Patent Application: US-55531300-A

Abstract:
the invention concerns an isolated polypeptide constitutive of splice variants for human serotonin receptor whereof the amino acid sequence is selected among the sequence seq id no . 2 of the 5 - ht 4 polypeptide variant or the sequence seq id no . 4 of the 5 - ht 4 polypeptide variant , or any polypeptide fragment or biologically active derivative thereof . the invention also concerns the inverse agonist effect of ml 10375 on 5 - ht4 and 5 - ht4 receptors .

Description:
the pcrs were carried out using a geneamp 2400 machine ( perkin elmer ). the hiraq dna polymerase and the associated buffer were obtained from bioprobe systems . in all the pcr reactions , the dntps and the specific primers were at a final concentration of 200 μm and 1 μm , respectively . the double - stranded dna was sequenced using a t7 polymerase dna sequencing kit ( pharmacia ) according to the manufacturer &# 39 ; s instructions . the sequence of the human receptor 5 - ht 4 ( a ) characterized by blondel et al ., 1997 was used to synthesize the two primers hht 4 5 [ 5 ′- cggtgcttatttcctgtmtg - 3 ′] ( seq id no . 11 ) and hts3 [ 5 ′- atggtcmcaagccctac - 3 ′] ( seq id no . 12 ), which correspond to the start of the sequence of the receptor and to the fifth transmembrane domain respectively . to obtain the cdnas from the 5 ′ ends generated by the 5 - ht 4 gene , the anchored - race extension technique was used as described in newton and graham , 1994 . 50 ng of total rna from human brain and ileum were subjected to a reverse transcription using an oligo ( dt ) primer containing two anchorage sequences , and the superscript reverse transcriptase ( gibco / brl ). the reaction products from these two tissues were then collected and used as a matrix for a race reaction using the hht 4 5 primer with the first anchor . the product from the first pcr reaction was used as a matrix for the nested - race reaction using the hts3 primer ( modified to include a hindiii restriction sequence ) with the second “ anchor ” ( containing an ecori restriction sequence ). these two pcr reactions were carried out at an average of 25 amplification cycles under the following conditions : denaturation for 1 minute at 94 ° c ., hybridation for 1 minute at 54 ° c . and extension for 2 minutes at 72 ° c ., with a final extension for 8 minutes . the hybridization temperatures were 52 ° c . for the race reaction and 54 ° c . for the nested - race reaction . the dna fragments were separated on a 1 . 5 % agarose gel , cloned into pgem - 7z ( cleaved with hindiii / ecori ) and sequenced . the total cdna corresponding to the splicing variants 5 - ht 4 ( b ) , 5 - ht 4 ( c ) and 5 - ht 4 ( d ) was amplified using the original reverse transcription pool from human brain and ileum and a nested - pcr strategy . the hht 4 5 primer ( modified to include a hindiii restriction site ) was used as sense primer in all the pcr reactions . the reverse primers for the first amplification cycle were f81 [ 5 ′- gcctcaggtgaagagaat - 3 ′] ( seq id no . 13 ), f61 [ 5 ′- tggcattaggatggtitggtca - 3 ′] ( seq id no . 14 ) and f71 [ 5 ′- gcaataagmttggccac - 3 ′] ( seq id no . 15 ) for 5 - ht 4 ( b ) , 5 - ht 4 ( c ) and 5 - ht 4 ( d ) respectively . the reverse primers for the second amplification cycle , which all contain an ecori restriction site at their 5 ′ end , were f82 [ 5 ′- gtcttctgggtcattgtc - 3 ′] ( seq id no . 16 ), f62 [ 5 ′- ttaggatggtttggtca - 3 ′] ( seq id no . 17 ) and f72 [ 5 ′- ctcaaggagctcaaaatc - 3 ′] ( seq id no . 18 ) for 5 - ht 4 ( b ) , 5 - ht 4 ( c ) and 5 - ht 4 ( d ) respectively . the pcr conditions were the same as those used for both of the amplification cycles of race - pcr . the fragments corresponding to the full length cdnas were purified on a 1 . 5 % agarose gel , subcloned in pgem - 7z ( cleaved with hindiii / ecori ) and sequenced to confirm the integrity of the coding sequence . 