Daily Moderate Exercise Is Beneficial and Social Stress Is Detrimental to Disease Pathology in Murine Lupus Nephritis.

Daily moderate exercise (DME) and stress management are underemphasized in the care of patients with lupus nephritis (LN) due to a poor comprehensive understanding of their potential roles in controlling the inflammatory response. To investigate these effects on murine LN, disease progression was monitored with either DME or social disruption stress (SDR) induction in NZM2410/J mice, which spontaneously develop severe, early-onset LN. SDR of previously established social hierarchies was performed daily for 6 days and DME consisted of treadmill walking (8.5 m/min for 45 min/day). SDR significantly enhanced kidney disease when compared to age-matched, randomly selected control counterparts, as measured by histopathological analysis of H&E staining and immunohistochemistry for complement component 3 (C3) and IgG complex deposition. Conversely, while 88% of non-exercised mice displayed significant renal damage by 43 weeks of age, this was reduced to 45% with exercise. DME also reduced histopathology in kidney tissue and significantly decreased deposits of C3 and IgG complexes. Further examination of renal infiltrates revealed a macrophage-mediated inflammatory response that was significantly induced with SDR and suppressed with DME, which also correlated with expression of inflammatory mediators. Specifically, SDR induced IL-6, TNF-α, IL-1ß, and MCP-1, while DME suppressed IL-6, TNF-α, IL-10, CXCL1, and anti-dsDNA autoantibodies. These data demonstrate that psychological stressors and DME have significant, but opposing effects on the chronic inflammation associated with LN; thus identifying and characterizing stress reduction and a daily regimen of physical activity as potential adjunct therapies to complement pharmacological intervention in the management of autoimmune disorders, including LN.

Estrogen-regulated STAT1 activation promotes TLR8 expression to facilitate signaling via microRNA-21 in systemic lupus erythematosus.

Recent studies implicate innate immunity to systemic lupus erythematosus (SLE) pathogenesis. Toll-like receptor (TLR)8 is estrogen-regulated and binds viral ssRNA to stimulate innate immune responses, but recent work indicates that microRNA (miR)-21 within extracellular vesicles (EVs) can also trigger this receptor. Our objective was to examine TLR8 expression/activation to better understand sex-biased responses involving TLR8 in SLE. Our data identify an estrogen response element that promotes STAT1 expression and demonstrate STAT1-dependent transcriptional activation of TLR8 with estrogen stimulation. In lieu of viral ssRNA activation, we explored EV-encapsulated miR-21 as an endogenous ligand and observed induction of both TLR8 and cytokine expression in vitro. Moreover, extracellular miR detection was found predominantly within EVs. Thus, just as a cytokine or chemokine, EV-encapsulated miR-21 can act as an inflammatory signaling molecule, or miRokine, by virtue of being an endogenous ligand of TLR8. Collectively, our data elucidates a novel innate inflammatory pathway in SLE.

A chimeric human-mouse model of Sjögren's syndrome.

Despite recent advances in the understanding of Sjögren's Syndrome (SjS), the pathogenic mechanisms remain elusive and an ideal model for early drug discovery is not yet available. To establish a humanized mouse model of SjS, peripheral blood mononuclear cells (PBMCs) from healthy volunteers or patients with SjS were transferred into immunodeficient NOD-scid IL-2rγ(null) mouse recipients to produce chimeric mice. While no difference was observed in the distribution of cells, chimeric mice transferred with PBMCs from SjS patients produced enhanced cytokine levels, most significantly IFN-γ and IL-10. Histological examination revealed enhanced inflammatory responses in the lacrimal and salivary glands of SjS chimeras, as measured by digital image analysis and blinded histopathological scoring. Infiltrates were primarily CD4+, with minimal detection of CD8+ T-cells and B-cells. These results demonstrate a novel chimeric mouse model of human SjS that provides a unique in vivo environment to test experimental therapeutics and investigate T-cell disease pathology.

