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.

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.

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.

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.

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.

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.

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.

Effects of tramadol and O-demethyl-tramadol on human 5-HT reuptake carriers and human 5-HT3A receptors: a possible mechanism for tramadol-induced early emesis.

([3H]5-HT)-uptake and patch-clamp techniques were used to study the actions of (+) and (-) tramadol and the active metabolites of tramadol, (+) and (-) O-demethyl-tramadol on the human serotonin (5-HT) transporter and the human 5-HT3A receptor, stably expressed in HEK-293 cells. The (+) and (-) enantiomers of tramadol suppressed the human 5-HT transporter concentration-dependently (IC50=1.0 and 0.8 microM, respectively), resulting in 97% and 87% transport inhibition at their respective initial plasma concentrations (9.5 microM). The (+) and (-) enantiomers of the active tramadol metabolite were less potent than tramadol in inhibiting the human 5-HT transporter (IC50=15 and 44 microM, respectively), resulting in 19.2% and 4.8% transport inhibition at their highest plasma concentrations (2.5 microM). In contrast to their potent suppression of the 5-HT transporter, both, (+) and (-) tramadol inhibited 5-HT (30 microM)-induced currents only at substantially higher concentrations (IC50=199 and 251 microM, respectively), resulting in only 6% and 4% inhibition at the initial maximum plasma concentration. A similar low potent inhibition of human 5-HT(3A) receptors was found for (+) and (-) O-demethyl-tramadol (IC50=158 and 63 microM, respectively). In conclusion, at clinical plasma concentrations tramadol potently suppresses the human 5-HT transporter, whereas it has only a slight effect on the human 5-HT3A receptor. The results are compatible with a possible mechanism for tramadol-induced early emesis involving the serotonergic system.

Interactions of metoclopramide and ergotamine with human 5-HT(3A) receptors and human 5-HT reuptake carriers.

The actions of metoclopramide and ergotamine, drugs which are used as a combined migraine medication, on human (h)5-HT3A receptors and 5-HT reuptake carriers, stably expressed in HEK-293 cells, were studied with patch-clamp- and ([3H]5-HT)-uptake techniques. At clinical concentrations, metoclopramide inhibited peak and integrated currents through h5-HT3A receptors concentration-dependently (IC50 = 0.064 and 0.076 microM, respectively) when it was applied in equilibrium (60 s before and during 5-HT (30 microM) exposure). The onset and offset time constants of metoclopramide action were 1.3 and 2.1 s, respectively. The potency of metoclopramide when exclusively applied during the agonist pulse decreased more than 200-fold (IC50 = 19.0 microM, peak current suppression). Metoclopramide (0.10 microM) did not alter the EC50 of 5-HT-induced peak currents. In contrast to the lack of competitive interaction between metoclopramide and 5-HT in this functional assay, metoclopramide inhibited specific [3H]GR65630 binding to human h5-HT3A receptors in a surmountable manner. This seeming discrepancy between functional studies and radioligand binding experiments may be accounted for by (1) the slow kinetics of inhibition of peak currents by metoclopramide compared with the fast onset and offset kinetics of 5-HT-induced currents and (2) the low efficacy of metoclopramide in inhibiting radioligand binding (e.g. only 20% binding inhibition compared to 79% peak current suppression by 200 nM metoclopramide). At low concentrations (1-10 nM), ergotamine had no effect on 5-HT (30 microM)-induced peak currents. Above clinical concentrations, ergotamine (>3 microM) inhibited them. When both drugs were applied together (0.10 microM metoclopramide +0.001 to 0.01 microM ergotamine), an inhibition of both, peak and integrated current responses was observed. Neither metoclopramide (< or =30 microM) nor ergotamine (< or =30 microM) had an effect on the 5-HT reuptake carrier as they did not alter the citalopram-sensitive [3H]5-HT uptake.

Decreased agonist, but not antagonist, binding to the naturally occurring Thr92Lys variant of the h5-HT7(a) receptor.

