.. ·tish
`J urnal of
`Pharmacology
`
`March 1994
`
`? l 7 7994
`
`V\,.,1t_,,t..,c_
`v L,u, i1'1R'(
`
`-: ~ :i
`
`.._, tL 11 ,
`(
`
`Volume 111
`
`Number 3
`
`pages 649-968
`
`

`

`OF PHARMACOLOGY
`.b t·ons in all fields of experimental
`BRITISH JOURNAL
`:i::~~a:tw:i;'.;:~~~~
`:;;;,~~ochemical, ce~i~:a~:,;'~
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`pharBmacodl:r ~di tors represents a wi?e range ?ft extp::th maintaining the overall quality of
`tly as possible cons1s en
`The oar
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`work is published as promp
`the journal.
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`0
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`h B ·1,·sh Pharmacological Society by
`fi
`Edited or t e ri
`A.T. Birmingham
`( Chairman)
`W.A. Large
`R.W. Horton
`( SecreTaries)
`
`J.A. Angus Melbourne. Australia
`M.L.J. Ashford Cambridge
`G.W. Benneu No11i11g/ra111
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`Alison F. Brading Oxford
`Lo11do11
`S.D. Brain
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`Helen M. Cox
`A.J. Cross London
`V. Crunelli Cardiff
`T.C. Cunnane Oxford
`Lo11do11
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`A. Dray London
`J.R. Docherty Dublin
`J.M. Edwardson Cambridge
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`C.A. Maggi Florence, Italy
`Janice M. Marshall Birmingham
`G. Martin Becke11/ra111
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`Corresponding Editors
`
`C.P. Page London
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`P . Sneddon Glasgo11
`K . Starke Freiburg, Germany
`R.J. Summers Melbourne. Australia
`P. v. Taberner Bn'.~tol
`J. Tamargo Madrid, Spain
`c. Thiemermann London
`M.D. Tricklebank Har/o,..
`M. B. Tyers Ware
`S.P. Watson Oxford
`K.J. Watling Cambridge
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`B.J.R. Whittle Beckenhrmt
`Eileen Winslow R,om. France
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`lt, Jolla, U.S.A .
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`R.F. Furchgou New York, U.S. A.
`T. Godfraind Br11ssels, Belgium
`S.Z. Langer Paris. France
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`R.J . Miller Chicago , U.S.A.
`R.C. Murphy Denver, U.S .A.
`E. Muscholl Main=, Germany
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`M. Otsuka Tokyo, Japan
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`S. Rosell Soderta/je, S11'l!(len
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`Netherlands
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`Sobmissio, of m.,..,,ipts, Ma, osc,ipts (two copies) sho•ld be sent to The Editodai Office, B,i tish
`lo•mal of Phannaeology, St. Geo,ge', Hospital Medical Scho0I, Cran me, Terrace, London SW 17 ORE.
`A•thorn sho•ld cons•lt the lnstrnotions to A•tho,s and the Nomendat"'e G•idelines fo, Amhorn
`in Vol. II I , 378- 38). These I nst'"otions and G • ideHnes also appea, with the jo• mal Index fo,
`Vol•mos 108- 110, 1993. A checkJ;s, oft he esse,rial res•i«ments is s.mma.ised in each iss.e of the
`Journal, or as the last page of the issue.
`
`

`

`Catalogue
`
`Supplement
`
`Now Available
`
`Full details of all the latest additions to our catalogue
`
`New compounds include:
`
`1-[4-(1-Adamantanecarboxamido)butyl]-4-(2-methoxy)phenylpiperazine - 5-HT1A antagonist
`with as high affinity for 5-HT1A sites as NAN-190, but much more selective
`4-(4-Fluorobenzoyl)-1 -( 4-phenylbutyl)piperidine - selective 5-HT 2 antagonist with almost
`as high affinity as ketanserin, but with a much lower affinity for 5-HT1c sites
`8-[3-(4-Fluorophenoxy)propyl]-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one, AMl-193 - binds
`to 5-HT 2 sites as strongly as spiperone but with even lower affinity for 5-HT1 c site. Also
`has high affinity for dopamine D2 sites
`3,4-Dihydro-1 '-(3-methylbut-2-enyl)spiro[1 H-indene-1,4'-piperidine] - high affinity o ligand
`with 10000 fold preference for o binding site over the dopamine D2 site
`1 '-Benzyl-3,4-dihydrospiro[1 H-indene-1 ,4'-piperidine] - high affinity o ligand with excellent
`selectivity over dopamine D2 receptor
`N-Acetylglycyl-D-glutamic acid - excitatory peptide which is more potent than L-glutamic
`acid in inducing seizures in mice
`H-9, N-(2-Aminoethyl)-5-isoquinolinesulphonamide - protein kinase inhibitor
`4-[N-( 4-Chlorophenyl)carbamoyloxy]-4-pent-2-ynyltrimethylammonium iodide - partial
`agonist with 16 times the power of the analogue McN-A-343 in increasing arterial blood
`pressure and 18 times the affinity of the latter for ganglionic M1 receptors
`
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`Look here first f or new ...
