Role of P2Y1 purinoceptor in ADP-induced platelet activation.

ADP acts as an agonist of platelet aggregation via specific receptors which are still to be characterised. Amplification by PCR of a human platelet cDNA library confirmed the presence of mRNA of the P2Y1 receptor in platelets. In order to determine if these P2Y1 receptors were involved in ADP-induced platelet activation, we determined the effects of A3P5PS, an antagonist of the P2Y1 receptor, on the binding of [33P]2-MeS-ADP, a potent analogue of ADP. We found that A3P5PS displaced about 27% of [33P]2-MeS-ADP binding, a receptor population which has been shown to be resistant to treatment with clopidogrel, a selective anti-ADP agent. A3P5PS specifically inhibited 2-MeS-ADP-induced shape change and calcium increase but did not affect adenylyl cyclase down-regulation. 2-MeS-ADP-induced platelet aggregation was also inhibited by A3P5PS but was restored when platelets were further activated by serotonin, a non-aggregating compound, therefore suggesting that P2Y1-mediated stimulation is an absolute prerequisite for ADP to induce platelet aggregation and a key event for platelet activation and aggregation to occur. These results therefore show that ADP-induced aggregation cannot be attributed to activation of P2Y1 alone, but must be attributed to the simultaneous activation of the high affinity receptor (P2Y1) and a low affinity receptor of ADP (still to be discovered), each of them essential, but neither able to trigger aggregation alone.

NPY receptor subtype in the rabbit isolated ileum.

1. The purpose of this work was to verify the hypothesis that the rabbit ileum is a selective preparation for the NPY Y5 receptor by using new selective antagonists recently synthesized. Spontaneous contractions of the rabbit isolated ileum were recorded and binding experiments were performed in cells expressing the human NPY Y1, Y2, Y4 or Y5 receptor subtype. 2. NPY analogues produced a concentration-dependent transient inhibition of the spontaneous contractions of the rabbit ileum with the following order of potency hPP > rPP > PYY > or = [Leu31,-Pro34]-NPY > NPY >> NPY13-36. Pre-exposure to rPP, PYY, [Leu31,Pro34]-NPY or NPY (but not NPY13-36) inhibited the effect of subsequent administration of hPP suggesting cross-desensitization of the preparation. The apparent affinity of the various agonists studied was correlated to the affinity reported for the human Y4 receptor subtype (and to a lesser extent for the rat Y4 subtype) but not to the affinity for the Y5 receptor subtype. 3. BIBO 3304, a selective NPY Y1 receptor antagonist, and CGP 71683A, a selective NPY Y5 receptor antagonist, did not affect the response to hPP. JCF 109, another NPY Y5 receptor antagonist, produced an inhibition of the response to hPP but only at the highest dose tested (10 microM) which also, by itself, produced intrinsic inhibitory effects. 4. 1229U91, a non-selective ligand for Y1, Y2, Y4 and Y5 receptors with high affinity toward the Y1 and Y4 receptor subtypes, produced a concentration-dependent transient inhibition of the spontaneous contractions of the rabbit ileum and a dose-dependent inhibition of the response to hPP (apparent pKB: 7.2). 5. These results suggest that in the rabbit ileum, the NPY receptor involved in the inhibition of the spontaneous contractile activity is a NPY Y4 receptor subtype.

[125I]-S36057: a new and highly potent radioligand for the melanin-concentrating hormone receptor.

Shortened, more stable and weakly hydrophobic analogues of melanin-concentrating hormone (MCH) were searched as candidates for radioiodination. Starting from the dodecapeptide MCH(6 - 17), we found that: (1) substitution of Tyr(13) by a Phe residue; (2) addition of a 3-iodo-Tyr residue at the N-terminus; and (3) addition of a hydrophilic spacer 8-amino-3,6-dioxyoctanoyl between the 3-iodo-Tyr and MCH(6 - 17) (compound S36057), led to an agonist more potent than MCH itself in stimulating [35S]-GTPgammaS binding at membranes from HEK293 cells stably expressing the human MCH receptor. Specific binding of [125I]-S36057 was found in HEK293 and CHO cell lines stably expressing the human MCH receptor. This radioligand recognized a similar number of binding sites (ca. 800 fmol mg(-1)) than [125I]-[3-iodo Tyr(13)]-MCH. However, the K(D) for [125I]-S36057 obtained from saturation studies (0.037 nM) or from binding kinetics (0.046 nM) was at least 10 fold higher to that of [125I]-[3-iodo Tyr(13)]-MCH (0.46 nM). Affinities determined for a series of MCH analogues were similar with both radioligands, S36057 being the most potent compound tested (K(i)=0.053 nM). Finally, [125I]-S36057 also potently labelled the MCH receptor in membranes from whole rat brain (K(D) 0.044 nM, B(max)=11 fmol mg(-1)). In conclusion, [125I]-S36057 is a more potent and more stable radioligand than [125I]-[3-iodo Tyr(13)]-MCH that will represent a reliable tool for binding assays in the search of novel MCH ligands. It should also provide great help for autoradiographic studies of the MCH receptor distribution in the central nervous system.

