAMP synthesized by 1 mg of membrane protein
`during 1 min of incubation.
`LLC-PKI cell culture and study of receptor binding and
`cAMPproduction
`in these cells. LLC-PK1 pig kidney cells
`(ATCC, CRL 1392) (19) were grown at 37°C in a 1:l
`mixture of Ham’s FlZ-Dulbecco’s modified Eagle’s me-
`dium with 10% fetal calf serum (FCS) under humidified
`95% 025% Co2 atmosphere. For the experiments,
`the
`cells were washed with phosphate-buffered saline, dis-
`
`0 w
`
`0
`
`I
`
`12
`
`I
`
`I
`
`1
`
`I
`
`I
`
`I
`6
`
`I
`5
`
`7
`9
`8
`10
`11
`M>
`( -
`Concentration
`Peptide
`(CGRP)-
`peptide
`calcitonin
`gene-related
`of human
`2. Effect
`FIG.
`calcitonin-
`CGRP-stimulated
`as well
`as human
`on human
`(8-37)
`cyclase
`activation
`in rat
`liver
`plasma membrane.
`adenylate
`stimulated
`membranes
`were
`incubated
`either
`with
`increasing
`plasma
`Rat
`liver
`of human
`CGRP
`(O--O
`), human
`CGRP-(8-37)
`concentrations
`with
`or human
`calcitonin
`( M)
`alone
`or
`in combination
`(H),
`human
`CGRP
`plus human
`CGRP-(8-37)
`(10m5 M;
`l - - -o)
`or human
`calcitonin
`plus human
`CGRP-(8-37)
`(low5 M; 0-
`- -0)
`for 10 min
`at
`37°C.
`Each
`value
`represents
`mean
`t SE of 5 separate
`experiments.
`Each
`experiment
`was done
`in duplicate.
`Statistical
`analysis
`was done
`by analysis
`of variance
`in conjunction
`with
`Duncan’s
`new multiple
`range
`test.
`*Fp < 0.01 vs. control
`value.
`
`log
`
`persed with 0.5% trypsin and 1 mM EDTA, and prein-
`cubated in HEPES-buffered Krebs-Ringer bicarbonate
`buffer with 1 mg/ml BSA and 2.5 mM glucose (HKRBG),
`pH 7.4, for 1 h. The binding study was carried out by
`
`Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (079.177.020.145) on November 6, 2018.
`
`Copyright © 1989 American Physiological Society. All rights reserved.
`
`2
`
`

`

`A SPECIFIC
`
`CGRP
`
`ANTAGONIST
`
`E333
`
`water-saturated diethylether. CAMP
`mined by a specific RIA (17).
`
`levels were deter-
`
`RESULTS
`
`The specific binding of 1251-[Tyro]-rat CGRP to rat
`liver plasma membrane was displaced by human CGRP,
`human CGRP-(8-37)
`and by human calcitonin
`in a
`dose-dependent manner with relative affinities as fol-
`lows: human CGRP > human CGRP-(8-37)
`> human
`(Fig. 1). Of these three peptides, both human
`calcitonin
`CGRP and human calcitonin significantly
`stimulated
`adenylate cyclase activation
`in a dose-dependent fashion
`in rat liver membrane, but human CGRP was clearly
`more potent
`than human calcitonin
`(Fig. 2). Further-
`more, the doses of these peptides necessary for producing
`a half-maximal effect on adenylate cyclase activation
`(human CGRP, 21.5 pM; human calcitonin, 0.46 PM)
`were comparable to those producing half-maximal
`inhi-
`bition of label binding to the membrane (32.1 pM and
`1.32 PM, respectively).
`In contrast, human CGRP-(8-
`37) did not have any effect on adenylate cyclase activity
`up to 10 -5 M, and moreover, in the presence of 10D5 M
`human CGRP-(8-37),
`the dose-response curve for ade-
`nylate cyclase activation
`induced not only by human
`CGRP but also by human calcitonin was shifted to the
`right
`(Fig. 2). Indeed, the inhibitory
`effect of human
`CGRP-(8-37)
`on human CGRP-induced as well as hu-
`man calcitonin-induced adenylate cyclase activation was
`dose-dependent (Fig. 3). However, human CGRP-(8-
`37) (10B5 M) had no effect on either epinephrine (10e6
`M)- or glucagon ( 10m7 M)-induced activation of adenylate
`cyclase (Table 1).
