Michael Brüss

Expression and pharmacological profile of the human organic cation transporters hOCT1, hOCT2 and hOCT3

Organic cation transporters (OCTs) are involved in the elimination of monoamines and cationic xenobiotics. To examine whether some cell lines express several different OCTs, we investigated seven human cell lines for the mRNA expression pattern of the human (h) transporters hOCT1, hOCT2 and hOCT3. hOCT1 mRNA was found in all cell lines, six additionally expressed hOCT3 and only two cell lines contained all three hOCTs. Among the three OCTs only for the OCT3 (also designated as ‘uptake2’ or ‘extraneuronal monoamine transporter’) ‘selective’ inhibitors are described in the literature. The affinities of the OCT3 inhibitors for the other two OCTs are largely unknown. Therefore, we compared the potencies of eight compounds as inhibitors of hOCT‐mediated uptake of the organic cation [3H]‐1‐methyl‐4‐phenylpyridinium ([3H]‐MPP+) in human embryonic kidney 293 (HEK293) cells stably expressing hOCT1, hOCT2 or hOCT3. Decynium‐22 inhibited hOCT3 with 10 fold higher potency than hOCT1 and hOCT2. Corticosterone was about 100 fold more potent as inhibitor of hOCT3 than of hOCT1 or hOCT2, and O‐methylisoprenaline (OMI) inhibited almost exclusively hOCT3. Progesterone and β‐Oestradiol preferentially inhibited hOCT3 and hOCT1, whereas prazosin was a potent inhibitor of hOCT1 and hOCT3. Phenoxybenzamine (PbA) inhibited with about equal apparent potency all three hOCTs, whereas the PbA derivative SKF550 ((9‐fluorenyl)‐N‐methyl‐β‐chloroethylamine) preferentially inhibited hOCT3 and hOCT2. PbA reversibly inhibited hOCT1 and irreversibly hOCT2 and hOCT3; SKF550 also irreversibly inhibited hOCT3 but hOCT2 in a reversible manner. These compounds enable a functional discrimination of the three hOCTs: hOCT1 is selectively inhibited by prazosin, reversibly inhibited by PbA and it is not sensitive to inhibition by SKF550 and OMI; hOCT2 is reversibly inhibited by SKF550, irreversibly by PbA and not by prazosin, β‐oestradiol and OMI, whereas hOCT3 is selectively inhibited by corticosterone, OMI and decynium22.

Direct inhibition by cannabinoids of human 5‐HT3A receptors: probable involvement of an allosteric modulatory site

Excised outside‐out patches from HEK293 cells stably transfected with the human (h) 5‐HT3A receptor cDNA were used to determine the effects of cannabinoid receptor ligands on the 5‐HT‐induced current using the patch clamp technique. In addition, binding studies with radioligands for 5‐HT3 as well as for cannabinoid CB1 and CB2 receptors were carried out. The 5‐HT‐induced current was inhibited by the following cannabinoid receptor agonists (at decreasing order of potency): Δ9‐THC, WIN55,212‐2, anandamide, JWH‐015 and CP55940. The WIN55,212‐2‐induced inhibition was not altered by SR141716A, a CB1 receptor antagonist. WIN55,212‐3, an enantiomer of WIN55,212‐2, did not affect the 5‐HT‐induced current. WIN55,212‐2 did not change the EC50 value of 5‐HT in stimulating current, but reduced the maximum effect. The CB1 receptor ligand [3H]‐SR141716A and the CB1/CB2 receptor ligand [3H]‐CP55940 did not specifically bind to parental HEK293 cells. In competition experiments on membranes of HEK293 cells transfected with the h5‐HT3A receptor cDNA, WIN55,212‐2, CP55940, anandamide and SR141716A did not affect [3H]‐GR65630 binding, but 5‐HT caused a concentration dependent‐inhibition. In conclusion, cannabinoids stereoselectively inhibit currents through recombinant h5‐HT3A receptors independently of cannabinoid receptors. Probably the cannabinoids act allosterically at a modulatory site of the h5‐HT3A receptor. Thus the functional state of the receptor can be controlled by the endogenous ligand anandamide. This site is a potential target for new analgesic and antiemetic drugs.