NPY receptor subtypes involved in the contraction of the proximal colon of the rat.

Experiments were designed to determine the receptor subtype(s) involved in the contraction of the rat proximal colon to NPY. In this tissue, mRNA of Y2 and Y4 NPY receptor subtypes were highly expressed, whereas Y5 mRNA levels were very low and Y1 mRNA levels were intermediate. NPY analogues induced contractions with the following order of potency: rPP > hPP = PYY = NPY = [Leu31,Pro34]NPY > NPY(2-36) = [D-Trp32]NPY > NPY(33-36). Responses to NPY, PYY and NPY(13-36) were not or partially affected by tetrodotoxin, in contrast to the responses to [Leu31,Pro34]NPY, rPP, hPP and [D-Trp32]NPY which were fully blocked. Atropine did not inhibit the contractions to NPY, PYY and [Leu31,Pro34]NPY but significantly affected those to NPY(13-36), [D-Trp32]NPY, rPP and hPP. The specific Y1 receptor antagonist BIBP 3226 was ineffective but JCF 104 and JCF 105 (two compounds with preferential affinity toward the hY5 receptor versus the hY1 or hY2 receptor) abolished the contractions provoked by the NPY analogues. These results suggest that NPY activates three receptor subtypes, a Y2 subtype possibly by a direct action on the smooth muscle cells, as well as a Y4 and a Y5 (or 'Y5-like') subtype which, respectively, release acetylcholine and an unknown neurotransmitter.

Establishment and characterisation of murine cells constitutively expressing the fusion, nucleoprotein and matrix proteins of measles virus.

To advance our understanding of the immunobiology of measles virus (MV) infections, we have investigated the possibility of establishing cell lines constitutively expressing the individual MV antigens. In contrast to previously published studies, we show that it is possible to establish cell lines expressing high levels of fusion (F), nucleoprotein (NP) and matrix (M) MV proteins. Once cloned, the cell lines were stable with high levels of expression for more than six months. The size and cell distribution of the NP and F proteins were similar to those observed in MV- or vaccinia-MV recombinant-infected cells. In contrast, the distribution of the M protein, although being similar to that of MV-infected cells, differed from that of Vaccinia-M recombinant virus-infected cells. Preliminary results suggest that these cell lines will be useful tools for studying the contribution of individual MV antigens to the cell-mediated immune response to this virus.

Binding of prostaglandins to human PPARgamma: tool assessment and new natural ligands.

The peroxisome proliferator-activated receptors (PPAR) form a family of nuclear receptors with a wide variety of biological roles from adipogenesis to carcinogenesis. More ligands (agonist and antagonist) are needed to explore the multiple functions of PPAR, particularly PPARgamma. In order to complete such ligand screening, a binding test should be assessed versus the classical transactivation reporter gene assay. In the present work, the full-length human PPARgamma protein as well as its ligand binding domain portion were expressed in Escherichia coli. Bacterial membrane preparations expressing those constructs were characterized using a classical binding competition assay [3H]rosiglitazone as the radioligand. When the receptor preparations were soluble, binding had to be measured with a new alternative method. The systems were assessed using a series of reference PPAR (alpha, beta and gamma) ligands. The full-length human PPARgamma fused to glutathione-S-transferase, expressed in E. coli and tested as a bacterial membrane-bound protein led to the most accurate results when compared to the literature. Furthermore, in an attempt to complete the panel of natural PPARgamma ligands, 29 commercially available prostaglandins were screened in the binding assay. Prostaglandins H(1) and H(2) were found to be modest ligands, however as potent as 15Delta(12-14 )prostaglandin J(2). These results were confirmed in the classical transactivation assay. The fact that these three prostaglandins were equally potent, suggests new pathways of PPARgamma-linked gene activation.