`On the other hand, all of human CGRP, human
`CGRP-(8-37)
`and human calcitonin displaced the spe-
`cific binding of 1251- [Tyr’] -rat CGRP as well as that of
`1251-human calcitonin
`to LLC-PK,
`cells in a dose-de-
`pendent fashion (Fig. 4). The relative potencies of these
`peptides for the displacement of 1251-[Tyro]rat CGRP and
`1251-human calcitonin were similar, and in contrast
`to
`the result obtained with rat
`liver plasma membrane,
`human calcitonin was most potent
`followed by human
`CGRP and then by human CGRP-(8-37)
`(Fig. 4). In
`striking contrast
`to the data obtained with
`rat
`liver
`plasma membrane,
`it was found
`that
`in addition
`to
`human CGRP and human calcitonin, human CGRP-(8-
`37) also evoked a significant and dose-dependent
`in-
`crease in CAMP production
`in LLC-PK1 cells (Fig. 5).
`Furthermore, human CGRP-(8-37)
`(low5 M) had no
`effect on either human CGRP- or human calcitonin-
`induced adenylate cyclase activation
`(Fig. 5). The rela-
`tive potencies of these peptides in stimulating CAMP
`production were similar to those in displacing the label
`bindings from LLC-PK1 cells.
`
`DISCUSSION
`
`In the previous report, we identified specific binding
`sites for CGRP in rat liver plasma membrane and showed
`that various kinds of calcitonin
`interacted with
`these
`CGRP receptors (20). The present study demonstrated
`that human CGRP as well as human calcitonin
`induced
`
`ow
`
`0
`
`I
`
`10
`
`I
`9
`(8-37)human
`
`I
`8
`
`I
`
`CGRP
`
`(-log
`
`I
`6
`M)
`
`I
`5
`
`gene-
`calcitonin
`of human
`concentration
`of increasing
`3. Effect
`FIG.
`cyclase
`on stimulation
`of adenylate
`(CGRP)-(8-37)
`peptide
`related
`liver
`plasma membrane
`induced
`by human
`CGRP
`in
`rat
`activity
`(10e8
`l ) or human
`M;
`calcitonin
`(10m6 M; 0). Rat
`liver
`plasma membranes
`were
`incubated
`with
`either
`human
`CGRP
`(10v7 M) or human
`calcitonin
`(lob6 M)
`in
`the presence
`or absence
`(control)
`of
`increasing
`concentra-
`tions
`of human
`CGRP-(8-37)
`for
`10 min at 37°C. Data
`are expressed
`as percentages
`of
`the
`increase
`in adenylate
`cyclase
`activity
`(from
`control)
`induced
`by
`a maximally
`effective
`concentration
`of human
`CGRP
`(low8 M).
`Results
`are means
`rt: SE of 5 separate
`experiments.
`Each experiment
`was done
`in duplicate.
`
`1. Effect of human CGRP-(8-37)
`(lo-’ M)
`TABLE
`on adenylate cyclase activation
`induced bY epinephrine
`or glucagon in rat liver plasma membrane
`
`Agents -
`
`Control
`Epinephrine
`Glucagon
`
`( low6 M)
`(lo-’ M)
`
`Activity,
`Cyclase
`Adenylate
`pmol CAMP. mg protein-l.
`
`min-l
`
`Alone
`
`6.220.5
`23.1t2.0
`56.8k4.6
`
`human
`With
`CGRP-(8-37)
`
`5.8kO.4
`23.5t1.8
`57.2k5.0
`
`plasma
`liver
`Rat
`experiments.
`& SE of 5 separate
`are means
`Values
`(10s6 M)
`or
`were
`incubated
`with
`or without
`epinephrine
`membranes
`CGRP-(8-37)
`(10m8 M)
`in presence
`or absence
`of human
`glucagon
`for 10 min at 37°C. Each
`experiment
`was done
`in duplicate.