Oral administration of nano-emulsion curcumin in mice suppresses inflammatory-induced NFκB signaling and macrophage migration.

Despite the widespread use of curcumin for centuries in Eastern medicine as an anti-inflammatory agent, its molecular actions and therapeutic viability have only recently been explored. While curcumin does have potential therapeutic efficacy, both solubility and bioavailability must be improved before it can be more successfully translated to clinical care. We have previously reported a novel formulation of nano-emulsion curcumin (NEC) that achieves significantly greater plasma concentrations in mice after oral administration. Here, we confirm the immunosuppressive effects of NEC in vivo and further examine its molecular mechanisms to better understand therapeutic potential. Using transgenic mice harboring an NFκB-luciferase reporter gene, we demonstrate a novel application of this in vivo inflammatory model to test the efficacy of NEC administration by bioluminescent imaging and show that LPS-induced NFκB activity was suppressed with NEC compared to an equivalent amount of curcumin in aqueous suspension. Administration of NEC by oral gavage resulted in a reduction of blood monocytes, decreased levels of both TLR4 and RAGE expression, and inhibited secretion of MCP-1. Mechanistically, curcumin blocked LPS-induced phosphorylation of the p65 subunit of NFκB and IκBα in murine macrophages. In a mouse model of peritonitis, NEC significantly reduced macrophage recruitment, but not T-cell or B-cell levels. In addition, curcumin treatment of monocyte derived cell lines and primary human macrophages in vitro significantly inhibited cell migration. These data demonstrate that curcumin can suppress inflammation by inhibiting macrophage migration via NFκB and MCP-1 inhibition and establish that NEC is an effective therapeutic formulation to increase the bioavailability of curcumin in order to facilitate this response.

Differences between human wild-type and C23S variant 5-HT2C receptors in inverse agonist-induced resensitization.

The aim of this study was to analyze functional properties of the naturally occurring C23S variant of the human 5-HT2C receptor. In HEK293 cells transiently expressing the unedited forms of the variant receptor (VR) or the wild-type receptor (WTR), surface expression was determined by [3H]mesulergine binding to membrane fragments. Function was examined by an aequorin luminescence-based Ca2+ assay. Surface expression of the VR was 116% of that of the WTR. The 5-HT-induced increase in cytosolic Ca2+ ([Ca2+]i), and its inhibition by the inverse agonist SB 206553 did not differ between VR- or WTR-expressing cells. Preexposure of VR- or WTR-expressing cells to 0.5 µM 5-HT (3 min-4.5 h) led to a practically identical time course and extent in the reduction of the 5-HT-induced increase in [Ca2+]i. In contrast, prolonged preexposure to the inverse agonist SB 206553 (1 µM) elevated the 5-HT-induced increase in [Ca2+]i for both isoreceptors. A preexposure time of 4.5 h was necessary to significantly elevate the Ca2+ response of the WTR, but the VR produced this elevation within 1 h with virtually no further effect after 4.5 h of preexposure. In conclusion, prolonged preexposure to 5-HT caused equally rapid and strong desensitization of both isoreceptors. The different time course of SB 206553-induced resensitization of the two isoreceptors might be therapeutically relevant for drugs exhibiting inverse agonist properties at 5-HT2C receptors, such as atypical antipsychotics and certain antidepressants.

Serotonin receptor diversity in the human colon: Expression of serotonin type 3 receptor subunits 5-HT3C, 5-HT3D, and 5-HT3E.