3 . primary structure of the splicing variants of the human receptor 5 - ht 4 . the deduced amino acid sequence of the various splicing variants is illustrated in fig1 starting from the splicing site leu 358 . within this region of the receptor , 5 - ht 4 ( a ) shares 93 % protein identity with the short form of the rat receptor 5 - ht 4s ( blondel et al ., 1997 ). the region between leu 358 and the last amino acid of 5 - ht 4 ( b ) presents 74 % protein identity with the corresponding region of the long form of the rat receptor 5 - ht 4l . however , the carboxy end of 5 - ht 4 ( b ) proved to have 18 amino acids less than its correspondent in rats , and the pkc phosphorylation consensus site described on the c - terminal side in the rat receptor 5 - ht 4l is missing ( gerald et al ., 1995 ). the splicing variants 5 - ht 4 ( c ) and 5 - ht 4 ( d ) have never been described in any species . it is interesting to note that the carboxy end of the receptor 5 - ht 4 ( c ) presents an unusually high number of putative phosphorylation sites : two casein kinase ii sites and one kinase c protein site , and a protein kinase a / protein kinase g phosphorylation consensus sequence . the 5 - ht 4 ( d ) isoform corresponds to an ultra - short form of the receptor , with a carboxy end truncation only two amino acids after the splicing site on the leu 358 . tissue specific expression of the receptor 5 - ht 4 splicing variants the expressions of the various 5 - ht 4 transcripts were analysed by amplification of cdna derived from rna isolated from diverse human tissues using a nested - rt - pcr technique . the tissue distribution was examined using two pairs of specific primers from each type of variant and two successive cycles of pcr amplification , in accordance with the conditions described in example 1 . the amplified products were identified using a specific internal oligonucleotide probe ( fig2 ). the 5 - ht 4 ( a ) , 5 - ht 4 ( b ) and 5 - ht 4 ( c ) isoforms are all expressed in the atrium , the brain and the intestines . the bladder and the liver each express detectable levels of only one receptor subtype ( 5 - ht 4 ( a ) and 5 - ht 4 ( b ) , respectively ). 5 - ht 4 ( d ) expression was detected only in the intestines . finally , the ventricles and the lungs do not express detectable amounts of any 5 - ht 4 isoform . the presence , in all the tissues , of cdna corresponding to the constitutively expressed β - actin gene and the absence of β - actin pcr products in control experiments without reverse transcriptase were also demonstrated ( fig2 ). pharmacological characterization of the receptor 5 - ht 4 splicing variants transiently expressed in cos - 7 cells pei ( polyethyleneimine mw 800 k d ) was obtained from fluka ( l &# 39 ; isle d &# 39 ; abeau chesnes , france ). ml 10302 ( 2 -( 1 - piperidyl ) ethyl 4 - amino - 5 - chloro - 2 - methoxy - benzoate ) and ml 10375 ( 2 -( cis - 3 , 5 - dimethyl - piperidino ) ethyl 4 - amino - 5 - chloro - 2 - methoxybenzoate ) were synthesized according to langlois et al ., 1994 ; yang et al ., 1997 . gr113808 (([ 1 -[ 2 -( methylsulphonyl ) amino ] ethyl ]- 4 - piperidyl ) methyl 1 - methyl - 1h - indole - 3 - carboxylate ) was obtained from the glaxo research group ( ware , hertfordshire , u . k .) and [ 3 h ] gr113808 was obtained from amersham ( arlington heights , ill .). the entire coding region of the cdnas of 5 - ht 4 ( a ) , 5 - ht 4 ( b ) , 5 - ht 4 ( c ) and 5 - t 4 ( d ) was subcloned into a mammalian expression vector prc / cmv ( invitrogen , carlsbad , calif .). the transfections were carried out using polyethyleneimine ( pei ) as described in boussif et al . ( 1995 ). the cells were transfected using a mixture of dna and of pei in a ratio of 20 μmol of pei / mg of dna in 0 . 9 % nacl . for the radioligand binding assays , the cos - 7 cells were seeded into culture flasks of 1 . 