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`

`

`British Journal of
`Pharmacology
`
`VO LUME 111 (3)
`
`MARCH 1994
`
`OBITUARY
`
`G.W. Bisset. Wilhelm Feldberg, CBE, F RS (1900- 1993)
`
`649
`
`SPECIAL REPORT
`I. Porsti, ~.T. Bara, R. Busse & M. Hecker. Release of ni tric oxide by
`ang1o tensm-(l-7) from porcine coronary endothelium: implications fo r
`652
`a novel angiotensin receptor
`
`PAPERS
`H. Moritoki, T. Hisayama, S. Takeuchi, W. Kondoh & M. lmagawa.
`Relaxation of ra t thoracic aorta induced by the ca>+ -A TPasc inhibitor,
`655
`cyclopiazonic acid, possibly through nit ric oxide fo rmation
`J. Cuevas & D.J. Adams. Local anaesthetic blockade of neuronal nicotinic
`663
`ACh receptor-channels in rat parasympathetic ganglion cells
`Y. Dohi, M. Kojima & K. Sato. Vasorelaxant effect of mexiletine in
`673
`mesenteric resistance arteries of ra ts
`M.K. Herbert & P. Holzer. Interleukin-IP enhances capsaicin-i nduced
`681
`neurogenic vasodilatation in the rat skin
`C.A. Maggi, S. Giuliani & P. Santicioli. Effect of cromakalim and
`glibenclamide on spontaneous and evoked motility of the guinea-pig
`687
`isolated renal pelvis and ureter
`P.B .
`. Clarke, M. Reuben & H. El-Bizri. Blockadeofnicotinic respon es
`by phy ostigmine, tacrine and other cholinesterase inhibitors in rat
`695
`stria tum
`K.K.C. Tan, M.J. Brown, J. Longmore, C. Plumpton & R.G. Hill .
`Demonstra tion of the neurotransmiller role of ca lciton in gene-related
`peptides (CGRP) by immunoblockade with anti-CG RP monoclonal
`antibodies
`703
`C. Jiang & P . Collins. Inhibition of hypoxia-induced relaxation of rabbit
`isolated coronary arteries by
`0 -monomethyl-t-arginine but not
`711
`glibenclamide
`0. Perez, C. Valenzuela, E. Delpon & J. Tamargo. Class I and Ill
`antiarrhythmic
`actions of prazosin
`in gui nea-pig papillary
`muscle
`717
`J .R. Purkiss, G.F. Wilkinson & M.R. Boarder. Differentia l regulation of
`inositol 1,4,5-trispho phate by co-existing P2y-purinoceptors and
`nucleotide recepto rs on bovine aortic endothelial cells
`723
`D.M. Pollock & T .J . O pgenorth. ETA receptor-mediated responses to
`729
`cndot helin-1 and big endothelin-1 in the rat kidney
`G.J . Molderings, E. Colling, J. Likungu, J. Jakschik & M. Gothert.