Differential pharmacological in vitro properties of organic cation transporters and regional distribution in rat brain

Organic cation transporters (OCTs) are polyspecific carriers implicated in low-affinity, corticosteroid-sensitive extraneuronal catecholamine uptake in peripheral tissues. The three main OCT subtypes, OCT1, OCT2 and OCT3, are also present in the brain, but their central role remains unclear. In the present study, we investigated by comparative in situ hybridization analysis the regional distribution of these transporters in rat brain and compared their functional properties in stably transfected HEK293 cells expressing human or rat OCTs. In rat brain, OCT2 and OCT3 mRNAs are expressed predominantly in regions located at the brain-cerebrospinal fluid border, with OCT3 mRNA expression extending to regions that belong to monoaminergic pathways such as raphe nuclei, striatum and thalamus. After normalization with MPP+ uptake, OCT2 and OCT3 subtypes share a similar monoamine preference profile, with higher transport efficacies for epinephrine and histamine than for the other monoamines. Interestingly, a significant level of epinephrine transport, previously only shown for rOCT2, is achieved by most OCTs subtypes. Finally, another novel finding was that OCTs are sensitive to 3,4-methylenedioxymetamphetamine (MDMA), phencyclidine (PCP), MK-801 and ketamine. Altogether, all our results suggest a functional specialization of OCT subtypes, based both on their intrinsic properties and their differential regional expression pattern in the brain.

Comparison of in vitro and in vivo reference genes for internal standardization of real-time PCR data

Real-time PCR is a powerful technique for gene expression studies, which have become increasingly important in a large number of clinical and scientific fields. The significance of the obtained results strongly depends on the normalization of the data to compensate for differences between the samples. The most widely used approach is to use endogenous reference genes (housekeeping genes) as internal standards. This approach is controversially discussed in the literature because none of the reference genes is stably expressed throughout all biological samples. Therefore, candidate reference genes have to be validated for each experimental condition. In our studies, we introduced and evaluated an in vitro synthesized reference cRNA for internal standardization of relative messenger RNA (mRNA) expression patterns. This reference, consisting of the in vitro transcribed coding sequence of aequorin, a jellyfish protein, was incorporated in the extracted RNA. The experimental significance of this approach was representatively tested for the expression of the neurotrophin-3 mRNA in distinct regions of mouse brains. A comparison to three stably expressed reference genes [beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and hypoxanthine phosphoribosyl-transferase 1 (HPRT1)] gave evidence that the spiking of template RNA with in vitro transcribed cRNA is a valuable tool for internal standardization of real-time PCR experiments.

Chrosomal mapping of the human gene for the tricyclic antidepressant-sensitive noradrenaline transporter

The neuronal Na+- and Cl--dependent transporter for the neurotransmitter noradrenaline (NA) is the primary target for the tricyclic antidepressant desipramine. The NA-transporter belongs to a new gene family of structurally related Na+- and Cl--dependent neurotransmitter transporters. In this study, the chromosomal localization for the gene encoding the human NA-transporter (h-NAT) was determined. By hybridization of a panel of somatic cell hybrids and by fluorescent in situ hybridization to metaphase chromosomes, the hNAT gene was localized on chromosome 16q12.2. In addition, evidence is presented for an intron-exon structure of the gene.

Cloning and expression of the bovine sodium‐ and chloride‐dependent noradrenaline transporter

A cDNA encoding a functional bovine, tricyclic antidepressant‐sensitive noradrenaline transporter has been identified by screening a λgt11 cDNA library of the bovine adrenal medulla using the cDNA of the human noradrenaline transporter [1991, Nature 350, 350‐354]. The sequence predicts a protein of 615 amino acids (M r, 68 900). The bovine transporter shares 93% amino acid identity with the human sequence, but displays two more consensus sites for phosphorylation by protein kinase C. Transient expression of the transporter in COS‐7 cells resulted in a sodium‐ and chloride‐dependent uptake of noradrenaline with a pharmacology typical for a neuronal noradrenaline transporter.

Systematic search for variation in the human norepinephrine transporter gene : Identification of five naturally occurring missense mutations and study of association with major psychiatric disorders

The complete coding region of the norepinephrine transporter (NET) gene was systematically screened for genetic variants in 137 unrelated individuals (including 46 probands with bipolar affective disorder and 45 schizophrenic probands, as well as 46 blood donors) using single-strand conformation analysis. We identified 13 DNA sequence variants, among them five missense substitutions. The missense substitutions Val69Ile, Thr99Ile, Val245Ile, Val449Ile, and Gly478Ser are located at putative transmembrane domains (TMD) 1, 2, 4, 9, and 10, respectively. The Thr99Ile substitution is at the 5th position of the putative leucine-zipper in TMD2. In a case-control study distribution of missense substitutions was found to be similar in 103 patients with bipolar affective disorder, in 228 schizophrenia patients and in 187 controls, indicating that presence of these variants is not causally related to major psychiatric diseases. The detection of a highly polymorphic silent 1287G/A polymorphism was utilized to demonstrate biallelic expression of the NET in adult human brain.