`(10B5 M)
`t test was used
`for statistical
`analysis.
`See text
`for definition
`Student’s
`of abbreviations.
`
`the cells (2 x 106/0.5 ml) with 1251-[Tyro]rat
`incubating
`CGRP (50 PM) or 12?-human calcitonin
`(25 PM) in the
`presence or absence of various unlabeled peptides at 37°C
`for 60 min. Cells were separated by centrifugation, and
`the radioactivity bound to the cells was counted. Specific
`binding was defined as total binding minus nonspecific
`in the presence of 10v6 M unlabeled human
`binding
`calcitonin.
`the cells (106) were
`In the CAMP production study,
`in 1 ml HKRBG with or without various
`incubated
`agents in the presence of IBMX
`(low4 M) at 37OC. After
`15 min, the incubations were terminated by adding 1 ml
`of ice-cold 12%
`trichloroacetic
`acid, centrifuging
`the
`mixture, and washing the supernatant
`three times with
`
`Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (079.177.020.145) on November 6, 2018.
`
`Copyright © 1989 American Physiological Society. All rights reserved.
`
`3
`
`

`

`’ 25 I-human
`
`Calcitonin
`
`A SPECIFIC
`
`CGRP
`
`ANTAGONIST
`
`I 1251-[ Tyr’]rat
`
`CGRP
`
`1
`
`80
`
`60
`
`E334
`
`100
`
`:
`F
`5
`E
`z .-
`$
`
`‘Y-
`0
`x
`
`80
`
`6o
`
`40
`
`20
`
`A
`_\:‘;
`
`0
`
`11
`
`10
`
`9
`
`8
`
`7
`
`6
`
`5
`
`0
`
`11
`
`10
`
`9
`
`8
`
`7
`
`6
`
`5
`
`Peptide
`
`Concentration
`
`( -log
`
`M)
`
`of
`
`inhibition
`4. Dose-response
`FIG.
`or 1251- [Tyr’]
`(A)
`1251-human
`calcitonin
`peptide
`rat
`calcitonin
`gene-related
`cells by
`(CGRP;
`B) binding
`to LLC-PK,
`human
`CGRP
`(o),
`human
`CGRP-(8-
`37)
`(B), and human
`calcitonin
`LLC-
`(0).
`PK1
`cells were
`incubated
`for 60 min
`at
`37°C with
`either
`25 pM
`1251-human
`cal-
`citonin
`or 50 pM
`1251- [ Tyr’]
`rat CGRP
`plus peptides
`indicated.
`Total
`and
`spe-
`cific
`bindings
`of 1251-human
`calcitonin
`were
`14.5
`t 1.1 and 12.6
`t 1.0%
`of total
`radioactivity
`added,
`respectively,
`and
`those
`of 1251-[Tyro]rat
`CGRP
`were
`2.7
`t
`0.4 and
`2.4
`t
`0.3%,
`respectively.
`Data
`are expressed
`as percentages
`of maximal
`specific
`binding
`in absence
`of unlabeled
`peptides.
`Values
`are means
`of 6 separate
`experiments
`differing
`by <6%.
`Each
`ex-
`periment
`was done
`triplicate.
`
`in
`
`receptors
`
`to CGRP
`
`that bind
`for the peptides
`antagonist
`in rat
`liver plasma membrane.
`cells have
`pig kidney
`On
`the other hand, LLC-PK1
`receptors
`for calcitonin,
`been reported
`to possess
`specific
`and CGRP
`appears
`to exert
`its action on
`these
`cells
`through
`the calcitonin
`receptors
`(19).