Since the first description of 5-HT3 receptors more than 50 years ago, there has been speculation about the molecular basis of their receptor heterogeneity. We have cloned the genes encoding novel 5-HT3 subunits 5-HT3C, 5-HT3D, and 5-HT3E and have shown that these subunits are able to form functional heteromeric receptors when coexpressed with the 5-HT3A subunit. However, whether these subunits are actually expressed in human tissue remained to be confirmed. In the current study, we performed immunocytochemistry to locate the 5-HT3A as well as the 5-HT3C, 5-HT3D, and 5-HT3E subunits within the human colon. Western blot analysis was used to confirm subunit expression, and RT-PCR was employed to detect transcripts encoding 5-HT3 receptor subunits in microdissected tissue samples. This investigation revealed, for the first time, that 5-HT3C, 5-HT3D, and 5-HT3E subunits are coexpressed with 5-HT3A in cell bodies of myenteric neurons. Furthermore, 5-HT3A and 5-HT3D were found to be expressed in submucosal plexus of the human large intestine. These data provide a strong basis for future studies of the roles that specific 5-HT3 receptor subtypes play in the function of the enteric and central nervous systems and the contribution that specific 5-HT3 receptors make to the pathophysiology of gastrointestinal disorders such as irritable bowel syndrome and dyspepsia.

RIC-3 exclusively enhances the surface expression of human homomeric 5-hydroxytryptamine type 3A (5-HT3A) receptors despite direct interactions with 5-HT3A, -C, -D, and -E subunits.

Although five 5-hydroxytryptamine type 3 (5-HT3) subunits (A-E) have been cloned, knowledge on the regulation of their assembly is limited. RIC-3 has been identified as a chaperone specific for the pentameric ligand-gated nicotinic acetylcholine and 5-HT(3) receptors. Therefore, we examined the impact of RIC-3 on differently composed 5-HT(3) receptors with the focus on 5-HT3C, -D, and -E subunits. The influence of RIC-3 on these receptor subtypes is supported by the presence of RIC3 mRNA in tissues expressing at least one of the subunits 5-HT3C, -D, and -E. Furthermore, immunocytochemical studies on transfected mammalian cells revealed co-localization in the endoplasmic reticulum and direct interaction of RIC-3 with 5-HT3A, -C, -D, and -E. Functional and pharmacological characterization was performed using HEK293 cells expressing 5-HT3A or 5-HT3A + 5-HT3B (or -C, -D, or -E) in the presence or absence of RIC-3. Ca(2+) influx analyses revealed that RIC-3 does not influence the 5-HT concentration-response relationship on 5-HT(3)A receptors but leads to differential increases of 5-HT-induced maximum response (E(max)) on cells expressing different subunits. Increases of E(max) were due to analogously enhanced B(max) values for binding of the 5-HT(3) receptor antagonist [(3)H]GR65630. The observed enhanced cell surface expression of the tested 5-HT3 subunit combinations correlated with the increased surface expression of 5-HT3A as determined by flow cytometry. In conclusion, we showed that RIC-3 can interact with 5-HT3A, -C, -D, and -E subunits and predominantly enhances the surface expression of homomeric 5-HT(3)A receptors in HEK293 cells. These data implicate a possible role of RIC-3 in determining 5-HT(3) receptor composition in vivo.

Thyroidal radioactive iodide uptake in the lactating rhesus monkey.

Although radioactive iodide uptake (RAIU) is one of the reliable diagnostic methods for thyroid function in adult humans, especially in the diagnosis of thyrotoxicosis, there are limited data for RAIU during pregnancy and lactation in humans and animals. Therefore, we proposed to validate RAIU for the lactating rhesus monkey to further human model studies in thyroid disease. RAIU was performed at 6 and 24 h using 100 microCi of (123)I orally in four lactating monkeys. The thyroid and thigh were counted using a scintillation probe and multichannel analyser. A dose/standard ratio of counts/minute was calculated to compensate for background, utilizing differences in the activity between the dose administered and a standard. Thyroidal RAIU varied significantly among monkeys: 6.71 +/- 2.40% for the 6 h uptake and 15.44 +/- 7.71% for the 24 h uptake. These data showed that the RAIU test may allow a rational clinical approach to thyroid function testing for lactating rhesus monkeys. Additional studies are needed for assessing thyroid function in rhesus monkeys of varying ages and gender with clinical abnormalities.

Characterization of the naturally occurring Arg344His variant of the human 5-HT 3A receptor.