Measles virus hemagglutinin induces an Ld-restricted CD8+ cytotoxic T lymphocyte response to two specific epitopes.

Balb/c mice (H-2d) immunized with a vaccinia virus recombinant expressing the measles virus hemagglutinin (MV HA) induced a strong CD8+ cytotoxic T lymphocyte (CTL) response which was Ld-restricted. With reference to the predictive motif for Ld-restricted epitopes, a number of nonameric peptides derived from the primary sequence of the MV HA were tested for their ability to be restricted to the CTLs by P815 cells. Two peptides corresponding to amino acids 343-351 and 544-552 of the HA primary sequence sensitized target cells for lysis by the HA-specific CTLs.

Measles virus fusion: role of the cysteine-rich region of the fusion glycoprotein.

Measles virus (MV) fusion requires the participation of both the fusion (F) and hemagglutinin (H) glycoproteins. The canine distemper virus fusion protein (CDVF) cannot substitute for the measles virus fusion protein (MVF) in this process. Introduction of restriction enzyme sites into the cDNAs of CDVF and MVF by site-directed mutagenesis facilitated the production of chimeric F proteins which were tested for their capacity to give fusion when coexpressed with MVH. Fusion resulted when the amino-terminal half of the MVF cysteine-rich region was transferred to CDVF.

Passively administered antibody suppresses the induction of measles virus antibodies by vaccinia-measles recombinant viruses.

We have used vaccinia-measles recombinant viruses to study vaccination in the presence of pre-existing antibody. When mice were vaccinated with recombinants expressing either the haemagglutinin (H) or fusion (F) measles virus (MV) proteins, the humoral response to the MV protein was suppressed by passively administered polyclonal antibody. However, individual monoclonal antibodies (H or F) did not affect the response. Mice whose anti-MV antibody response to H or F was initially suppressed by passive administration of anti-MV antibody were revaccinated 120 days later and gave a normal humoral response to the MV proteins. The VV-H recombinant induces a strong class I CTL response in Balb/c mice. This was not affected by the presence of levels of anti-MV antibody which inhibited the humoral response.

A leucine zipper structure present in the measles virus fusion protein is not required for its tetramerization but is essential for fusion.

The biological role of a leucine zipper motif present in the measles virus fusion (F) protein has been investigated. This motif is present in all paramyxovirus F proteins, all coronavirus spike proteins and many if not all retrovirus envelope proteins. By analogy to its role in certain transcription factors, it has been suggested that the motif may be responsible for the oligomerization of these viral membrane proteins. In this study, one, two or four heptadic leucines in the motif were substituted using site-directed mutagenesis. We found that fusion is prevented when all four heptadic leucines present in the motif are mutated whereas cellular transport and the oligomeric state of the F protein are unaffected.

Measles virus antigens induce both type-specific and canine distemper virus cross-reactive cytotoxic T lymphocytes in mice: localization of a common Ld-restricted nucleoprotein epitope.

We have studied the induction of the cytotoxic T lymphocyte (CTL) response to measles virus (MV) antigens expressed as vaccinia virus (VV) recombinants in a murine model. In C3H mice (H-2k) only the nucleoprotein (NP) induced a CTL response and this was shown to be cross-reactive with the closely related canine distemper virus (CDV). The presentation of this antigen was shown to be Kk-restricted. In BALB/c mice (H-2d), both the haemagglutinin (HA) and the NP induced a strong CTL response, the former being serotype-specific, whereas the latter cross-reacted with CDV. Both responses were found to be Ld-restricted. Based on the prediction for Ld T cell motifs, we tested a number of MV NP-derived nonapeptides for their capacity to sensitize P815 cells (H-2d) for lysis by spleen cells from VV-NP-immunized mice. One of these peptides, comprising amino acids 281 to 289 (Tyr-Pro-Ala-Leu-Gly-Leu-His-Glu-Phe) was as effective as cells expressing the complete NP protein. This motif is conserved in the CDV NP.

Serotype-specific and canine distemper virus cross-reactive H-2Kk-restricted cytotoxic T lymphocyte epitopes in the measles virus nucleoprotein.