`In
`the present
`experiment,
`human
`calcitonin
`was more potent
`than
`human CGRP not only
`in displacing
`the specific bindings
`of
`lz51- [ Tyr’]
`rat CGRP
`and ?-human
`calcitonin
`but
`also
`in activating
`adenylate
`cyclase
`in LLC-PK1
`cells,
`thus
`confirming
`previous
`reports
`(19). We,
`therefore,
`examined whether
`this CGRP
`receptor
`antagonist,
`hu-
`man CGRP-(8-37),
`also antagonizes
`the actions of hu-
`man CGRP and human calcitonin
`on calcitonin
`receptors
`in LLC-PK1
`cells, and it was
`found
`that
`this
`fragment
`not only stimulated
`adenylate
`cyclase activation
`but also
`had no inhibitory
`effect on adenylate
`cyclase activation
`induced by either human CGRP
`or human
`calcitonin.
`Thus
`it is evident
`that
`this CGRP
`receptor
`antagonist,
`human CGRP-(8-37),
`acts as an agonist
`for calcitonin
`receptors
`in LLC-PK1
`cells.
`liver plasma
`rat
`In a previous
`report of a study using
`membranes,
`we suggested
`that
`the COOH-terminal
`por-
`tion of CGRP
`is essential
`for binding
`to its
`receptors,
`since an NH2-terminal
`fragment
`of human CGRP
`could
`not bind
`to CGRP
`receptors,
`whereas
`COOH-terminal
`fragments
`were able to displace
`label binding
`(20). The
`present
`data add new evidence
`to support
`the
`fact
`that
`in spite of
`their
`ability
`to bind
`to CGRP
`receptors,
`COOH-terminal
`fragments
`of CGRP
`lacking a ring struc-
`ture with a disulfide bond may not be sufficient
`to induce
`a subsequent
`intracellular
`signal
`transduction
`event,
`i.e.,
`adenylate
`cyclase
`activation.
`Of particular
`interest
`in
`this study, however, was
`that
`this COOH-terminal
`frag-
`ment of human CGRP was able to promote
`adenylate
`cyclase activation,
`probably
`through
`specific
`calcitonin
`receptors
`in LLC-PK1
`cells. This
`is rather
`surprising,
`since
`this COOH-terminal
`portion of human CGRP has
`only slight amino acid sequence homology with human
`calcitonin. Goltzman
`(5), on the other hand, has reported
`that various modifications
`of the NHg-terminal
`portion
`
`ow
`
`0
`
`I
`11
`
`I
`10
`Peptide
`
`I
`9
`Concentration
`
`I
`8
`
`( -log
`
`I
`7
`M)
`
`I
`6
`
`r
`5
`
`(CGRP)-
`
`peptide
`gene-related
`calcitonin
`of human
`5. Effect
`FIG.
`in-
`as well
`as calcitonin-induced
`CGRP-induced
`on human
`(8-37)
`production
`in LLC-PK1
`cells.
`LLC-PK1
`cells were
`in CAMP
`crease
`increasing
`concentrations
`of human
`CGRP
`(M),
`incubated
`with
`alone
`human
`CGRP-(8-37)
`or human
`calcitonin
`(w)
`(H),
`human
`(10e5
`with
`or in combination
`CGRP
`plus human
`CGRP-(8-37)
`calcitonin
`(10m5 M;
`M)
`(a-
`- -0)
`or human
`plus human
`CGRP-(8-37)
`represents
`Each
`- -0).
`Each
`value
`mean
`k SE of 5 experiments.
`0-
`in duplicate.
`data were
`experiment
`was done
`For
`statistical
`analysis,
`in
`variance
`tested
`by
`analysis
`of
`conjunction
`with
`Duncan’s
`new
`multiple
`range
`test.
`*p < 0.01 vs. control
`value.
`
`in rat
`cyclase
`of adenylate
`activation
`a dose-dependent
`that
`receptors
`liver plasma membrane.
`Thus,
`it appears
`are
`linked
`to
`for CGRP
`in rat
`liver plasma membrane
`the adenylate
`cyclase-CAMP
`system. However,
`in con-
`trast
`to the effects of human CGRP
`and human
`calci-
`tonin and despite
`its ability
`to bind
`to CGRP
`receptors,
`the present
`data clearly
`demonstrated
`that a human
`CGRP
`fragment,
`human CGRP-(t&37),
`had no effect
`on adenylate
`cyclase activity
`in rat
`liver plasma mem-
`brane and that, moreover,
`it significantly
`inhibited
`the
`activation
`of adenylate
`cyclase
`induced not only by hu-
`man CGRP
`but also by human
`calcitonin.