The present study aimed at examining the function and pharmacological properties of the naturally occurring Arg344His variant of the human 5-HT(3A) receptor, identified in a schizophrenic patient. In intact human embryonic kidney (HEK) 293 cells expressing the wild-type (WT) or the variant receptor, the function was analyzed by indirect measurement of agonist-induced Ca(2+) current through the 5-HT(3A) receptor channel by an aequorin luminescence-based Ca(2+) assay. In cell membrane patches cation currents were determined electrophysiologically including technically demanding single channel analyses. The pharmacological properties were analyzed by [(3)H]GR65630 binding to cell membrane fragments. The density of [(3)H]GR65630 binding sites in cells expressing the variant receptor was reduced to 55% of that in cells expressing the WT receptor, which, however, was not accompanied by an analogous decrease in 5-HT-induced Ca(2+) influx through the receptor channel. However, the single channel analysis suggests an increase in single receptor channel mean open time (which is known to be subject of many variables) but not in unitary current amplitude. Radioligand competition experiments revealed that the affinity of five 5-HT(3) receptor agonists and four antagonists for the variant receptor did not differ from that for the WT receptor. In conclusion, the variant receptor resembles the WT receptor in that it forms functional homopentameric 5-HT(3A) receptors with identical pharmacological properties. In view of the lack of reduction in Ca(2+) flux through the variant receptor channels in spite of the decrease in its density on the cell membrane, the increase in single receptor channel mean open time appears to compensate for the reduction in variant receptor density.

Effects of intravenous administration of pirfenidone on horses with experimentally induced endotoxemia.

OBJECTIVE: To characterize effects of IV administration of pirfenidone on clinical, biochemical, and hematologic variables and circulating tumor necrosis factor (TNF)-alpha concentrations in horses after infusion of a low dose of endotoxin. ANIMALS: 18 healthy adult horses. PROCEDURES: Horses were randomly assigned to 3 groups (n = 6 horses/group) and administered an IV infusion of 30 ng of endotoxin/kg or saline (0.9% NaCl) solution during a 30-minute period. Lipopolysaccharide-pirfenidone horses received endotoxin followed by pirfenidone (loading dose of 11.6 mg/kg and then constant rate infusion [CRI] at 9.9 mg/kg/h for 3 hours). Lipopolysaccharide-saline horses received endotoxin followed by infusion (loading dose and CRI for 3 hours) of saline solution. Saline-pirfenidone horses received saline solution followed by pirfenidone (loading dose and then CRI for 3 hours). Physical examination variables were recorded and blood samples collected at predetermined intervals throughout the 24-hour study period. Blood samples were used for CBCs, biochemical analyses, and determinations of TNF-alpha concentrations. RESULTS: IV infusion of pirfenidone after administration of a low dose of endotoxin failed to attenuate the clinical, clinicopathologic, or cytokine alterations that developed secondary to endotoxin exposure. Intravenous infusion of pirfenidone after administration of saline solution induced mild transient clinical signs, but associated clinicopathologic changes were not detected. CONCLUSIONS AND CLINICAL RELEVANCE: IV administration of pirfenidone was tolerated with only mild transient clinical adverse effects during infusion. However, administration of pirfenidone did not protect horses from the systemic effects of experimentally induced endotoxemia. Further studies of related, but more potent, drugs may be warranted.

Association of major depression with rare functional variants in norepinephrine transporter and serotonin1A receptor genes.