Immunization of C3H mice with a vaccinia virus (VV) recombinant expressing the measles virus (MV) nucleoprotein (NP) induces an H-2Kk-restricted cytotoxic T lymphocyte (CTL) response. With reference to the predicted peptide epitope motifs binding to this MHC class I molecule, we have used synthetic peptides derived from the primary sequence of the MV NP to establish the identity of the Kk-restricted CTL epitopes. Two octameric peptides, LDRLVRLI (aa 52-59) and VESPGQLI (aa 81-88) sensitized P815-Kk cells to lysis by MV NP-induced CTLs. In contrast to LDRLVRLI, the sequence VESPGQLI is also present in the primary sequence of the NP from the closely related canine distemper virus (CDV). In vitro stimulation of spleen cells from VV NP-immunized mice with the peptides showed that peptide VESPGQLI induced CTLs which could also lyse CDV-infected cells, whereas peptide LDRLVRLI could only lyse cells presenting the MV protein. Different concentrations of peptides were used, and the lysis efficiencies for both epitopes were shown to be of the same order. The value of predicative motifs for determining MHC class I CTL epitopes is discussed.

Cholera toxin B stimulates systemic neutralizing antibodies after intranasal co-immunization with measles virus.

An efficient mucosal vaccination has a number of obvious advantages over invasive routes of immunization. The immune response to measles virus (MV) was investigated after intranasal and intragastric co-immunization of mice with cholera toxin B (CTB) as an adjuvant. High titres of virus-specific IgG antibodies and a transient IgA response were detected in the sera after intranasal but not after intragastric immunization when CTB was used. In the presence of CTB, higher titres were reached with less antigen and fewer intranasal boosts. Neutralizing antibodies were found in all animals only after co-immunization with MV and CTB. In the nasal wash and the saliva, IgG and IgA titres were significant only in the MV plus CTB groups; IgG levels were comparable to those found after intraperitoneal (i.p.) immunization with complete Freund's adjuvant. Specific IgA was detected in the mucosal fluids only after intranasal immunization with MV plus CTB but not after i.p. or intragastric immunization. The antibody response consisted of 99% IgG1 after MV immunization. In the CTB groups 10% IgG2b and 1% IgG2a were detected in addition to the predominant IgG1 antibodies.

Formaldehyde inactivation of measles virus abolishes CD46-dependent presentation of nucleoprotein to murine class I-restricted CTLs but not to class II-restricted helper T cells.

To induce an MHC-restricted specific CTL or Th response, an antigen must be delivered into the appropriate cellular compartment. We explored the role of CD46 in the presentation of measles virus (MV) nucleoprotein (NP) to murine NP-specific and MHC Class I-restricted polyclonal CTLs and the effect of inactivating MV by uv or formaldehyde. CD46(-)- and CD46(+)-transfected murine cells were used as target cells. After MV infection, only the targets which expressed CD46 were lysed by NP-specific class I-restricted CTLs. When MV was uv-inactivated, NP presentation by MHC class I molecules was retained but could be blocked by fusion inhibitors which block virus cell entry. When MV was inactivated with formaldehyde, NP was no longer presented by MHC class I molecules, although it was still presented by MHC class II molecules to a NP-specific class II-restricted T cell hybridoma. These data show that MV binding to the CD46 molecule is a prerequisite for virus-to-cell fusion and that cytosolic delivery of NP is necessary for presentation by class I molecules. Moreover, formaldehyde inactivation of virus induces the loss of class I-restricted presentation of NP due to selective abrogation of fusion and cytosolic delivery of NP.

Analysis of the contribution of CTL epitopes in the immunobiology of morbillivirus infection.

In Balb/c (H-2d) mice, the nucleoprotein (NP) of measles virus (MV) induces a MHC class I restricted-CTL response to a single 9-amino-acid epitope (aa 281--289). This L(d)-restricted epitope is also present in the NP of the closely related canine distemper virus (CDV). To investigate whether this epitope is immunologically effective when it is present within the primary sequence of a nonviral protein, we have incorporated the 281--289 motif into the human CD36 protein. When cells are infected with vaccinia virus (VV) recombinants expressing this protein, CD36NP, the MV epitope is correctly processed and the cells are lysed by MVNP-specific CTLs. In vivo, VV-CD36NP induced CTLs which protected mice from a lethal dose of CDV, but did not block virus replication. The MVNP contains four other potential L(d)-restricted motifs. To investigate if these could be utilized in the absence of the dominant epitope, a mutant NP was produced in which one of the anchor residues in the aa 281--289 motif was mutated. Cells infected with a VV recombinant expressing this protein (VV-NP F289S) were only poorly lysed by MVNP-specific CTLs. Similarly, immunization of Balb/c mice with VV-NP F289S induced a lower level of CTL activity compared to the VV-NP, but the activity was now directed to three other epitopes. When mice were vaccinated with VV-NP F289S they were only partially protected from a lethal CDV challenge. The significance of these results for MV vaccine development is discussed.