5 × 10 4 mm 2 one day before the transfection , at a density of 1 × 10 7 cells / flask , incubated for six hours with plasmid dna ( 150 μg / flask ) and collected 48 hours after transfection . for the measurement of camp formation , the cos - 7 cells were seeded onto twelve - well plates one day before transfection , at a density of 5 × 10 5 cells / well , incubated for six hours with plasmid dna ( 4 to 8 μg / well according to the experiments ) and assayed 24 hours after the transfection . the cells transfected with the receptor 5 - ht 4 cdna constructs were compared with the “ blank ”- transfected cells , which were exposed to the unmodified prc / cmv plasmid . each flask of cells intended for the radioligand binding assays was washed twice with a phosphate buffer ( pbs ). the cells were then scraped , collected and centrifuged at 300 g for five minutes . the pellet was resuspended in 2 . 5 ml of ice - cold hepes buffer ( 50 mm , ph 7 . 4 ) and homogenized with an ultraturax tissue grinder . the lysate was then centrifuged at 40 , 000 g for 20 minutes at 4 ° c . the resulting pellet was resuspended in 15 volumes of hepes buffer ( 50 mm , ph 7 . 4 ). the membrane preparations were maintained in ice and used within two hours for radioligand binding assays . the protein concentrations were determined by the method of lowry et al ., 1951 , using bovine serum albumin as standard . the radioligand binding studies were carried out in 500 μl of buffer ( 50 mm hepes , ph 7 . 4 ) containing 20 μl of competitor agent ( for the drug competition studies ), or of ml 10375 to give a final concentration of 10 μm ( for determining the nonspecific binding ), or of a buffer ( for determining total binding ), and 20 μl of [ 3 h ] gr113808 to give a final concentration of 50 % of k d and 50 μl ( 100 to 200 μg ) of membrane preparation . the saturation studies were performed using [ 3 h ] gr113808 at 9 different concentrations ranging from 0 . 01 to 3 . 5 nm . the tubes were incubated at 25 ° c . for 30 minutes . the reaction - was stopped by rapid filtration , under vacuum , through whatman gf / b filter paper using the brandel 48r cell harvester . the filters were presoaked in a solution of pei ( 0 . 1 %) to reduce the binding to the filters . the filters were then washed with an ice - cold buffer ( 50 mm tris - hcl , ph 7 . 4 ) and placed in 4 ml of “ ready safe ” scintillation cocktail ( beckman , fullerton , calif .) overnight . the radioactivity was measured using a beckman ls 6500 c liquid scintillation counter . the binding data were analysed by computer - assisted nonlinear regression analysis ( graph pad prism program , graph pad software , inc ., san diego , calif .). the saturation analysis using [ 3 h ] gr113808 revealed unique high affinity sites which can be saturated for the four splicing variants of the receptor ( fig3 ). similar k d values for [ 3 h ] gr113808 were found between the four isoforms ( 5 - ht 4 ( a ) , 0 . 23 ± 0 . 06 ; 5 - ht 4 ( b ) 0 . 62 ± 0 . 05 ; 5 - ht 4 ( c ) 0 . 30 ± 0 . 08 ; 5 - ht 4 ( d ) 0 . 14 ± 0 . 05 nm ). however , the density of the transiently expressed receptors in the cos - 7 cells ( b max ) varies considerably between the transfection assays ( 5 - ht 4 ( a ) , 214 ± 10 ; 5 - ht 4 ( b ) 1411 ± 55 ; 5 - ht 4 ( c ) 77 . 5 ± 5 . 8 ; 5 - ht 4 ( d ) 31 . 4 ± 2 . 8 fmol / mg of protein ). the nonspecific binding increases in nonlinear fashion with the increase in ligand concentration ( fig3 ). a series of 5 - ht 4 agonists and antagonists completely inhibits the specific binding of [ 3 h ] gr113808 to all the cloned isoforms of the receptor 5 - ht 4 . all the displacement curves are monophasic , giving a hill coefficient of 0 . 6 to 1 . 1 . the data summarized in table 1 demonstrate that the pharmacological profiles of all the cloned isoforms of the receptors 5 - ht 4 , in terms of order of potencies of the ligands tested , are very similar to those found for the receptors 5 - ht 4 studied in situ in the human atrium ( kaumann et al ., 1996 ) and the piglet atrium ( kaumann et al ., 1995 ); the human striatum ( reynolds et al ., 1995 ) and the human caudate nucleus ( waeber et al ., 1993 ), the rat striatum ( langois et al ., 1994 ; yang et al ., 1997 ) and the guinea - pig striatum ( ansanay et al ., 1996 ), and mouse colliculi ( ansanay et al ., 1996 ), or after expression of the cloned isoforms in cultured fibroblasts ( r5 - ht 4l ( gérald et al ., 1995 ; adham et al ., 1996 ); m5 - ht 4l ( claeysen et al ., 1996 )). it may also be noted that while each of the compounds tested bound to the isoforms a , b and c of the receptor 5 - ht 4 with similar affinity , the affinity for the 5 - ht 4 ( d ) isoform was usually higher by a factor of 2 to 4 ( table i ). stimulation of camp production by the 5 - ht 4 splicing variants transiently expressed in cos - 7 cells dmem was obtained from gibco - brl . pertussis toxin ( ptx ) was obtained from calbiochem . bimul ( endo - n - 8 - methyl - 8 - azabicyclo [ 3 . 