`Thus
`it
`is
`strongly
`suggested
`that human CGRP-(8-37)
`acts as an
`
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`
`Copyright © 1989 American Physiological Society. All rights reserved.
`
`4
`
`

`

`A SPECIFIC
`
`CGRP
`
`ANTAGONIST
`
`E335
`
`and
`
`the disulfide bridge
`including
`of the calcitonin molecule
`reduction
`of its capacity
`to
`did not result
`in substantial
`binding
`and
`to stimulate
`inhibit
`125 I-salmon
`calcitonin
`renal membranes.
`In agree-
`adenylate
`cyclase
`in rabbit
`ment with his data, we found
`that
`the potency of human
`CGRP-(8-37),
`a COOH-terminal
`fragment
`of human
`CGRP,
`both
`for
`the binding
`the stimulation
`of
`and
`adenylate
`cyclase
`in LLC-PK,
`cells was not substantially
`different
`from
`that of human CGRP.
`Taken
`together,
`the COOH-terminal
`portion
`of the calcitonin molecule
`appears
`to be more
`important
`than
`the NH2-terminal
`portion both
`in binding
`to its receptor and in stimulating
`adenylate
`cyclase, although
`the calcitonin
`receptor may
`have a broad specificity
`in recognizing
`the COOH-ter-
`minal portions
`of binding agonists.
`of
`localization
`Recent
`studies
`have
`revealed distinct
`(14, 18) as well as
`immunoreactive
`CGRP and calcitonin
`localization
`of specific
`receptors
`for these peptides
`(5, 7,
`11, 19, 20).
`In
`the brain,
`however,
`specific
`receptors
`appear
`to exist
`for both CGRP
`and calcitonin
`(6) and
`these peptides are known
`to cross-react
`with each other
`(12, 19, 20). Thus,
`although
`it has
`on their
`receptors
`been reported
`that
`intracerebroventricular
`administra-
`tion of CGRP and calcitonin
`often exerts
`similar effects
`(10) and
`such as
`inhibition
`of gastric
`acid secretion
`reduction
`of food intake
`(9), it is still uncertain whether
`the actions of these peptides are mediated
`through
`cal-
`citonin
`receptors
`or CGRP
`receptors.
`It
`is,
`therefore,
`tempting
`to predict
`that
`this newly
`discovered
`CGRP
`antagonist,
`human CGRP-(8-37),
`could be a useful
`tool
`for precise characterization
`of the actions of CGRP and
`calcitonin
`at the levels of their
`respective
`receptors.
`
`Address
`Medicine,
`Chuo-ku,
`
`reprint
`for
`Kobe University
`Kobe
`650, Japan.
`
`requests:
`School
`
`T. Chiba,
`of Medicine,
`
`Dept.
`Third
`Kusunoki-cho
`
`of
`
`Internal
`7-chome,
`
`Received
`
`8 June
`
`1988; accepted
`
`in
`
`final
`
`form
`
`1 November
`
`1988.
`
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`
`Copyright © 1989 American Physiological Society. All rights reserved.