Dysregulations of central noradrenergic and serotonergic neurotransmission have been suggested to contribute to the pathogenesis of neuropsychiatric disorders such as depression. The norepinephrine transporter (NET; SLC6A2) and the serotonin (5-HT)(1A) receptor (5-HT(1A) receptor; HTR1A) play an important role in central nervous monoaminergic homeostasis. As shown previously, variations in the human NET and 5-HT(1A) receptor genes can alter noradrenergic and serotonergic signaling in the brain: a single nucleotide polymorphism (SNP) in the coding region of the NET gene resulting in a F528C substitution increased plasma membrane expression of this NET variant, and a SNP in the human 5-HT(1A) receptor gene leading to the R219L receptor variant almost abolished cellular signal transduction subsequent to receptor activation. The present study aimed at investigating whether these NET and 5-HT(1A) receptor variants are associated with major depression (MD). The sample comprised 426 patients suffering from unipolar MD as well as 643 healthy control subjects for the variants of the 5-HT(1A) receptor and the NET. Both SNPs were shown to be associated with MD. In conclusion, our results favor the hypothesis that monoaminergic neurotransmission in general and the F528C NET and R219L 5-HT(1A) receptor variants in particular are involved in the pathogenesis of depression.

Pharmacokinetics and clinical effects of pirfenidone administered intravenously in horses.

OBJECTIVE: To characterize the plasma pharmacokinetics and clinical effects of pirfenidone administered IV in healthy horses. ANIMALS: 6 adult horses. PROCEDURES: A 15 mg/kg dose of pirfenidone was administered IV over 5 minutes. Physical variables were recorded and blood samples collected prior to infusion; 2.5 minutes after beginning infusion; at the end of infusion; and at 3, 6, 9, 12, 15, 20, 25, 30, 40, 50, 60, 75, and 90 minutes and 2, 2.5, 3, 4, 6, 8, 12, and 24 hours after completion of infusion. Plasma concentrations of pirfenidone and its metabolites were determined. RESULTS: Mild clinical effects, including tachycardia and muscle fasciculations, were observed during drug administration but stopped at the end of the infusion. Pirfenidone and 2 metabolites, hydroxypirfenidone and carboxypirfenidone, were detected by the end of the 5-minute infusion. Mean peak plasma concentration of pirfenidone was 182.5 micromol/L, detected at the end of the infusion. Mean peak plasma concentrations of hydroxypirfenidone and carboxypirfenidone were 1.07 and 3.4 micromol/L, respectively, at 40 minutes after infusion. No parent drug or metabolites were detected at 24 hours. Distribution of pirfenidone best fit a 2-compartment model, and the drug had mean +/- SEM elimination half-life of 86.0 +/- 4.7 minutes, mean body clearance of 6.54 +/- 0.45 mL/kg/min, and apparent volume of distribution at steady state of 0.791 +/- 0.056 L/kg. CONCLUSIONS AND CLINICAL RELEVANCE: Intravenous administration of pirfenidone was tolerated with transient adverse affects during infusion, and drug clearance was rapid.

Pharmacokinetics and metabolism of intravenous pirfenidone in sheep.

Pirfenidone, a promising antifibrotic agent, was administered intravenously to six female sheep at 30 mg/kg. Four sheep received 14C-pirfenidone simultaneously. Plasma and urine were obtained for assay of pirfenidone and its metabolites over two days, and tissues were obtained via necropsy. Samples were analysed for pirfenidone and metabolites using HPLC-MS and flow scintillation spectrometry. Plasma pirfenidone disappeared with first order kinetics with a clearance of 1.2 l/kg/h, half-life of 24 min, and distribution volume of 0.71 l/kg. After 48 h, the organs containing the largest quantity of 14C were lungs, liver and intestinal wall. Tissues with the highest concentration of 14C were lung, kidney, brain, liver, lymph node and adipose. Metabolites found in plasma and urine were hydroxypirfenidone (half-life of 44 min) and carboxypirfenidone. Additional metabolites found in urine were hydroxypirfenidone glucuronide and acetoxypirfenidone. Approximately, 80% of the tracer eventually appeared in the urine, and approximately 50% of it was in the form of identifiable metabolites. Less than 1% of the dose appeared in the urine in the form of the parent drug. Quantitatively, most of the metabolites appeared in the urine within 2 h. Thus, the drug is rapidly and completely metabolized.

Molecular cloning and functional expression of the murine noradrenaline transporter.

