Functional expression of L- and T-type Ca2+ channels in murine HL-1 cells.

In the search for a readily available source of native cardiac cells, we investigated the molecular and pharmacological properties of the immortalized cardiac atrial myocyte cell line, HL-1 cells. This work focused on the expression pattern of voltage-gated Ca2+ channels (VGCC). Reverse transcription-polymerase chain reaction analysis revealed that HL-1 cells have mRNA for several types of Ca2+ channels including the L-types, alpha1C and alpha1D, as well as T-types, alpha1H and alpha1G, but are lacking N-type, alpha1B and the T-type, alpha1I. Western blot analysis demonstrated significant alpha1C protein subunit expression, with less alpha1D subunit apparent, while alpha1A, alpha1B and alpha1E subunit expression was undetectable. Immunocytochemical staining showed that the alpha1C protein subunit is expressed predominantly on the cell surface, whereas the alpha1D protein is expressed mostly intracellularly. Whole-cell patch-clamp measurements demonstrated the presence of low (ICa,T) and high (ICa,L) voltage-activated Ca2+ currents, with preferential sensitivity to mibefradil and nimodipine, respectively. Addition of increasing external Ca2+ concentrations, [Ca2+]o, resulted in Ca2+ influx measured by fluorometric imaging with an EC50 of 0.8 mM [Ca2+]o. At a fixed [Ca2+]o of 0.125 mM, Ca2+ influx was also triggered by increasing the extracellular K+ concentration, [K+]o, with an EC50 of 3.7 mM [K+]o. As increasing [K+]o depolarizes the cell, this latter result is consistent with Ca2+ influx through a voltage-dependent mechanism. L-type (nimodipine and verapamil) and T-type (mibefradil and pimozide) Ca2+ channel blockers inhibited Ca2+ influx with IC50s of 1, 2, 0.4 and 0.2 microM, respectively. Antagonists of N-type (omega-conotoxins GVIA) and P/Q-type (MVIIC or omega-agatoxin IVA) did not inhibit Ca2+ influx, consistent with the lack of expression of N-, P-, or Q-type channels observed in the molecular studies. Taken together, these findings indicate that HL-1 cells express L- and T-subtypes of VGCC and are a unique in vitro model system for the study of native, mammalian cardiac Ca2+ channels.

Expression of cysteinyl leukotriene synthetic and signalling proteins in inflammatory cells in active seasonal allergic rhinitis.

BACKGROUND: Cysteinyl leukotrienes (CysLTs) are bioactive lipids that have been shown to contribute to allergic and inflammatory diseases. Eosinophils and mast cells have the capacity to produce large amounts of CysLTs after allergic or non-allergic stimulation. Molecular identification of both the synthetic and signalling proteins in the CysLT pathway allows the investigation of expression of the CysLT enzymes and receptors in active allergic rhinitis. OBJECTIVE: We examined the expression of the proteins involved in the synthesis of CysLTs and the cysteinyl leukotriene-1 (CysLT1) and cysteinyl leukotriene-2 (CysLT2) receptors in inflammatory cells from patients with active seasonal allergic rhinitis. METHODS: Nasal lavage samples were obtained from patients during active seasonal allergic rhinitis. Specific cellular immunocytochemical techniques were used to detect the cysteinyl leukotriene synthetic proteins, namely 5-lipoxygenase (5-LO), 5-lipoxygenase-activating protein (FLAP) and leukotriene C4 synthase (LTC4S). In situ hybridization and immunocytochemical techniques were used to identify the mRNA and proteins for the CysLT1 and CysLT2 receptors. RESULTS: 5-LO, FLAP and LTC4S, and the CysLT1 and CysLT2 receptors were expressed in the majority of eosinophils and in subsets of mast cells and mononuclear cells. 5-LO, FLAP and the CysLT1 receptor, but not LTC4S or the CysLT2 receptor, were expressed in a subset of nasal neutrophils. CONCLUSIONS: Our study demonstrates the presence of CysLT pathway proteins in key allergic and inflammatory cells from the upper airway of patients with active seasonal allergic rhinitis. Our expression data highlight the potential of CysLT-modifying agents to treat both upper and lower airway symptoms in patients suffering from allergic rhinitis and asthma.

The murine cysteinyl leukotriene 2 (CysLT2) receptor. cDNA and genomic cloning, alternative splicing, and in vitro characterization.