2 . 1 ] oct - 3 - yl )- 2 , 3 - dihydro - 3 - ethyl - 2 - oxo - 1h - benzimidazole - 1 - carboxamide ) and zacopride ( 4 - amino - 5 - chloro - 2 - methoxy - n -( 1 - azabicyclo -[ 2 , 2 , 2 ] octo - 3 - yl ) benzamide , hydrochloride ) were synthesized in the laboratory by the authors of the present invention . renzapride ( brl 24924 ) ((+) endo - 4 - amino - 5 - chloro - 2 - methoxy - n -( 1 - azabicyclo [ 3 . 3 . 1 ] non - 4 - yl ) benzamide , hydrochloride ) was obtained from smithkline beecham . 5 - hydroxylryptamine ( 5 - ht ) and 5 - methoxytryptamine ( 5 - meot ) were obtained from aldrich ( l &# 39 ; isle d &# 39 ; abeau chesnes , france ) and all the other drugs were obtained from sigma ( l &# 39 ; isle d &# 39 ; abeau chesnes , france ). to measure intracellular camp accumulation , the transiently transfected cos - 7 cells were incubated , 24 hours after transfection , in dulbecco &# 39 ; s modified eagle medium ( dmem ) containing 5 mm theophylline , 10 mm hepes and 10 μm pargyline for 15 minutes at 37 ° c ., in the presence of 5 % co 2 . 5 - ht ( 1 μm ) or other serotoninergic agents ( 1 μm ) or forskolin ( 10 μm ) were added , and incubated for a further 15 minutes at 37 ° c . in the presence of 5 % co 2 . the reaction was stopped by aspiration of the medium and addition of 500 μl of ice - cold ethanol . after one hour at room temperature , the cells were scraped , and everything was collected and freeze - dried . the pellet was resuspended in 350 μl of pbs and centrifuged for 5 min at 300 g . the camp was quantified in the supernatant using a radioimmunological assay ( e . r . i . a . assay kit from pasteur diagnostics 79830 ). student &# 39 ; s t tests were carried out using the program quick test . to examine and compare the capacity of the 5 - ht 4 receptors to be coupled to adenylate cyclase , camp synthesis was assayed in the cos - 7 cells transiently transfected with the 5 - ht 4 ( a ) , 5 - ht 4 ( b ) , 5 - ht 4 ( c ) and 5 - ht 4 ( d ) cdnas . the basal camp values were not significantly different in the “ blank ”- transfected cells and the cells expressing the receptors 5 - ht 4 ( a ) , 5 - ht 4 ( b ) and 5 - ht 4 ( d ) , indicating that these expressed isoforms of the receptor had no intrinsic activity , regarding camp formation , in transiently transfected cells in the absence of agonists ( fig5 a ). however , the transient expression of the 5 - ht 4 ( c ) isoform led to a significant increase in the basal activity level of adenylate cyclase in the absence of agonists 5 - ht 4 ( fig5 a ). the latter result indicates that the expression of 5 - ht 4 ( c ) in the system described generates a spontaneously active receptor state . 5 - ht ( 1 μm ) has no effect on the basal activity of adenylate cyclase in the “ blank ”- transfected cos - 7 cells ( fig5 a ), indicating that the endogenous adenylate cyclase - coupled serotoninergic receptors are not present in these cells . in the cells expressing 5 - ht 4 ( a ) , 5 - ht 4 ( b ) , 5 - ht 4 ( c ) and 5 - ht 4 ( d ) the addition of 5 - ht ( 1 μm ) significantly increases the camp concentration by 82 %, 85 %, 64 % and 77 % respectively ( fig5 a ). forskolin ( 10 μm ), which is a direct activator of adenylate cyclase , induces similar increases in camp concentrations in the cells expressing the isoforms of the receptor 5 - ht 4 and in the “ blank ”- transfected cos - 7 cells ( fig5 a ), indicating that the maximum activation potential of the adenylate cyclase has not been affected in the cells expressing the