The cDNA of the murine noradrenaline transporter (mNAT) was cloned from the RNA of the placenta of a C57BL/6 mouse. The cloned mNAT differs from a previously published sequence in two amino acids within the C-terminal region. A cDNA obtained from an inbred mouse strain showed a further amino acid exchange (Ile(505)Val) within the fifth intracellular loop. The pharmacological properties of both, the wild-type mNAT and the variant (mNAT-I(505)V), were studied in human embryonic kidney HEK293 cells transfected with the corresponding cDNA. The kinetic constants for transport (K (m), V (max)) of [(3)H]noradrenaline ([(3)H]-NA) and binding (K (D), B (max)) of the selective NAT inhibitor [(3)H]nisoxetine were not different between the two isoforms; the mean kinetic constants amounted to about 4 microM and 120pmol/mg protein for K (m) and V (max) and 6nM and 18pmol/mg protein for K (D) and B (max), respectively. [(3)H]-NA transport by both isoforms showed the typical properties of an NAT because it was dependent on sodium and chloride and inhibited with almost identical K (i) values by various NAT substrates and inhibitors. The only significant pharmacological difference identified between the two mNAT isoforms was an about threefold higher affinity for cocaine of the very rare mNAT-I(505)V variant.

Multiple novel alterations in Kit tyrosine kinase in patients with gastrointestinally pronounced systemic mast cell activation disorder.

OBJECTIVE: Sequencing efforts to discover mutations in the tyrosine kinase Kit related to systemic mast cell disorders have so far been focused mainly on only a few of the 21 exons of the encoding gene c-kit, thus considerably limiting the possibility to quantitatively reveal pathogenetic relationships. The purpose of this study was to analyze and compare the total sequence of Kit tyrosine kinase at the level of the mRNAs obtained from patients with clear systemic signs of a pathologically increased mast cell mediator release and those from healthy volunteers. MATERIAL AND METHODS: Kit encoding mRNA isolated from mast cell progenitors in peripheral blood from 17 patients with a mast cell activation disorder and from 5 healthy volunteers as well as from the human mast cell leukemia cell line HMC1 was analyzed for alterations. RESULTS: Multiple novel point mutations and six isoforms of Kit which are due to alternative mRNA splicing were detected. One isoform, the insertion of a glutamine residue at amino acid position 252, was found to be a new splice variant expressed in all patients but in none of the healthy volunteers. CONCLUSIONS: Systemic mast cell activation disorder was pathogenetically characterized by two or more alterations in the Kit tyrosine kinase providing not only a means of confirming the diagnosis, but also of assessing prognosis and of starting adequate therapeutic interventions. The insertion of Q252 appears to be pathognomic for that disease, providing a novel means for the identification of chronic non-specific gastrointestinal symptoms as manifestations of a systemic mast cell activation disorder.

Aequorin luminescence-based assay for 5-hydroxytryptamine (serotonin) type 3 receptor characterization.

The classical electrophysiological method to measure the function of the 5-hydroxytryptamine (serotonin) type 3 (5-HT(3)) receptor, a cation-permeable ligand-gated ion channel, is time-consuming and not suitable for high-throughput screening. Therefore, we have optimized the conditions for a sensitive assay suitable to measure 5-HT(3) receptor responses in cell suspension based on aequorin bioluminescence caused by Ca(2+) influx. The assay, carried out in 96-well plates, was applied for the pharmacological characterization of 5-HT(3) receptors on human embryonic kidney (HEK) 293 cells transiently coexpressing apoaequorin and either the human homopentameric 5-HT(3A) receptor or the human heteromeric 5-HT(3A/B) receptor in the same subset of cells. Thus, the luminescence signal originates exclusively from transfected cells, leading to a high signal/noise ratio, a major advantage compared with fluorescence techniques using Ca(2+)-sensitive dyes. The potencies of two 5-HT(3A) receptor agonists and two antagonists as well as the potency and efficacy of serotonin at the heteromeric 5-HT(3A/B) receptor were comparable to those reported using other functional methods. In conclusion, the aequorin assay described here provides a convenient and highly sensitive method for functional characterization of 5-HT(3) receptors that is well suited for high-throughput screening.