In the present study on transfected human embryonic kidney (HEK)293 cells, we aimed at establishing whether expression of the naturally occurring Thr92Lys variation of the Gs-coupled h5-HT7(a) receptor leads to changes of ligand binding properties, of agonist-evoked cAMP formation and/or of antagonist-mediated blockade of the latter. Binding of [3H]5-carboxamidotryptamine ([3H]5-CT) to membranes and stimulated [3H]cAMP accumulation in whole cells were determined. Saturation binding experiments in membranes of transiently transfected cells expressing either the wild-type or the variant receptor revealed a single binding site in both cases and no difference in Bmax between both receptor isoforms. In competition binding experiments in membranes of stably transfected cells, the Thr92Lys variant exhibited a 2.8-11 times lower binding affinity of the ligands 5-hydroxytryptamine (5-HT), 5-CT, 5-methoxy-3-(1,2,3,6-tetrahydropyridin-4yl)-1H-indole (RU24969), (+/-)-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT) and sumatriptan compared to the wild-type receptor. However, the variant did not differ from the wild-type with respect to the binding properties of the antagonists (R)-3-(2-(2-(4-methylpiperidin-1-yl)ethyl)-pyrrolodine-1-sulfonyl)phenol hydrochloride (SB-269970), risperidone, mesulergine and clozapine. In agreement with the decreased binding affinity of 5-HT, 5-CT, RU24969 and 8-OH-DPAT for the variant receptor, these agonists were less potent in stimulating [3H]cAMP accumulation in cells stably expressing the Thr92Lys h5-HT7(a) receptor. Sumatriptan did not stimulate cAMP accumulation in spite of its affinity for both receptor isoforms pointing to a putative weak antagonistic property of this drug at the h5-HT7 receptor. SB-269970 and clozapine were equipotent at both the variant and the wild-type receptor in producing a rightward shift of the 5-HT concentration-response curve for its stimulant effect on [3H]cAMP accumulation. In view of, e.g., the purported involvement of the 5-HT7 receptor in the regulation of circadian rhythm, it may be concluded that the decrease in affinity of 5-HT and other 5-HT receptor agonists at the (Thr92Lys) h5-HT7 receptor may be associated with changes of sleep physiology and of actions of new 5-HT7 receptor agonists designed to treat circadian dysregulation.

Impairment of signal transduction in response to stimulation of the naturally occurring Pro279Leu variant of the h5-HT7(a) receptor.

The aim of this study, performed in stably transfected HEK293 cells, was to investigate whether expression of the naturally occurring Pro279Leu variant of the h5-HT7(a) receptor (located in the third intracellular loop) is associated with changes in the pharmacological properties and/or second messenger formation compared to the wild-type receptor. Radioligand binding of [3H]5-carboxamidotryptamine ([3H]5-CT) to membranes and stimulation of [3H]cAMP formation in whole cells evoked by 5-HT receptor agonists were determined. Maximum binding (B(max)) to, and affinity (K(D)) of [3H]5-CT for, the variant receptor and the wild-type receptor were equal. All agonists and antagonists investigated exhibited no differences in affinity between the variant receptor and the wild-type receptor. However, the intrinsic activity of the 5-HT receptor agonists 5-HT, 5-CT, RU24969 and 8-OH-DPAT in stimulating [3H]cAMP accumulation in the cells expressing the Pro279Leu variant was almost abolished and their potency was 2.9-4.3-fold lower. Despite its affinity for both receptor isoforms, sumatriptan did not stimulate the accumulation of cAMP. In individuals expressing the Pro279Leu variant of the h5-HT7(a) receptor, a considerable attenuation of its function may be predicted. This may have relevance for the action of new classes of drugs which affect circadian rhythm or psychiatric diseases, such as schizophrenia.

Pharmacological and electrophysiological properties of the naturally occurring Pro391Arg variant of the human 5-HT3A receptor.

The 5-HT3A receptor, a ligand-gated ion channel, is involved in pain pathways, nausea and emesis, and irritable bowel syndrome, and may play a role in the pathogenesis of psychiatric diseases such as schizophrenia and depression. Recently, a naturally occurring variation (ProArg) in the second intracellular loop of the human (h) 5-HT3A receptor was identified in a schizophrenic patient. Because the substitution of proline, an alpha-imino acid, by arginine may affect the conformation of the whole receptor, the aim of the present study was to determine the pharmacological and functional properties of this variant compared to the wild-type receptor in stably transfected HEK293 cells. Studies of binding of [H]GR65630, a 5-HT3 receptor antagonist, to membranes (saturation and competition experiments with 5-HT3 receptor ligands) and patch-clamp studies of agonist-induced currents in outside-out patches were carried out. In comparison to the wild-type, the variant receptor exhibited no changes in the receptor density and the affinities for nine representative ligands (five agonists and four antagonists). The potencies and efficacies of three 5-HT3 receptor agonists in inducing currents through the ion channel and the potencies of two 5-HT3 receptor antagonists in blocking 5-HT-evoked currents did not differ between wild-type and variant receptors. In addition, there were no differences in the desensitization kinetics of both receptor isoforms. In conclusion, the ArgPro variation of the h5-HT3A receptor does not change ligand binding to the h5-HT3A receptor, nor does it modify current through the receptor channel.