`Modula tion of noradrenali ne release from the sympathetic nerves of the
`human sa phenous vein and pulmona ry a rtery by presynaptic EP,- and
`DP-receptors
`733
`S. Meini, R. Patacchini &. C.A. Maggi. Tachykinin NK, recepto r
`subtypes in the ra t urina ry bladder
`739
`S.S. Kelly & C.B. Ferry. The o rigin of the effects of an anticholinesterase
`747
`on the latencies of action potentials in mouse skeletal muscles
`J.M. Hunt, R.S. Redman & E.M. Silinsky. Reduction by intracellular
`calcium chelation of acetylcholine secretion without occluding the
`753
`effects of adenosine at frog motor nerve endings
`
`A. Floch, V. Fardin & I. Ca,·ero. Characterization of K1 and N K,
`tachykinin receptors in guinea-pig a nd rat bronchopulmonary and
`vascula r systems
`759
`
`J. Cortijo, C.M. Sanz, V. Villagrasa, E.J. Morcillo & R.C. Small . The
`effects of phorbol 12, 13-diacetate on responses of guinea-pig isolated
`769
`trachea to methylxanthines. isoprenaline a nd ryanodinc
`C.M. Yang, Y.-L. Yo, J.-T. Hsieh & R. Ong. 5- Hyd roxyiryptami ne
`receptor-mediated phosphoinosi1ide hydrolysis 111 cani ne cultured
`777
`tracheal smooth muscle cells
`R. Atcheson, D.G. Lambert, R.A. Hirst & D.J. Rowbotham. St udies on
`the mechanism of [' HJ-noradrenaline release from SH-SY SY cells: the
`role of Cal+ and cyclic AMP
`787
`
`C.J. Bailey & K.J. Myoett. In ulin requirement fo r the antihyper(cid:173)
`793
`glycaemic effect of metform in
`
`G.A. Kennett, M.D. Wood, A. Glen, S. Grewal, I. Forbes, A. Gadrc &
`T. P. Blackburn. /11 vivo properties of SB 200646A, a 5- HT2 128 receptor
`antagonist
`797
`D. Bowie & T.G. Smart. Species-dependent functional properties of
`non-NMDA receptor expre sed in Xe11op11s /ae,•isoocytes injcc1cd with
`803
`mammalian and avian brain mR A
`M.M. Teixeira, S. Rcynia, M. Robinson, A. Shock, T.J. Williams, f.
`.
`William , A.G. Rossi & P.G. Hellewell . Role of CD 18 in the accumula(cid:173)
`tion of eosinophils and neulrophi ls and local oedema formation in
`in fl ammatory reactions in guinea-pig skin
`81 I
`
`A.M .. Elhawary & C.C.Y. Pang. a w Adrenoceptor mediate renal
`819
`tubular sodium and water reabsorption in the rat
`
`M.G. Persson, P. Agvald & L.E. Gustafsson. Detection of nitric oxide in
`ex haled air during administration of nitroglycerin in ••ivo
`825
`W.J. Wieczorek & Z.L. Kruk. Differential ac1ion of ( + )-amphetamine
`o n electrically evoked dopamine overflow in rat brain slice containing
`corpu striatum and nucleu accumbens
`829
`
`M. Santiago, A. Machado & J. Cano. Effect of L-arginine/ nitric oxide
`837
`pathway on MMP +-induced cell injury in the striatum of rats
`A. Barber, G_D. Bartoszyk, H.E. Griener, f . Mauler, R.D. Murray, C.A.
`Seyfried, M. Simon, R. Gottschlich, J. Harting & I. Lues. Central
`and peripheral acti ons of the novel ,c-opioid receptor agonist, EM D
`843
`60400
`P. P. McDonald, S.R. McColl, P. Braq uct & P. Borgcat. Autocrine
`enhancement of leuko triene synthesis by endogenous leukotriene B, and
`852
`platelet-activating factor in human neutrophils
`O.G. Garcia Hermida, T. Fontela, M. Ghiglionc & L.O. Uttcnthal. Effect
`of lithium on plasma glucose, insulin and glucagon in normal and
`streptozotocin-diabetic rats: role of glucagon in the hyperglycaemic
`861
`response
`M.N. Sillence & M.L. Matthews. Classical and atypica l binding sites for
`P-adrenoceptor ligands and activation of adenylyl cyclase in bovine
`866
`skeletal muscle and adipose tissue membranes
`D.K. Martin, Y. Nakaya, K.R. Wyse & T.J. Campbell. Effects of
`disopyramide and flecainide on the kinetics of inward rectifier potas(cid:173)
`873
`sium channels in rabbit heart muscle
`
`

`

`w .-M. f'u & f'.-L. Huang. Poten1ia1_ion by endogenously released ~ ~p
`of spontaneous transmi11er secreuon at developing neuromuscula~
`88
`synapses in Xe11opus cell cultures
`A.C. Kappellc, G. Biessd s, B. Bra,·enbocr, T. van Burcn, J . T!aber, O.J. 0c
`Wildt & W.H. Gispen. Beneficial effect of the Ca·
`antagomsl,
`887
`nimodipine. on existing diabetic ncuropathy in the BB{Wor rat
`E. Pipili-Synctos, E. Sakkoula, G. Haralabopoulos, P. _A?drio~ul~u, P.