S1P-receptors in PC12 and transfected HEK293 cells: molecular targets of hypotensive imidazoline I(1) receptor ligands.

The present study aimed at elucidating the molecular identity of the proposed "I(1)-imidazoline receptors", i.e. non-adrenoceptor recognition sites via which the centrally acting imidazolines clonidine and moxonidine mediate a major part of their effects. In radioligand binding experiments with [(3)H]clonidine and [(3)H]lysophosphatidic acid on intact, alpha(2)-adrenoceptor-deficient PC12 cells, moxonidine, clonidine, lysophosphatidic acid and sphingosine-1-phosphate (S1P) competed for the specific binding sites of both radioligands with similar affinities. RNA interference with the rat S1P(1)-, S1P(2)- or S1P(3)-receptor abolished specific [(3)H]lysophosphatidic acid binding. [(3)H]Clonidine binding was markedly decreased by siRNA targeting S1P(1)- and S1P(3)-receptors but not by siRNA against S1P(2)-receptors. Finally, in HEK293 cells transiently expressing human S1P(3)-receptors, sphingosine-1-phosphate, clonidine and moxonidine induced increases in intracellular calcium concentration, moxonidine being more potent than clonidine; this is in agreement with the known properties of the "I(1)-imidazoline receptors". The present results indicate that the "I(1)-imidazoline receptors" mediating effects of clonidine and moxonidine in PC12 and the transfected HEK293 cells belong to the S1P-receptor family; in particular, the data obtained in PC12 cells suggest that the I(1) imidazoline receptors represent a mixture of S1P(1)- and S1P(3)-receptors and/or hetero-dimers of both.

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

5-Hydroxytryptamine-induced contraction of human temporal arteries coexpressing 5-HT2A receptors and wild-type or variant (Phe124Cys) 5-HT1B receptors: increased contribution of 5-HT1B receptors to the total contractile amplitude in arteries from Phe124Cys heterozygous individuals.

OBJECTIVES: Expression studies of the rare Phe124Cys sequence variant of the human 5-HT1B receptor in HEK293 cells demonstrated that 5-hydroxytryptamine (5-HT) and sumatriptan had both three times higher binding affinity and agonist potency at the variant receptor than wild-type receptor. We examined whether in-vivo expression of the variant compared to the wild-type Phe/Phe genotype at codon 124 of the 5-HT1B receptor in human temporal arteries modifies their agonist-induced contraction. METHODS: Rings of arteries, coexpressing 5-HT1B and 5-HT2A receptors, from 98 patients undergoing neurosurgery were set up to measure contraction. Blood sample genotyping was performed by PCR using a mutagenic primer which induces a NheI restriction site in the Cys but not in the Phe allele. RESULTS: Three patients exhibited the Cys/Phe genotype, probably yielding coexpression of both the 124Phe and the 124Cys 5-HT1B receptors. In 95 Phe/Phe patients, exclusively the 124Phe receptor was expressed. The contractile potencies of 5-HT and sumatriptan were not significantly different in arteries from Cys/Phe or Phe/Phe individuals. The 5-HT1B receptor-selective antagonist SB224289 was five-fold more potent in blocking the effects of 5-HT in arteries from three Cys/Phe than from 30 Phe/Phe individuals (P < 0.03). The fraction of 5-HT effects via 5-HT1B receptors, related to the total contractile amplitude via 5-HT1B and 5-HT2A receptors, was enhanced from 0.42 +/- 0.03 in 88 Phe/Phe individuals to 0.75 +/- 0.10 in three Cys/Phe individuals (P < 0.05). CONCLUSIONS: Although the potency of 5-HT1B receptor agonists does not differ between arteries from Phe/Phe and Cys/Phe individuals, the contribution of 5-HT1B receptors to the mediation of the effects of 5-HT is increased in Cys/Phe individuals.

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.