Characterization of the Novel Human Serotonin Receptor Subunits 5-HT3C,5-HT3D, and 5-HT3E

Within the family of serotonin receptors, the 5-hydroxytryptamine-3 (5-HT3) receptor is the only ligand-gated ion channel. It is composed of five subunits, of which the 5-HT3A and 5-HT3B subunits are best characterized. Several studies, however, have reported on the functional diversity of native 5-HT3 receptors, which cannot solely be explained on the basis of the 5-HT3A and 5-HT3B subunits. After our discovery of further putative 5-HT3 serotonin receptor-encoding genes, HTR3C, HTR3D, and HTR3E, we investigated whether these novel candidates and the isoform 5-HT3Ea are able to form functional 5-HT3 receptor complexes. Using immunofluorescence and immunoprecipitation studies of heterologously expressed proteins, we found that each of the respective candidates coassembles with 5-HT3A. To investigate whether the novel subunits modulate 5-HT3 receptor function, we performed radioligand-binding assays and calcium-influx studies in human embryonic kidney 293 cells. Our experiments revealed that the 5-HT3C,5-HT3D, 5-HT3E, and 5-HT3Ea subunits alone cannot form functional receptors. Coexpression with 5-HT3A, however, results in the formation of functional heteromeric complexes with different serotonin efficacies. Potencies of two agonists and antagonists were nearly identical with respect to homomeric 5-HT3A and heteromeric complexes. However, 5-HT showed increased efficacy with respect to 5-HT3A/D and 5-HT3A/E receptors, which is consistent with the increased surface expression compared with 5-HT3A receptors. In contrast, 5-HT3A/C and 5-HT3A/Ea receptors exhibited decreased 5-HT efficacy. These data show for the first time that the novel 5-HT3 subunits are able to form heteromeric 5-HT3 receptors, which exhibit quantitatively different functional properties compared with homomeric 5-HT3A receptors.

The rat norepinephrine transporter: molecular cloning from PC12 cells and functional expression

The rat norepinephrine transporter (rNET) cDNA from the PC12 pheochromocytoma cell line has been cloned by RT-PCR and characterized. The cDNA encodes an integral membrane protein consisting of 617 amino acids which contains twelve putative transmembrane domains, two potential N-glycosylation sites, two potential phosphorylation sites for protein kinase C and one phosphorylation site for casein kinase II. The nucleotide and deduced amino acid sequence shows a high level of homology to the human and the bovine norepinephrine transporter and less homology to the rat dopamine transporter (rDAT). Heterologous expression of rNET in HEK293 cells revealed that uptake of [3H]norepinephrine is sodium- and chloride-dependent and highly sensitive to the selective norepinephrine transporter inhibitors desipramine and nisoxetine. The cloned rNET cDNA provides the opportunity to investigate this transporter in heterologous expression systems and adds a new member to the family of sodium- and chloride-dependent neurotransmitter transporters.

The human noradrenaline transporter gene contains multiple polyadenylation sites and two alternatively spliced C-terminal exons.

Sequencing downstream of the C-terminal exon 14 of the human noradrenaline transporter (hNAT) gene reveals 5 consensus polyadenylation signals, several adenylate/uridylate-rich elements (AREs) and a new C-terminal exon, designated as exon 15. The tandemly arranged polyadenylation sites are in good conformity with the 3.6- and 5.8-kb hNAT mRNA transcripts. Expression of the alternatively spliced C-terminal exon 15 is shown by RT-PCR. This alternative splicing event proposes additional hNAT mRNA species of 2.4-3 kb in size. Corresponding NAT transcripts are found by Northern analysis of human SKN and rat PC12 cell RNA. Sequence comparison of the hNAT gene to two bovine NAT cDNAs shows the interspecies conservation of this alternative splicing event, the close relationship of human and bovine NAT genes, and implicates a functional role for the transporters C-terminal domain.