`
`5
`
`

`

`American Journal of Physiology;
`Endocrinology and
`Metabolism
`
`FEBRUARY 1989/VOIume 19, Number 2
`
`Physiological attributes of endogenous bioactive luteinizing hormone secretory
`bursts in man
`J. D, Veldhuis, M. L. Johnson, and M. L. Dufau
`Insulin degradation products from perfused rat kidney
`W. C. Duckworth, F. G. Hamel, J. Liepnieks, D. Peavy, B. Frank, and FE. Rubkin
`
`Myornetrial afadrenoceptors in pregnant guinea pig: their distribution
`and increased number
`8'. J. Arkinstall and C. T. Jones
`
`Effect of epoxyeicosatrienoic acids on growth hormone release from somatotrophs
`G. D. Snyder, P. Yadagiri, and J. R. Falch
`Exercise and insulin stimulate skeletal muscle glucose transport
`through different mechanisms
`E. Sternlicht, R. J. Barnard, and G. K. Grimditch
`Hypothalamic noradrenergic and sympathoadrenal control of glycemia after stress
`G. A. Smythe, W. S. Pascoe, and L. H. Storlien
`Hypothalamic monoamines associated with the cephalic phase insulin response
`L. J. Holmes, L. H. Storlien, and G. A. Smythe
`Chronic exercise compensates for insulin resistance induced by a high—fat diet in rats
`E. W. Kraegen, L. H. Storiien, A. B. Jenkins, and D. E. James
`Microdialysis of intercellular adenosine concentration in subcutaneous tissue in humans
`P. LOnnroth, P.~A. Jansson, B. B. Fredholm, and U. Smith
`Evidence for functional thromboxane Ag-prostaglandin H2 receptors in human placenta
`A. Hedberg, P. F. Manta, E. C.-K. Liu, A. M. Hollander, and B. M. Wilkes
`In vitro correction of impaired Na‘-K*-ATPase in diabetic nerve by protein
`kinase C agonists
`S. A. Lattimer, A. A. F. Sima, and D, A. Greene
`Attenuation of the osmotic release of vasopressin by enkephalins in dogs
`K. Matsui, T. Kimura, K. 0m, M. Shoji, M. Inoue, K. Iitahe, and K. Yoshinaga
`Phlorizineinduced normoglycemia partially restores glucoreg'ulation in diabetic dogs
`G. Hetenyi, Jr., C. Gauthier, M. Byers, and M. Vranic
`DOPA, norepinephrine, and dopamine in rat tissues: no effect of sympathectomy
`on muscle DOPA
`E. Eldrup, E. A. Richter, and N. J. Christensen
`Leucine metabolism in aging humans: effect of insulin and substrate availability
`N. K. Fuhagawa, K. L. Minaker, V. R. Young, 1). E. Matthews,
`D. M. Bier, and J. W. Rowe
`Insulin sensitivity and responsiveness during lactation and dry period in goats
`E. Debras, J. Grizard, E. Aina, S. Tesseraud, C. Champredon, and M. Arnal
`
`E199
`
`E208
`
`E215
`
`E221
`
`E227
`
`E23]
`
`E236
`
`E242
`
`E250
`
`E256
`
`E264
`
`E270
`
`E277
`
`E284
`
`E288
`
`E295
`(Continued)
`
`This Journal is printed on “acid~free" paper.
`
`6
`
`

`

`. Contents continued)
`
`E303
`
`Proinsulin mRNA levels in fasting and fed ADX rats: evidence for an indirect
`effect of glucocorticoids
`F. T. Fiedorek, Jr., and M. A. Per-mutt
`Effect of hypercalcemia-producing tumor on 1,25(0H)2D. biosynthesis in athymic mice
`S. C. Kuhreja, P. A. York, C. Nalbantian—Brandt, D. II. Shevrin, and M. J. Fauus
`Palmitate oxidation by isolated working fetal and newborn pig hearts
`J. C. Werner, R. E. Sicard, and H. G. Schiller
`Central effects of fi-endorphins on glucose homeostasis in the conscious dog
`E322
`P. M. Radoseuich, D. B. Lacy, L. L. Brown, P. E. Williams, and N. N. Aburm‘acl
`__—_—__—_—
`
`E309
`
`E315
`
`RAPID COMMUNICATIONS
`
`Calcitonin gene-related peptide receptor antagonist human CGRP-(8—37)
`T. Chiba, A. Yamaguchi, T. Yamatani, A. Nahamura, T. Marishita, T. Inui,
`E331
`M. Fukase. T. Nada, and T. Fujita
`______———_———
`
`LETTERS TO THE EDITOR
`
`On the measurement of lactate turnover in humans
`J. Katz; M. D. Jensen and J. M. Miles
`Retraction
`P. E. Cryer
`
`E336
`
`E338
`
`7
`
`