`Perist<ris & M.E. Maragoudakis. Evidence that mine oxide ts an
`894
`endogenous antiangiogenic mediator
`J .C. Ellory, S.J . Culliford, P.A. Smith, the late M.W. Wolowyk & E.E.
`Knaus. Specific inhibition of Ca-activated K channels in red cells by
`selectc<I dihydropyridine derivatives
`903
`S.-M. Yu, T.-S. Wu & C.-M. Teng. Pharmacological characterization of
`cinnamophilin, a novel dual inhibitor of thromboxane synthase and
`thromboxane A, receptor
`906
`
`G.J. McLaren, G. Lambrecht, E. Mutschler, H.G. Baumert, P. Sneddon
`& C. Kennedy. Investigation of the actions of PPADS. a novel P,x(cid:173)
`purinoccptor antagonist, in the guinea-pig isolated vas deferens 913
`G.M. Nichol, C.H. Parsons & K.f . Chung. Effect of sodium metabisul(cid:173)
`phitc on bronchial blood How in conscious sheep: pharmacological
`918
`modulation
`
`A.U. Ziganshin, C.H.V. Hoyle, G. Lambrcct, E. Mutschler, H.G.
`Biiumert & G. Burnstock. Selective antagonism by PPADS al P,x(cid:173)
`923
`purinoceptors in rabbit isolated blood vessels
`
`M. Gue, C. G leizes-Escala, C. Del Rio-Lacheze, J.-1.,. Junien & L. Bueno.
`Reversal of C RF- and d opamine-induced stimulatio n o f colonic
`930
`motility by CCK a nd igmesine (JO 1784) in the rat
`Y. Nakazato, Y. Tani, H. Tcraoka, T. Sugawara, T. Asano, T. Ohta & s.
`Ito. Inhibitory effects of caffeine on secretagogue-induced catechol(cid:173)
`amine secretion from adrenal chromaffin cells of the guinea-pig 935
`
`W. Norenberg, J.M. Langosch, P.J. Gebicke-Hacrter & P. mes.
`Characterization and possible functio n of adenosine 5' -1riphosphate
`942
`receptors in activated rat microglia
`
`M.A. S huker, F. Bowser-Riley & S.N. Davies. Possible NMDA
`antagonist properties of drugs tha t affect high p ressure neurological
`951
`syndrome
`
`M. Derrico, C. Ourieux & B.P. Roques. Antidepressant-like effects of
`956
`CCK8 antagonists in mice: antagonism by naltrindole
`
`E.M. van Geldercn & P.R. Saxena. Inhibition of nitric oxide biosynthcsis
`961
`and carotid arteriovenous anastomotic shunting in the pig
`
`ERRATUM
`Br. J. Pharmacol. (1993), ll0, 1483- 1490
`R.G. Pertwec, L.A. S tevenson & G. Griffin. C ross-tolerance between
`delta· 9 · tetrahydrocannabinol and
`the cannabimimetic agents,
`CP 55,940, WIN 55,2 12-2 and ana ndamide
`
`968
`
`

`

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`Br. J. Pharmaco/. (1994), 111 , 703 - 710
`
`© Macmillan Press Ltd, 1994
`
`Demonstration of the neurotransmitter role of calcitonin
`gene-related peptides (CGRP) by immunoblockade with
`anti-CGRP monoclonal antibodies
`
`1K.K.C. Tan, MJ. Brown, *J. Longmore, C. Plumpton & *R.G. Hill
`
`Clinical Pharmacology Unit, Addenbrooke's Hospital, Cambridge and *Merck Sharp and Dohme Resea rch Laboratorie ,
`Neuro cience Research Centre, Terlings Park, Harlow
`
`Monoclonal antibodies (MAbs) against rat o:-calcitonin gene-related peptide (o:CGRP) were pro(cid:173)
`duced. Those which bound CGRP in a radioimmunoassay and inhibited the binding of 2-[ml](cid:173)
`iodohistidyl 10-CGRP in a receptor binding assay were selected for immunoblockade ex periments.