receptors 5 - ht 4 . the receptor 5 - ht 4 agonists ml10302 and renzapride and the receptor 5 - ht 4 antagonist ml10375 have no significant effect on the basal camp levels in the “ blank ”- transfected cos - 7 cells ( fig5 b ). in the cells expressing the isoforms of the receptor 5 - ht 4 , ml10302 behaves as a weak 5 - ht 4 agonist , and shows only 28 to 34 % of the stimulatory effect of 5 - ht ( fig5 c ), despite a high affinity for the receptor ( table 1 ). in addition , the preincubation of the cells with 1 μm ml10302 , prior to the addition of 5 - ht ( 1 μm ), significantly antagonizes the capacity of 5 - ht to increase basal camp levels ( fig5 c ). as described by blondel et al . ( 1997 ), renzapride also behaves like a weak 5 - ht 4 agonist in the cells expressing the receptor isoform 5 - ht 4 ( a ) , and increases camp formation by only 56 % in the cells ( fig5 d ), despite an affinity for the receptor 5 - ht 4 ( a ) which is similar to that of 5 - ht ( table 1 ). however , renzapride behaves like a total agonist in the cells expressing the 5 - ht 4 ( b ) , 5 - ht 4 ( c ) and 5 - ht 4 ( d ) isoforms , in that the renzapride - induced camp formation mediated by these splicing variants of the receptor is not significantly different from the 5 - ht - induced camp formation ( fig5 d ). the 5 - ht 4 antagonist ml10375 ( yang et al ., 1997 ), at a concentration of 1 μm , had no significant effect on the basal camp levels in the cells expressing the receptor isoforms 5 - ht 4 ( a ) and 5 - ht 4 ( b ) , but significantly reduced the basal camp levels in the cells expressing the receptor isoforms 5 - ht 4 ( c ) and 5 - ht 4 ( d ) ( by 21 % and 24 % respectively , fig5 e ). in addition , the preincubation of - t : he cells expressing the receptor 5 - ht 4 isoforms with 1 μm ml10375 , prior to the addition of 5 - ht ( 1 μm ), antagonizes the capacity of 5 - ht to significantly increase the basal camp levels ( fig5 e ). the experiments correspond to the competition of various compounds for [ 3 h ] gr113808 binding to membranes of transiently transfected cos - 7 cells . for each isoform of the human receptor 5 - ht 4 , the [ 3 h ] gr113808 concentration was adjusted to 50 % of the k d value . the estimations of affinity are given as k i values in nm , and are determined from ic 50 values obtained by computer - assisted nonlinear curve analysis ( graphpad , prism software ). the k i values are representative of at least two determinations . the order of potencies of the drugs tested is identical for the four 5 - ht 4 isoforms , and is : gr113808 & gt ; ml10375 & gt ; ml10302 & gt ; bimul & gt ; renzapride = 5 - ht & gt ; 5 - meot & gt ; zacopride . ml10375 is capable of reducing the camp levels in the cells expressing the 5 - ht 4 ( c ) and 5 - ht 4 ( d ) isoforms in the absence of any stimulation of the receptor by 5 - ht 4 agonists . to test the hypothesis that ml10375 may constitute a reverse agonist for the human receptor 5 - ht 4 , the 5 - ht 4 ( c ) isoform was overexpressed in cos - 7 cells in order to increase the frequency of the spontaneously active state of the receptor . the cos - 7 cells were transfected with 4 to 8 μg of plasmid dna per well in order to obtain an increase in the expression levels of the receptor 5 - ht 4 ( c ) . the transfection of the cos - 7 cells using 4 μg and 8 μg of plasmid dna increased the basal camp level by 59 % and by 235 % respectively ( fig6 a ). 5 - ht ( 1 μm ) induced an increase of 64 % in the basal camp level in the cells transfected with 4 μg of plasmid dna , but did not significantly increase the basal camp level in the cells transfected with 8 μg of plasmid dna ( fig6 a ). the latter result indicates that in the cells overexpressing the receptor isoform 5 - ht 4 ( c ) , the frequency of the spontaneously active receptor prevents the stimulatory effect of the 5 - ht on adenylate cyclase activity . forskolin ( 10 μm ) induced similar increases in camp concentrations in the cells transfected with either 4 μg or 8 μg of plasmid dna and in the “ blank ”- transfected cells ( fig6 a ), indicating that the maximum activation potential of adenylate cyclase was not affected in the cells overexpressing the 5 - ht 4 ( c ) isoform . in the cos - 7 cells transfected with 4 μg and 8 μg of plasmid dna , ml10375 ( 1 μm ) decreased the basal camp values by 24 % and 62 % respectively ( fig6 b ). in the cells transfected with 4 μg of plasmid dna , the preincubation with ml10375 ( 1 μm ) antagonized the stimulatory effect of 5 - ht ( 1 μm ) on the basal camp ( fig6 b ). in the cells transfected with 8 μg of plasmid dna , the preincubatio : n with ml10375 ( 1 μm ) induced a decrease of 47 % in the basal camp level , even in the presence of 1 μm 5 - ht . ( fig6 b ). to test whether the effect of ml10375 on the camp level corresponds to an activation of the spontaneously active receptors 5 - ht 4 ( c ) or to an inhibition of adenylate cyclase activity via the gi protein - mediated regulatory pathway , the effect of ml10375 was examined in the cos - 7 cells transfected with 6 pg of 5 - ht 4 ( c ) plasmid dna , in the presence or absence of ptx ( 100 ng / ml ). the ptx treatment modified neither the basal camp level nor the 5 - ht - induced and forskolin - induced stimulation of camp in the transfected cells ( fig6 c ). in addition , the ptx treatment did not significantly modify the ml10375 - induced reduction in basal camp in the transfected cells ( fig6 d ). pharmacological characterization of the receptor 5 - ht 4 splicing variants stably expressed in cho cells ham - f12 medium was obtained from gibco - brl . ml 10375 ( 2 -( cis - 3 , 5 - dimethylpiperidino ) ethyl 4 - amino - 5 - chloro - 2 - methoxybenzoate ) was synthesized according to langlois et al ., 1994 ; yang et al ., 1997 . gr113808 (([ 1 -[ 2 -( methylsulphonyl )] amino ] ethyl ]- 4 - piperidyl )- methyl 1 - methyl - 1h - indole - 3 - carboxylate ) was obtained from the glaxo research group ( ware , hertfordshire , u . k .) and [ 3 h ] gr113808 was obtained from amersham ( arlington heights , ill .). the electroporator used was a gene pulser obtained from biorad . the entire coding region of the 5 - ht 4 ( c ) and 5 - ht 4 ( d ) cdnas was subcloned into a mammalian expression vector prc / cmv ( invitrogen , carlsbad , calif .). the cho cells are stably transfected by electroporation ( 250 v , 960 μf ) with the plasmid prc / cmv , containing the clone h5 - ht 4 ( c ) or h5 - ht 4 ( d ) , cleaved with the restriction enzyme kpn1 ( 10 μg of plasmid for 10 7 cells ). the cells are cultured in ham - f12 medium supplemented with 10 % of heat - inactivated foetal calf serum . after 48 hours , geneticin ( 1 . 25 mg / ml ) is added to the medium . twelve days later , the clones are individualized and cultured in 12 - well plates . clone selection is carried out by studying the stimulation of the camp formed in the presence of 1 μm serotonin . for the radioligand binding assays or for the measurement of camp formation , the cells transfected with the receptor 5 - ht 4 cdna constructs were compared with the “ blank ”- transfected cells , which were exposed to the unmodified prc / cmv plasmid . each flask of cells intended for the radioligand binding assays was washed twice with a phosphate buffer ( pbs ). the cells were then scraped , collected and centrifuged at 300 g for five minutes . the pellet was resuspended in 2 . 5 ml of ice - cold hepes buffer ( 50 mm , ph 7 . 4 ) and homogenized with an ultraturax tissue grinder . the lysate was then centrifuged at 40 , 000 g for 20 minutes at 4 ° c . the resulting pellet was resuspended in 15 volumes of hepes buffer ( 50 mm , ph 7 . 4 ). the membrane preparations were maintained in ice and used within two hours for radioligand binding assays . the protein concentrations were determined by the method of lowry et al ., 1951 , using bovine serum albumin as standard . the radioligand binding studies were carried out in 500 μl of buffer ( 50 mm hepes , ph 7 . 