Two classes of cysteinyl leukotriene receptor, CysLT(1) and CysLT(2), have been identified and pharmacologically characterized in human tissues. Although the CysLT(1) receptor mediates the proinflammatory effects of leukotrienes in human asthma, the physiological roles of CysLT(2) receptor are not defined, and a suitable mouse model would be useful in delineating function. We report here the molecular cloning and characterization of the mouse CysLT(2) receptor (mCysLT(2)R) from heart tissue. mCysLT(2)R cDNA encodes a protein of 309 amino acids, truncated at both ends compared with the human ortholog (hCysLT(2)R). The gene resides on the central region of mouse chromosome 14 and is composed of 6 exons with the entire coding region located in the last exon. Two 5'-untranslated region splice variants were identified with the short form lacking exon 3 as the predominant transcript. Although the overall expression of mCysLT(2)R is very low, the highest expression was detected in spleen, thymus, and adrenal gland by ribonuclease protection assay, and discrete sites of expression in heart were observed by in situ hybridization. Intracellular calcium mobilization in response to cysteinyl leukotriene administration was detected in human embryonic kidney 293T cells transfected with recombinant mCysLT(2)R with a rank order of potency leukotriene C(4)(LTC(4) ) = LTD(4)>>LTE(4). [(3)H]LTD(4) binding to membranes expressing mCysLT(2)R could be effectively competed by LTC(4) and LTD(4) and only partially inhibited by LTE(4) and BAYu9773. The identification of mCysLT(2)R will be useful for establishing CysLT(2)R-deficient mice and determining novel leukotriene functions.

Identification and characterization of a second melanin-concentrating hormone receptor, MCH-2R.

Melanin-concentrating hormone (MCH) is a 19-aa cyclic neuropeptide originally isolated from chum salmon pituitaries. Besides its effects on the aggregation of melanophores in fish several lines of evidence suggest that in mammals MCH functions as a regulator of energy homeostasis. Recently, several groups reported the identification of an orphan G protein-coupled receptor as a receptor for MCH (MCH-1R). We hereby report the identification of a second human MCH receptor termed MCH-2R, which shares about 38% amino acid identity with MCH-1R. MCH-2R displayed high-affinity MCH binding, resulting in inositol phosphate turnover and release of intracellular calcium in mammalian cells. In contrast to MCH-1R, MCH-2R signaling is not sensitive to pertussis toxin and MCH-2R cannot reduce forskolin-stimulated cAMP production, suggesting an exclusive G(alpha)q coupling of the MCH-2R in cell-based systems. Northern blot and in situ hybridization analysis of human and monkey tissue shows that expression of MCH-2R mRNA is restricted to several regions of the brain, including the arcuate nucleus and the ventral medial hypothalamus, areas implicated in regulation of body weight. In addition, the human MCH-2R gene was mapped to the long arm of chromosome 6 at band 6q16.2-16.3, a region reported to be associated with cytogenetic abnormalities of obese patients. The characterization of a second mammalian G protein-coupled receptor for MCH potentially indicates that the control of energy homeostasis in mammals by the MCH neuropeptide system may be more complex than initially anticipated.

Expression of the cysteinyl leukotriene 1 receptor in normal human lung and peripheral blood leukocytes.

The cysteinyl leukotrienes (CysLTs) are important mediators of human asthma. Pharmacologic and clinical studies show that the CysLTs exert most of their bronchoconstrictive and proinflammatory effects through activation of a putative, 7-transmembrane domain, G-protein-coupled receptor, the CysLT1 receptor. The initial molecular characterization of the CysLT1 receptor showed by in situ hybridization, the presence of CysLT1 receptor messenger RNA (mRNA) in human lung smooth-muscle cells and lung macrophages. We confirmed the results of these in situ hybridization analyses for the CysLT1 receptor, and produced the first immunohistochemical characterization of the CysLT1 receptor protein in human lung. The identification of the CysLT1 receptor in the lung is consistent with the antibronchoconstrictive and antiinflammatory actions of CysLT1 receptor antagonists. We also report the expression of CysLT1 receptor mRNA and protein in most peripheral blood eosinophils and pregranulocytic CD34+ cells, and in subsets of monocytes and B lymphocytes.

A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy.

Stargardt-like macular dystrophy (STGD3, MIM 600110) and autosomal dominant macular dystrophy (adMD) are inherited forms of macular degeneration characterized by decreased visual acuity, macular atrophy and extensive fundus flecks. Genetic mapping data suggest that mutations in a single gene may be responsible for both conditions, already known to bear clinical resemblance. Here we limit the minimum genetic region for STGD3 and adMD to a 0.6-cM interval by recombination breakpoint mapping and identify a single 5-bp deletion within the protein-coding region of a new retinal photoreceptor-specific gene, ELOVL4, in all affected members of STGD3 and adMD families. Bioinformatic analysis of ELOVL4 revealed that it has homology to a group of yeast proteins that function in the biosynthesis of very long chain fatty acids. Our results are therefore the first to implicate the biosynthesis of fatty acids in the pathogenesis of inherited macular degeneration.

The human inward rectifier K(+) channel subunit kir5.1 (KCNJ16) maps to chromosome 17q25 and is expressed in kidney and pancreas.