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

1. 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. 2. Among the three OCTs only for the OCT3 (also designated as 'uptake(2)' 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 [(3)H]-1-methyl-4-phenylpyridinium ([(3)H]-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 beta-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-beta-chloroethylamine) preferentially inhibited hOCT3 and hOCT2. 3. PbA reversibly inhibited hOCT1 and irreversibly hOCT2 and hOCT3; SKF550 also irreversibly inhibited hOCT3 but hOCT2 in a reversible manner. 4. 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, beta-oestradiol and OMI, whereas hOCT3 is selectively inhibited by corticosterone, OMI and decynium22.

Identification and pharmacological characterization of a specific agmatine transport system in human tumor cell lines.

Specific accumulation of [(14C)]agmatine in six human intestinal tumor cell lines and in the glioma cell line SK-MG-1 was inhibited by phentolamine, idazoxan, clonidine, 1,3-di-(2-tolyl)guanidine, histamine, putrescine, spermine and spermidine. Corticosterone, desipramine, O-methylisoprenaline, cirazoline, moxonidine, l-arginine, l-lysine, verapamil, nifedipine, CdCl(2), ondansetron, and l-carnitine failed to inhibit specific [(14)C]agmatine accumulation, thus excluding that it is mediated by amino acid or monoamine carriers, by the putrescine carrier, by 5-HT(3) receptor channels, by Ca(2+) channels or by the organic cation transporters OCT1, OCT2, OCT3, OCTN1, or OCTN2. This conclusion is supported by the finding that transfection of HEK293 cells with cDNA encoding either hOCT1, hOCT2, or hOCT3 did not enhance specific [(14)C]agmatine accumulation compared to nontransfected cells. The data suggest that agmatine is accumulated by a specific agmatine transporter. Since incubation with exogenous agmatine for 24 hours increased intracellular agmatine content in all cell lines by a multiple of the basal endogenous content, the agmatine uptake system may be relevant for the regulation of the intra- and extracellular concentration of agmatine in humans.

Alpha(2A)-adrenergic versus imidazoline receptor controversy in rilmenidine's action: alpha(2A)-antagonism in humans versus alpha(2A)-agonism in rabbits.

At the alpha(2A)-autoreceptors on the sympathetic nerve terminals of the human atrial appendages and rabbit pulmonary artery, rilmenidine and oxymetazoline exhibit different properties (antagonism and agonism, respectively). These opposite pharmacodynamic properties of alpha(2)-adrenoceptor ligands seem to be due to substantial differences in the nucleotide and amino acid sequences between human and rabbit alpha(2A)-adrenoceptors. Hence, the rabbit alpha(2A)-adrenoceptor is not reliably predictive for the action of ligands at the human alpha(2A)-adrenoceptor.

Agmatine (decarboxylated arginine), a modulator of liver cell homeostasis and proliferation.

Agmatine interferes with polyamine metabolism and cell growth in cell culture. When absorbed from the gastrointestinal tract, it is accumulated in the liver. Based on these facts, the aim of the present study was to investigate the effects of agmatine on the proliferation of rat and human hepatoma cells and on regeneration of rat liver after partial two-thirds hepatectomy. In cultured cells of the rat and human hepatoma cell lines McRH7777 and HepG2 respectively, agmatine reduced ATP and protein contents (50% inhibition in the range of 169-569 microM) which were determined as estimates for the cell number. This antiproliferative action was not due to an effect on ornithine decarboxylase antizyme, since its intracellular concentration was not significantly changed by agmatine. In addition, agmatine metabolism by diamine oxidase seems not to be involved in the inhibition of cell growth and proliferation because the diamine oxidase inhibitor aminoguanidine did not alter agmatine's antiproliferative action. After two-thirds hepatectomy, daily administration of 250 mg and 500 mg agmatine to rats for 6 days by gavage reduced the weight gain of the remnant liver on day 7 by 19.5+/-0.9% and 22.3+/-1.5% respectively. The decrease in regeneration correlated with a pronounced accumulation of agmatine in the liver. Hence, the present data provide evidence of an involvement of agmatine in liver cell growth.