4 ) [ lacuna ] 20 μl of ml 10375 , to give a final concentration of 10 μm , ( for determining the nonspecific binding ), or of a buffer ( for determining total binding ), and 20 μl of [ 3 h ] gr113808 to give a final concentration of 50 % of k d and 50 μl ( 100 to 200 μg ) of membrane preparation . the saturation studies were performed using [ 3 h ] gr113808 at 9 different concentrations ranging from 0 . 01 to 3 . 5 nm . the tubes were incubated at 25 ° c . for 30 minutes . the reaction was stopped by rapid filtration , under vacuum , through whatman gf / b filter paper using the brandel 48r cell harvester . the filters were presoaked in a solution of pei ( 0 . 1 %) to reduce the binding to the filters . the filters were then washed with an ice - cold buffer ( 50 mm tris - hcl , ph 7 . 4 ) and placed in 4 ml of “ ready safe ” scintillation cocktail ( beckman , fullerton , calif .) overnight . the radioactivity was measured using a beckman ls 6500 c liquid scintillation counter . the binding data were analysed by computer - assisted nonlinear regression analysis ( graph pad prism program , graph pad software , increase ., san diego , calif .). the saturation analysis using [ 3 h ] gr113808 revealed unique high affinity sites which can be saturated for the two splicing variants of the receptor ( fig7 ). similar k d values for [ 3 h ] gr113808 were found between the two isoforms ( 5 - ht 4 ( c ) , 0 . 42 ± 0 . 04 nm ; 5 - ht 4 ( d ) , 0 . 23 ± 0 . 03 nm ). the density of the receptors expressed in the cho cells ( b max ) is 568 . 1 ± 17 . 4 fmol / mg of protein for 5 - ht 4 ( c ) and 179 . 9 ± 5 . 8 fmol / mg of protein for 5 - ht 4 ( d ) . the nonspecific binding increases in nonlinear fashion with the increase in ligand concentration ( fig7 ). stimulation of camp production by the 5 - ht 4 splicing variants stably expressed in cho cells dmem and ham - f12 media were obtained from gibco - brl . 5 - hydroxytryptamine ( 5 - ht ) was obtained from aldrich ( l &# 39 ; isle d &# 39 ; abeau chesnes , france ) and all the other drugs were obtained from sigma ( l &# 39 ; isle d &# 39 ; abeau chesnes , france ). to measure intracellular camp accumulation , the stably transfected cho cells were incubated in the ham - f12 medium containing 5 mm theophylline , 10 mm hepes and 10 μm pargyline , for 15 minutes at 37 ° c . in the presence of 5 % co 2 . 5 - ht ( 1 μm ) was added , and the cells were incubated for a further 15 minutes at 37 ° c . in the presence of 5 % co 2 . the reaction was stopped by aspiration of the medium and addition of 500 μl of ice - cold ethanol . after one hour at room temperature , the cells were scraped , and everything was collected and freeze - dried . the pellet was resuspended in 350 μl of pbs and centrifuged for 5 min at 300 g . the camp was quantified in the supernatant using a radio - immunological assay ( e . r . i . a . assay kit from immunotech , marseilles ). student &# 39 ; s t tests were carried out using the program quick test . to examine and compare the capacity of the 5 - ht 4 receptors to be coupled to adenylate cyclase , camp synthesis was assayed in the cho cells stably transfected with the h5 - ht 4 ( c ) and h5 - ht 4 ( d ) cdnas . the basal camp values were higher in the cells expressing the receptors h5 - ht 4 ( c ) ( 9 . 8 ± 1 . 6 pmol / well ) and h5 - ht 4 ( d ) ( 12 . 1 ± 0 . 9 pmol / well ) then in the “ blank ”- transfected cells ( 3 . 6 ± 0 . 4 pmol / well ), indicating that these expressed isoforms of the receptor possess an intrinsic activity , regarding camp formation , in stably transfected cells , in the absence of agonists . in the cells expressing h5 - ht 4 ( c ) and h5 - ht 4 ( d ) , the addition of 5 - ht ( 1 μm ) significantly increases the camp concentration ( by 91 % for h5 - ht 4 ( c ) and by 118 % for h5 - ht 4 ( d ) respectively , fig8 ), whereas 5 - ht has no effect on the “ blank ”- transfected cho cells ( fig8 ). production of rabbit polyclonal antibodies against synthetic peptides derived from the sequences of the receptor h5 - ht 4 isoforms . the table below presents the sequence of the synthetic peptides prepared from the receptor 5ht 4 isoforms : rabbits were immunized with 250 μg of the peptides f26v and g21v , which correspond to