A novel human Kir5.1 (inward rectifier K+ channel subunit, gene name KCNJ16) was identified through database searches. This human KCNJ16 was mapped to chromosome 17q25. The full-length cDNA was identified and its genomic structure was determined. Tissue distribution studies showed that human KCNJ16 is significantly expressed in human kidney, pancreas and thyroid gland. In situ hybridization revealed expression in convoluted tubule cells of kidney and in the acinar and ductal cells of pancreas. These suggest that human Kir5.1 may be involved in the regulation of fluid and pH balance, thus making it a potential therapeutic target for hypertension, renal failure, or pancreatic disease.

Expression of the type I diabetes-associated gene LRP5 in macrophages, vitamin A system cells, and the Islets of Langerhans suggests multiple potential roles in diabetes.

LRP5 is a novel member of the low-density lipoprotein receptor family that is genetically associated with Type 1 diabetes. As a start to defining the normal function of LRP5 and to generate testable hypotheses of its potential role in Type 1 diabetes pathogenesis, we carried out an extensive expression analysis of this gene at the mRNA and protein levels in normal human, monkey, and mouse, as well as in non-obese diabetic (NOD) mice at several stages of diabetes development. In all species, expression of LRP5 was found in four functionally important cell types: the distributed mononuclear phagocyte system, the islets of Langerhans, vitamin A-metabolizing cells, and CNS neurons. Given the critical role of macrophages in the onset and progression of islet cell destruction in Type 1 diabetes and the hypothesized role of retinoids as modifiers of diabetes progression, these findings suggest that LRP5 may confer Type 1 diabetes risk by altering the normal functioning of one or more of these regulatory systems. Specifically, given that the LRP5 polymorphisms associated with diabetes are in the promoter region of the gene, alterations in LRP5 expression may be responsible for diabetes susceptibility and therefore may be potential targets for therapeutic intervention. (J Histochem Cytochem 48:1357-1368, 2000)

Characterization of the human cysteinyl leukotriene 2 receptor.

The contractile and inflammatory actions of the cysteinyl leukotrienes (CysLTs), LTC(4), LTD(4), and LTE(4), are thought to be mediated through at least two distinct but related CysLT G protein-coupled receptors. The human CysLT(1) receptor has been recently cloned and characterized. We describe here the cloning and characterization of the second cysteinyl leukotriene receptor, CysLT(2), a 346-amino acid protein with 38% amino acid identity to the CysLT(1) receptor. The recombinant human CysLT(2) receptor was expressed in Xenopus oocytes and HEK293T cells and shown to couple to elevation of intracellular calcium when activated by LTC(4), LTD(4), or LTE(4). Analyses of radiolabeled LTD(4) binding to the recombinant CysLT(2) receptor demonstrated high affinity binding and a rank order of potency for competition of LTC(4) = LTD(4) LTE(4). In contrast to the dual CysLT(1)/CysLT(2) antagonist, BAY u9773, the CysLT(1) receptor-selective antagonists MK-571, montelukast (Singulair(TM)), zafirlukast (Accolate(TM)), and pranlukast (Onon(TM)) exhibited low potency in competition for LTD(4) binding and as antagonists of CysLT(2) receptor signaling. CysLT(2) receptor mRNA was detected in lung macrophages and airway smooth muscle, cardiac Purkinje cells, adrenal medulla cells, peripheral blood leukocytes, and brain, and the receptor gene was mapped to chromosome 13q14, a region linked to atopic asthma.

Cloning and functional expression of two families of beta-subunits of the large conductance calcium-activated K+ channel.

We report here a characterization of two families of calcium-activated K(+) channel beta-subunits, beta2 and beta3, which are encoded by distinct genes that map to 3q26.2-27. A single beta2 family member and four alternatively spliced variants of beta3 were investigated. These subunits have predicted molecular masses of 27. 1-31.6 kDa, share approximately 30-44% amino acid identity with beta1, and exhibit distinct but overlapping expression patterns. Coexpression of the beta2 or beta3a-c subunits with a BK alpha-subunit altered the functional properties of the current expressed by the alpha-subunit alone. The beta2 subunit rapidly and completely inactivated the current and shifted the voltage dependence for activation to more polarized membrane potentials. In contrast, coexpression of the beta3a-c subunits resulted in only partial inactivation of the current, and the beta3b subunit conferred an apparent inward rectification. Furthermore, unlike the beta1 and beta2 subunits, none of the beta3 subunits increased channel sensitivity to calcium or voltage. The tissue-specific expression of these beta-subunits may allow for the assembly of a large number of distinct BK channels in vivo, contributing to the functional diversity of native BK currents.

Retina-specific nuclear receptor: A potential regulator of cellular retinaldehyde-binding protein expressed in retinal pigment epithelium and Müller glial cells.