Identification of the melatonin-binding site MT3 as the quinone reductase 2.

The regulation of the circadian rhythm is relayed from the central nervous system to the periphery by melatonin, a hormone synthesized at night in the pineal gland. Besides two melatonin G-coupled receptors, mt(1) and MT(2), the existence of a novel putative melatonin receptor, MT(3), was hypothesized from the observation of a binding site in both central and peripheral hamster tissues with an original binding profile and a very rapid kinetics of ligand exchange compared with mt(1) and MT(2). In this report, we present the purification of MT(3) from Syrian hamster kidney and its identification as the hamster homologue of the human quinone reductase 2 (QR(2), EC ). Our purification strategy included the use of an affinity chromatography step which was crucial in purifying MT(3) to homogeneity. The protein was sequenced by tandem mass spectrometry and shown to align with 95% identity with human QR(2). After transfection of CHO-K1 cells with the human QR(2) gene, not only did the QR(2) enzymatic activity appear, but also the melatonin-binding sites with MT(3) characteristics, both being below the limit of detection in the native cells. We further confronted inhibition data from MT(3) binding and QR(2) enzymatic activity obtained from samples of Syrian hamster kidney or QR(2)-overexpressing Chinese hamster ovary cells, and observed an overall good correlation of the data. In summary, our results provide the identification of the melatonin-binding site MT(3) as the quinone reductase QR(2) and open perspectives as to the function of this enzyme, known so far mainly for its detoxifying properties.

Characterization and regulation of a CHO cell line stably expressing human serotonin N-acetyltransferase (EC 2.3.1.87).

Current melatonin research is essentially based on the finding of new molecular tools, including synthetic or natural agonists and antagonists for the melatonin receptors and synthetic inhibitors of the enzymes involved in its biosynthesis. Indeed, the use of these compounds will improve our understanding of some of the numerous mechanisms of action of melatonin. The present report deals with the establishment and description of a new cell line expressing in a stable manner human arylalkylamine-N-acetyltransferase (AANAT, E.C.2.3.1.87). This new cellular system permits one to check the capacity of newly discovered inhibitors to penetrate the cell and reach their target. Some emphasis is put on inhibitors of the bromoacetyltryptamine family since these precursor compounds form in situ bisubstrate inhibitors with strong affinity for the human enzyme. AANAT is known to undergo complex and rapid regulation by a subtle balance between extremely fast catabolism and protection against it, both due to serine phosphorylation. In the present report, this phosphorylation is shown to occur in vitro after incubation with several kinases (rho-kinase, chk-1, protein kinase A) but not with protein kinase C. Phosphorylation enhances the specific activity of the enzyme by a factor of two to five. This phosphorylation is also shown to occur after treatment of the cell with compounds such as forskolin and rolipram that enhance or protect the intracellular pool of cAMP or the cell-permeable cAMP analogue, dioctanoyl-cAMP. The specificity of the cellular model was assessed using a series of substrates and inhibitors of AANAT already described in the literature, and the characteristics of this cellular system are shown to correspond with those reported for the purified enzyme. This cell line was used to screen libraries of compounds in a living system and led to the discovery of several potent specific and non-toxic AANAT inhibitors.

Development of new assays and improved procedures for the purification of recombinant human chymase.

Chymase mediates a major alternative way of angiotensin II production from angiotensin I beside angiotensin converting enzyme in the final step of the renin-angiotensin system. This enzyme is also involved in other physio-pathological processes such as angiogenesis, atherosclerosis and inflammation. Several purification attempts of natural or recombinant chymase were reported in the literature. Most of these reports were not successful in obtaining the recombinant enzyme in a highly active form and in large quantity. In the present study, we describe a facile route for the purification of the human recombinant chymase. Chymase being produced as inactive prochymase, to be cathepsin C-activated, newly raised anti-chymase Ig were used to follow the purification. In order to complete the available tools for the search of chymase inhibitors, we developed and assessed a new 96-well plate based assay for the measurement of enzyme activity, as well as a low throughput, HPLC-based one. The assays used an original derivative of angiotensin I, or the native hormone. Chymase was produced in CHO cells and appropriately matured. The amount of enzyme obtained at the end of the process is compatible with the medium-throughput screening (up to 10,000 points per day), about 800 microg x L(-1) of culture medium with a specific activity of 6.16 mmol of angiotensin I cleaved per minute per mg of protein. All the biological and technical tools are now available for the discovery of new classes of chymase inhibitors.