the first and the second extracellular loop of the receptor h5 - t 4 , and with 250 μg of peptides c24t and c21s , which correspond respectively to the c - terminal portion of the h5 - ht 4 ( a ) and h5 - ht 4 ( c ) isoforms . these peptides where injected in their original state in the presence of 3 mg of methylated bovine serum albumin and 1 ml of complete freund &# 39 ; s adjuvant . after three weeks , the rabbits received the same immunogens in the presence of incomplete freund &# 39 ; s adjuvant , followed by the same immunization one month later . the antisera were sampled one week after the final injection . the peptides y23f , which corresponds to the c - terminal portion of the h5 - ht 4 ( b ) isoform , and c7f , which corresponds to the c - terminal portion of the h5 - t 4 ( d ) isoform , were coupled to bromoacetylated serum albumin , before immunization with 2 . 3 μmol of peptide coupled to 3 . 4 mg of modified bovine serum albumin . the first immunization , in complete freund &# 39 ; s adjuvant , is followed by two other injections in incomplete freund &# 39 ; s adjuvant as described in the previous paragraph . the antisera were sampled one week after the final injection . fig9 gives the optical density values of an immunoenzymatic assay of elisa type on the respective peptides adsorbed ( 5 μg / ml in a carbonate buffer at ph = 9 . 5 ) onto maxisorb plates ( nunc , denmark ). the antisera were incubated for one hour at 37 ° c ., and revealed with a peroxidase - coupled goat anti - rabbit igg antibody conjugate ( dilution { fraction ( 1 / 10 , 000 )}) and the substrate h 2 o 2 - abts . demonstration , using the anti - g21v antibody , of the presence of the receptor 5 - ht 4 ( c ) in the stably transfected cho cells the presence of the receptor 5 - ht 4 ( c ) in the cho cells stably expressing this isoform of the receptor was demonstrated by western blot using the common g21v antibody developed in the context of this study . [ lacuna ] the 5ht 4 ( a ) ( fig1 , line 2 ) and 5 - ht 4 ( c ) ( fig1 , line 3 ) isoforms . the cho cells were transfected with the expression vector encoding the form ( c ) of the receptor 5 - ht 4 , and were selected for their neomycin resistance . for comparison , other cho cells were transfected with the expression vector encoding the form ( a ) of the receptor 5 - ht 4 . fifty μg of proteins originating from membrane extracts are separated on a 10 % polyacrylamide gel , and then transferred onto nitrocellulose membrane . after a 16 - h incubation in the presence of 60 μg of anti - 5 - ht 4 ( g21v ) antibodies , the blot is revealed by chemiluminescence ( ecl , amersham , arlington heights , ill .) and scanned . line 1 of fig1 indicates the result obtained on the control cho cells , in which no labelling was detected ; lines 2 and 3 come from clones of cho cells overexpressing respectively the receptors 5 - ht 4 ( a ) and 5 - ht 4 ( c ) . a band which migrates approximately with the size of 60 kda is visualized . demonstration , using the anti - c21s and anti - c7f antibodies , of the presence respectively of the receptors h5 - ht 4 ( c ) and h5 - ht 4 ( d ) in the stably transfected cho cells using antibodies directed against the c - terminal sequence specific for the receptor h5 - ht 4 ( c ) ( anti - c21s ) and for the receptor h5 - ht 4 ( d ) ( anti - c7f ), the presence of these respective receptors on homogenates of cells expressing the two isoforms could be demonstrated . the specificity of the protein bands recognized was determined by the extinction of the response on immunoblot in the presence of inhibiting peptides ( fig1 a and 11 k ), second lines ). the h5 - ht 4 ( c ) isoform is recognized in its glycosylated and nonglycosylated form ( corresponding to molecular weights of 60 and 44 kda ); the h5 - ht 4 ( d ) isoform is recognized in its nonglycosylated form ( corresponding to a molecular weight of 40 kda ) ( fig1 a and 11 b , first lines ). adham n ., gerald c ., schechter l ., vaysse p ., weinshank r ., and branchek t . 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