`2 The effect of MAbs on CGRP inhibition of electrically stimulated contractions of the rat i olated vas
`deferens was characterized. Four out of 11 MAbs tested shifted the concentration-re ponse curve of
`CGRP to the right compared with vehicle or irrelevant MAb control. MAb C4.19 produced equip tent
`blockade of rat o:CGRP and rat PCGRP and was chosen for further studies. MAb C4. 19 had no
`pharmacologically significant effect on the concentration-response relationship of i oprenaline, rat
`P-endorphin or somatostatin.
`3 We demonstrated that the pharmacological response to CGRP in the presence of MAb C4.19 could
`be predicted when the dissociation constant and concentration of binding sites of the antibody were
`known. Comparison of experimental and computer simulated data showed good agreement for EC50 and
`maximum effect of CGRP in the presence of MAb C4.19.
`4 Capsaicin at I µM inhibited the electrically stimulated contractions by 60.8% (95% confidence
`interval 51.8% to 69.9%). This effect was significantly attenuated by MAb C4.19 to 26.0% (95%
`confidence interval 15.2% to 36.8%; P <0.0003).
`5 The immunoblockade of exogenous and endogenous CGRP described here, together with comp(cid:173)
`lementary evidence from other studies, strongly suggest that CGRP has a major neurotransmitter role at
`the neuroeffector junction of the rat vas deferens.
`Keywords: Calcitonin gene-related peptide; monoclonal antibodies, rat isolated vas deferens; immunoblockade; neurotransmis(cid:173)
`sion; capsaicin
`
`Introduction
`
`Calcitonin gene-related peptide (CG RP) is produced by alter(cid:173)
`native processing of the primary mRNA transcripts of the
`calcitonin gene (Rosenfeld et al., 1983). A second CG RP
`gene encoding another 37-ami no acid peptide was subse(cid:173)
`quently identified (Amara et al., 1985; Steenbergh et al.,
`1985). Thi peptide (PCGRP) differs from the originally dis(cid:173)
`covered CGRP (o:CG RP) by only one amino acid at position
`35 in the rat. Unlike calcitonin, CG RP is primarily localized
`in the brain and peripheral nervous tissue. Diverse biological
`effects have been attributed to CGRP but its physiological
`importance remains to be established in many organ systems.
`The localization of CGRP-like immunoreactivity in primary
`afferent neurone
`innervating many different tissues and the
`wide di tribution of CGRP binding sites suggest that CG RP
`may be a physiologically important neurotransmitter.
`One important criterion that must be fulfilled for any
`neurotransmiuer i
`that modulation of the effects of the
`exogenous putative neurotran milter by drugs should have
`corresponding effects on responses to nerve stimulation .
`Pharmacological blockade is normally achieved through the
`use of specific recepto r an tago nists. A number of C-terminal
`fragment of CGRP have been demonstrated to behave as
`receptor antagonists (Mimeault et al., 1991). The C-terminal
`(8 - 37) fragment of human <XCGRP has been well charac(cid:173)
`terized and is commerciall y available. However, CG RP (8-
`37) demonst rate variable antagonistic potency in different
`tissues and is a relatively poor antagonist in the rat isolated
`va deferens preparation (Dennis et al., I 990). This has led to
`the postulation that multiple receptor subtypes exist for
`
`1 Author for correspondence.
`
`CG RP. An alternative approach to pharmacological block(cid:173)
`ade is the use of antibodies which bind to and inhibit the
`biological activities of putative neurotran mitter peptide .
`lmmunoblockade may be a more general approach to the
`elucidation of the physiological roles of neuropeptides since
`no assumptions have to be made concerning receptor multi(cid:173)
`plicity and the relative selectivity of receptor antagonists.
`The majo r objective of the pre ent tud y wa
`to inve tigate
`the role of CG RP as a principal neurotransmitter, using the
`neuroeffector junction of the rat isolated vas deferens as a
`model for neurotransmission. The release of endogenous
`CG RP from nerves was achieved through the use of cap(cid:173)
`saicin. Capsaicin is the pungent ingredient of hot peppers of
`the genus Capsicum which selectively stimulates a ub-pop(cid:173)
`ulation of primary afferent neurones (reviewed by Holzer,
`1991). It has been widely used as a pharmacological tool to
`investigate the 'efferent' function of primary afferent neur(cid:173)
`ones (reviewed by Maggi & Meli, 1988). Stimulation of
`capsaicin-sensitive sensory neurone leads to release of neuro(cid:173)
`peptides stored in nerve terminals which may, in turn, med(cid:173)
`iate various efferent functions. Both capsaicin and CGRP
`inhibit the nerve-mediated contractions of the isolated vas
`deferens. It is therefore reasonable to hypothesize tha t CG RP
`may be involved in neurotransmission at the neuroeffector
`junction of the rat vas deferens.