Truncated isoforms inhibit [3H]prazosin binding and cellular trafficking of native human alpha1A-adrenoceptors.

We have identified from human liver eight alpha(1A)-adrenoceptor (alpha(1A)-AR) splice variants that were also expressed in human heart, prostate and hippocampus. Three of these alpha(1A)-AR isoforms (alpha(1A-1)-AR, alpha(1A-2a)-AR and alpha(1A-3a)-AR) gave rise to receptors with seven transmembrane domains (7TMalpha(1A)-AR). The other five (alpha(1A-2b)-AR, alpha(1A-2c)-AR, alpha(1A-3c)-AR, alpha(1A-5)-AR and alpha(1A-6)-AR) led to truncated receptors lacking transmembrane domain VII (6TMalpha(1A)-AR). The 7TMalpha(1A)-AR isoforms transiently expressed in COS-7 cells bound [(3)H]prazosin with high affinity (K(d) 0.2 nM) and mediated a noradrenaline (norepinephrine)-induced increase in cytoplasmic free Ca(2+) concentration, whereas the 6TMalpha(1A)-AR isoforms were incapable of ligand binding and signal transduction. Immunocytochemical studies with N-terminal epitope-tagged alpha(1A)-AR isoforms showed that the 7TMalpha(1A)-AR isoforms were present both at the cell surface and in intracellular compartments, whereas the 6TMalpha(1A)-AR isoforms were exclusively localized within the cell. Interestingly, in co-transfected cells, each truncated alpha(1A)-AR isoform inhibited [(3)H]prazosin binding and cell-surface trafficking of the co-expressed 'original' 7TMalpha(1A-1)-AR. However, there was no modification of either the [(3)H]prazosin-binding affinity or the pharmacological properties of alpha(1A-1)-AR. Immunoblotting experiments revealed that co-expression of the alpha(1A-1)-AR with 6TMalpha(1A)-AR isoforms did not impair alpha(1A-1)-AR expression. Therefore the expression in human tissues of many truncated isoforms constitutes a new regulation pathway of biological properties of alpha(1A)-AR.

Genomic organization and characterization of splice variants of the human histamine H3 receptor.

In the present paper we report the genomic organization of the human histamine H3-receptor gene, which consists of four exons spanning 5.5 kb on chromosome 20. Using PCR, six alternative splice variants of the H3 receptor were cloned from human thalamus. These variants were found to be coexpressed in human brain, but their relative distribution varied in a region-specific manner. These isoforms displayed either a deletion in the putative second transmembrane domain (TM), H3(DeltaTM2, 431aa) or a variable deletion in the third intracellular loop (i3), H3(Deltai3, 415aa), H3(Deltai3, 365aa), H3(Deltai3, 329aa) and H3(DeltaTM5+Deltai3, 326aa). In order to determine the biological role of the H3 receptor variants compared with the 'original' H3(445aa) receptor, three isoforms, namely H3(445aa), H3(DeltaTM2, 431aa) and H3(Deltai3, 365aa), were expressed in CHO cells and their pharmacological properties were investigated. Binding studies showed that H3(DeltaTM2, 431aa) transiently expressed in CHO cells was unable to bind [125I]iodoproxyfan, whereas both the H3(445aa) and H3(Deltai3, 365aa) receptors displayed a high affinity for [125I]iodoproxyfan [K(d)=28+/-5 pM (n=4) and 8+/-1 pM (n=5) respectively]. In addition, H3(Deltai3, 365aa) possessed the same pharmacological profile as the H3(445aa) receptor. However, in CHO cells expressing H3(Deltai3, 365aa), H3 agonists did not inhibit forskolin-induced cAMP production, stimulate [35S]guanosine 5'-[gamma-thio]triphosphate ([35S]GTP[S]) binding or stimulate intracellular Ca(2+) mobilization. Therefore the 80-amino-acid sequence located at the C-terminal portion of i3 plays an essential role in H3 agonist-mediated signal transduction. The existence of multiple H3 isoforms with different signal transduction capabilities suggests that H3-mediated biological functions might be tightly regulated through alternative splicing mechanisms.