In an effort to identify nuclear receptors important in retinal disease, we screened a retina cDNA library for nuclear receptors. Here we describe the identification of a retina-specific nuclear receptor (RNR) from both human and mouse. Human RNR is a splice variant of the recently published photoreceptor cell-specific nuclear receptor [Kobayashi, M., Takezawa, S., Hara, K., Yu, R. T., Umesono, Y., Agata, K., Taniwaki, M., Yasuda, K. & Umesono, K. (1999) Proc. Natl. Acad. Sci. USA 96, 4814-4819] whereas the mouse RNR is a mouse ortholog. Northern blot and reverse transcription-PCR analyses of human mRNA samples demonstrate that RNR is expressed exclusively in the retina, with transcripts of approximately 7.5 kb, approximately 3.0 kb, and approximately 2.3 kb by Northern blot analysis. In situ hybridization with multiple probes on both primate and mouse eye sections demonstrates that RNR is expressed in the retinal pigment epithelium and in Müller glial cells. By using the Gal4 chimeric receptor/reporter cotransfection system, the ligand binding domain of RNR was found to repress transcriptional activity in the absence of exogenous ligand. Gel mobility shift assays revealed that RNR can interact with the promoter of the cellular retinaldehyde binding protein gene in the presence of retinoic acid receptor (RAR) and/or retinoid X receptor (RXR). These data raise the possibility that RNR acts to regulate the visual cycle through its interaction with cellular retinaldehyde binding protein and therefore may be a target for retinal diseases such as retinitis pigmentosa and age-related macular degeneration.

Characterization of the human cysteinyl leukotriene CysLT1 receptor.

The cysteinyl leukotrienes-leukotriene C4(LTC4), leukotriene D4(LTD4) and leukotriene E4(LTE4)-are important mediators of human bronchial asthma. Pharmacological studies have determined that cysteinyl leukotrienes activate at least two receptors, designated CysLT1 and CysLT2. The CysLT1-selective antagonists, such as montelukast (Singulair), zafirlukast (Accolate) and pranlukast (Onon), are important in the treatment of asthma. Previous biochemical characterization of CysLT1 antagonists and the CysLT1 receptor has been in membrane preparations from tissues enriched for this receptor. Here we report the molecular and pharmacological characterization of the cloned human CysLT1 receptor. We describe the functional activation (calcium mobilization) of this receptor by LTD4 and LTC4, and competition for radiolabelled LTD4 binding to this receptor by the cysteinyl leukotrienes and three structurally distinct classes of CysLT1-receptor antagonists. We detected CysLT1-receptor messenger RNA in spleen, peripheral blood leukocytes and lung. In normal human lung, expression of the CysLT1-receptor mRNA was confined to smooth muscle cells and tissue macrophages. Finally, we mapped the human CysLT1-receptor gene to the X chromosome.

TECK: a novel CC chemokine specifically expressed by thymic dendritic cells and potentially involved in T cell development.

A novel CC chemokine was identified in the thymus of mouse and human and was designated TECK (thymus-expressed chemokine). TECK has weak homology to other CC chemokines and maps to mouse chromosome 8. Besides the thymus, mRNA encoding TECK was detected at substantial levels in the small intestine and at low levels in the liver. The source of TECK in the thymus was determined to be thymic dendritic cells; in contrast, bone marrow-derived dendritic cells do not express TECK. The murine TECK recombinant protein showed chemotactic activity for activated macrophages, dendritic cells, and thymocytes. We conclude that TECK represents a novel thymic dendritic cell-specific CC chemokine that is possibly involved in T cell development.

Similar RANTES levels in healthy and asthmatic airways by immunoassay and in situ hybridization.

To determine if RANTES expression is unregulated in the airways of asthmatic subjects, we performed bronchial mucosal biopsies and airway lavage in seven atopic asthmatic subjects and eight healthy subjects. Immunohistochemistry was used to reveal RANTES protein expression in the airway biopsies. An ELISA was used to quantitate RANTES in lavage. In three subjects in each group, we also used in situ hybridization to reveal mRNA for RANTES in airway biopsies. We found that the mean (+/- SD) percent expression for RANTES in the epithelium and submucosa was 26 +/- 9% and 26 +/- 10% in the asthmatic and healthy tissue samples, respectively. RANTES mRNA was demonstrable in the bronchial mucosa of both healthy and asthmatic subjects, predominantly in the epithelial cells but also in the submucosa. We also found that there was no significant difference in the median RANTES concentrations between the groups (healthy: 2.9 pg/ml [range: 0.0 to 28.7 pg/ml]; asthma: 1.8 pg/ml [range: 0.0 to 82.1 pg/ml], p > 0.05) despite a trend for higher concentrations of eosinophil cationic protein (ECP) in the asthmatic group (p = 0.08). In summary, this study confirms that cells in airway mucosal tissue produce RANTES but that the level of production in mild stable asthma is not different from that of healthy control subjects.