`Analysis of the effects of individual endogenous neuropep(cid:173)
`tides is often difficult because of the co-release of several
`neuropeptides by capsaicin in many tissues. In particular,
`tachykinins co-released with CGRP often produce a similar
`biological response (e.g. dilatation of arteries). Ln the elec(cid:173)
`trically stimulated isolated vas defe{ens, neurokinin A and
`susbtance P enhance contractions (Moritoki et al., 1987) in
`
`

`

`Tween 20 0.05% between each step of the ELISA. Wells were
`incubated with 100 µI porcine gelatin 0.25% in PBS pH 7.4
`(blocking step) for 2 h. Fifty µI serum or hybridoma culture
`supernatant, diluted as appropriate, was added to each well
`and incubated for 2 h. Serial dilutions were made in PBS/
`Tween 20 0.05%/gelatin 0.25%. Controls used were normal
`mouse immunoglobulin (10 µg ml- 1), culture medium and
`Sp2 myeloma cell culture supernatant. Wells were incubated
`with 50 µI horseradish peroxidase-conjugated rabbit anti(cid:173)
`mouse antibody (I in 1000 in PBS/Tween 20 0.05%/gelatin
`0.25%) for I h. One hundred µI substrate reagent (0.006%
`hydrogen peroxide, 0. I M acetate-citrate buffer pH 6) con(cid:173)
`taining tetramethylbenzidine (100 µg ml - 1) as chromogen
`(Bos e1 al., 198 I) was added to each well. Positive wells
`developed a blue colour when 25 µI of 2 M su lphuric acid was
`added to give a yellow colour with grea ter intensity. Absor(cid:173)
`bance was measured at 450 nm using an ELISA plate reader
`(Titertek Multiskan Plus Mk II , Flow Laboratories).
`Selectivity of antibodies for different CG RPs was assessed
`by coating plates with the ex and P forms of rat and human
`CGRP. Relative affinity of antibodies for CGRP was assess(cid:173)
`ed by serial dilution of hybridoma supernatants. The validity
`of this simple method of determining relative affinity (van
`Heyningen et al., 1983) depends on prior knowledge that the
`variation of immunoglobulin concentration in supernatants
`from confluent hybridomas is generally less than ten fold.
`
`Receptor binding assay
`
`in
`Livers di~sected from Wistar rats were homogenized
`50 mM Tns HCI pH 7.4 containing 0. 1 mM phenyl-methyl(cid:173)
`sulphonyl-fluonde (PMSF). The homogenate was centrifuged
`at 15000 g for 20 min at 4"C. Supernatant was decanted and
`centrifuged at 48000 g for 60 min at 4"C. The pellet was
`resuspended m buffer and centrifuged at 48000 g for 60 min
`at 4'C. _The_ su_pernatant was discarded and pellet resus(cid:173)
`pended m bmdmg assay buffer containing aprotinin (20 u
`, bacitracin (0: I%), PMSF (0.1 mM), MgCI, (5 mM},
`mf -_
`bovme serum albumm (0.5%) in 50 mM Tris HCI pH 7 4 (at
`.
`~q.
`T~e binding assay incu?ation mixture consisted of 50 µI
`?Ybndoma supernatant (diluted as appropriate), 50 µI bind(cid:173)
`mg buffer, 50 µI (2-l''.'TJ-iodohistidyl10-human cxCG RP (40,000
`c.p.m.) and l~0 µ I hver membrane suspension (3 mg ml - 1).
`~o~;5pecific bmdmg was assessed using excess rat ex CG RP
`( 0 M). Cell cultu~e medium, Sp2 myeloma supernatant
`and normal mouse 1_mmunoglobulin ( IO µg mI - 1) were used
`as controls. The mixture was incubated at 4•c ~
`2 h
`vacuum . filtered through Whatman GF/C filter pa;~ pre:
`soaked m polyethyleneimine (250 µI in 200 ml 50 mM Tris
`1 pH 7-~), and washed thrice with 3 ml ice-cold buffer
`.
`·
`b
`t· · ·
`mM Tns HCI pH 7 4 c
`on ,11111ng ovme serum albumin
`·
`. .
`.
`0 S'¼ )
`• Radioacuv1ty on the filter paper was counted in a
`0
`•
`Beckman Gamma 5500 counter.
`
`C0
`(H5
`
`1)
`
`Radioimmunoassay ( RIA )
`
`.
`.
`For screening procedures th RIA .
`mcubauon mixture con
`e
`,
`.
`s!dste10 of SO µI hybridoma supernatant. 50 µI 2-[' 2' 1]-iodohis:
`)
`ti yl -human cxCG RP (40 000
`c.p.m. and 400 µI of assay
`'
`buffer (50 M d.
`10 mM ED~A :~ ,um. phosphate buffer pH 7.4 containing
`d o.3 1/o bovine serum albumin) Tissue cul(cid:173)
`d.
`t
`; ~e rne 1Um, myelon_ia (Sp2) culture superna t~nt normal
`'
`bl
`d
`use 1mmunoglobuhn (10 µg mJ- ')
`ank without
`an a
`supernatant were u d
`se as controls. The assay was performed
`. h
`at 4"C
`·
`·
`a 4 day inc b
`w11
`u ation penod. Bound and free
`CGRP
`coated ~::r~:t~~~f tb~Scharco_al precipitation. One dextran(cid:173)
`ter~ntl Separex; Steranti Research}
`e
`was stirred in I 5 1
`ing 0.25% gelatin)fo:ef~ra~ion b~ffer (assay buffer contain(cid:173)
`µ! of the dextran/charco~'" at 4 C.· Two hundred and fifty
`s_ufspen_Sion was added to each
`assay tube followed b
`Y centn ugation at 2000 g for 20 min.
`
`704
`
`K. K.C. TAN et al.
`
`contrast to the inhibitory effect of CGRP (Goto et al., 1987).
`Tissue concentrations of tachykinins are low in the rat vas
`deferens and substance P-like and neurokinin-like immuno(cid:173)
`reactivity were undetectable by immunocytochemistry in this
`tissue (Saito e1 al., 1987). In this respect, the rat vas deferens
`is a particularly useful model for the investigation of CGRP
`as a major neurotransmitter.
`lmmunoblockade of biologically-active peptides is a rela(cid:173)
`tive new pharmacological tool which has not been subject to
`rigorous scientific evaluation. Therefore a further objective of
`the present investigations was to validate the use of immuno(cid:173)
`blockade as an experimental tool for probing the physio(cid:173)
`logical role of CGRP. We have done this by the use of
`monoclonal antibodies (MAbs) and appropriate experimental
`designs to confirm the specificity of blockade. In addition, we
`have fitted appropriate models to the observed data and
`performed limited simulations in order to compare observa(cid:173)
`tions with theoretical predictions.
`
`Methods
`
`Production of 111011oclona/ a111ibodies
`
`Rat cxCGRP was conjugated to bovine serum albumin using
`1-ethyl-3(3-dimethylaminopropyl)-carbodiimide hydrochlor(cid:173)
`ide (O'Shaughnessy, 1982). The conjugate was mixed with
`Freund's adjuvant to produce a water-in-oil emulsion. Two
`hundred µI of the mixture, containing 35 µg of the immuno(cid:173)
`gen, was injected into each mouse by the intraperitoneal
`route. Ten female Balb/C mice were immunized. Freund's
`complete adjuvant was used for the priming dose and Freund's
`incomplete adjuvant used for the first and second booster
`injections. Tl!e first and second booster injections were given
`al 4 weekly mtervals. A final aqueous injection was given 3
`days prior to fusion.
`. Sera collected 8 days following the second booster injec(cid:173)
`tion were screened by indirect enzyme-linked immunoadsor(cid:173)
`bent assay (ELISA; described below) and were found to be
`positive for antibodies against CG RP. Fusion between Sp2
`myel_oma _cells and spleen cells from mice was performed by a
`mod1ficat1on of the method described by Galfre & Milstein
`(1981)._ Hybridoma cells were co-cultured with feeder layer
`cells (irradiated MRC-5 human lung diploid fibroblasts;
`Long e! a(., 1986). Supernatants from the fusion were screen(cid:173)
`ed by md1rect ELISA. Prior to cloning, 100 positive hybri(cid:173)
`domas detected by the_ primary ELISA screen were subjected
`further
`included
`to secondary s