Ca2؉ elevations in Chinese hamster ovary cells stably expressing OX 1 receptors were measured using fluorescent Ca 2؉ indicators fura-2 and fluo-3. Stimulation with orexin-A led to pronounced Ca 2؉ elevations with an EC 50 around 1 nM. When the extracellular [Ca 2؉ ] was reduced to a submicromolar concentration, the EC 50 was increased 100-fold. Similarly, the inositol 1,4,5-trisphosphate production in the presence of 1 mM external Ca 2؉ was about 2 orders of magnitude more sensitive to orexin-A stimulation than in low extracellular Ca influx and (ii) a direct stimulation of phospholipase C, and that these two responses converge at the level of phospholipase C where the former markedly enhances the potency of the latter.The recently described hypothalamic peptides called orexins (1) or hypocretins (2) mediate their effects through G proteincoupled receptors called OX 1 and OX 2 receptors (1). The peptides and their receptors are widespread in the hypothalamus, cortex, and brainstem (2-5). The orexin/hypocretin peptides are encoded by a single mRNA giving rise to a 33-residue orexin-A peptide containing disulfide bridges and a linear 28-residue orexin-B (1). Orexin-A has a 10 -100-fold higher affinity and potency for OX 1 receptor as compared with orexin-B, whereas no preference is displayed by the OX 2 receptor (1). The orexins cause robust increases in intracellular Ca 2ϩ both in neurons cultured from rat medial and lateral hypothalamus (6) and spinal cord (7), and when studied using recombinant receptors (1). This has led to the suggestion that the receptors are coupled to the G q family G proteins. Interestingly, the response in neurons is partially dependent on extracellular Ca 2ϩ , which may suggest that the receptors are connected to a Ca 2ϩ influx pathway in neurons (6). Several different pathways for receptor-stimulated Ca 2ϩ entry have been suggested based on functional studies with other G protein-coupled receptors. Suggested pathways include store-operated Ca 2ϩ channels, second messenger-operated channels, as well as Ca 2ϩ -activated Ca 2ϩ channels (reviewed in Refs. 8 and 9). The aim of this study was to examine in detail the Ca 2ϩ mobilizing actions of orexins on recombinant OX 1 receptors expressed in CHO 1 -K1 cells. The results reveal the presence of a novel amplification mechanism at the level of phospholipase C that is dependent on activation of Ca 2ϩ influx pathway upstream of phospholipase C. EXPERIMENTAL PROCEDURESCell Cultures-To prepare the CHO-hOX 1 -C1 cells used in this study CHO-K1 cells were transfected with a bicistronic vector containing the coding sequence of human OX 1 receptor as described previously for chemokine receptors (10). Neomycin resistant clones were then isolated by limited dilution. They were grown in nutrient mixture (Ham's F-12) medium (Life Technologies, Inc., Paisley, United Kingdom) supplemented with 100 units/ml penicillin G (Sigma), 80 units/ml streptomycin (Sigma), 400 g/ml Geneticin (G418; Life Technologies, Inc.) and 10% (v/v) fetal calf serum (Life Te...

In this study, we have compared the abilities of orexin-A and orexin-B and variants of orexin-A to activate different Ca 2ϩ responses (influx and release) in human OX 1 and OX 2 receptorexpressing Chinese hamster ovary cells. Responses mediated by activation of both receptor subtypes with either orexin-A or -B were primarily dependent on extracellular Ca 2ϩ , suggesting similar activation of Ca 2ϩ influx as we have previously shown for orexin-A and OX 1 receptors. Amino acid-wise truncation of orexin-A reduced its ability to activate OX 1 and OX 2 receptors, but the response mediated by the OX 2 receptor was more resistant to truncation than the response mediated by the OX 1 receptor. We also performed a sequential replacement of amino acids 14 to 26 with alanine in the truncated orexin-A variant orexin-A 14 -33 . Replacement of the same amino acids produced a fall in the potency for each receptor subtype, but the reduction was less prominent for the OX 2 receptor. The most marked reduction was produced by the replacement of Leu20, Asp25, and His26 with alanine. Interestingly, extracellular Ca 2ϩ dependence of responses to some of the mutated peptides was different from those of orexin-A and -B. The mutagenesis also suggests that although the determinants required from orexin-A for binding to and activation of the receptor are highly conserved between the orexin receptor subtypes, the OX 2 receptor requires fewer determinants. This might in part explain why orexin-B has the affinity and potency equal to orexin-A for this subtype, although it has 10-to 100-fold lower affinity and potency for the OX 1 receptor.Recently, two novel hypothalamic peptides were isolated and subsequently named orexin-A and orexin-B (Sakurai et al., 1998) or hypocretin-1 and hypocretin-2 (de Lecea et al., 1998). Despite some initial confusion, orexin-A should now be considered identical to hypocretin-1 and orexin-B to hypocretin-2. Orexins act as agonists on two G-protein-coupled receptors called OX 1 and OX 2 receptors. Increased wakefulness and reduced sleep is a well demonstrated response to central administration of orexin, and disruption of central orexinergic signaling leads to the sleep disorder narcolepsy in animal models and probably also in man (reviewed in Beuckmann and Yanagisawa, 2002;Kukkonen et al., 2002;Sutcliffe and de Lecea, 2002). The other physiological roles for orexins may be regulation of energy homeostasis and stress response, probably both via central and peripheral mechanisms (reviewed in Willie et al., 2001;Beuckmann and Yanagisawa, 2002;Kirchgessner, 2002;Kukkonen et al., 2002;Smart and Jerman, 2002).The two orexin peptides, orexin-A and -B, are both products of the same precursor peptide, preproorexin, cleavage of which results in equimolar amounts of orexin-A and orexin-B. Orexin-A is composed of 33 amino acids and it contains two disulfide bridges, whereas orexin-B is a linear peptide of 28 residues (Sakurai et al., 1998). Although a product of a different part of the precursor peptide, orexin-B sho...

We have performed genome-wide expression profiling of endocrine regulation of genes expressed in the mouse initial segment (IS) and distal caput of the epididymis by using Affymetrix microarrays. The data revealed that of the 15 020 genes expressed in the epididymis, 35% were enriched in one of the two regions studied, indicating that differential functions can be attributed to the IS and the more distal caput regions. The data, furthermore, showed that 27% of the genes expressed in the IS and/or distal caput epididymidis are under the regulation of testicular factors present in the duct fluid, while bloodborne androgens can regulate for 14% of them. This is in line with the high testis dependency of epididymal physiology. We then focused on genes with moderate or strong expression, showing strict segment enrichment and strong dependency on testicular factors. Analyses of the 59 genes, including upregulated and downregulated genes, fulfilling the criteria indicated that the expression of 18 (17 downregulated genes; 1 upregulated gene) of 19 gonadectomy-responsive genes enriched in the IS was not maintained by the androgen treatment, whereas the expression of all six downregulated genes enriched in the distal caput and the majority of those with no strict segment enrichment of expression (28 of 34; consisting of 23 downregulated and 5 upregulated genes) were maintained by androgens. Hence, it is evident that testicular factors other than androgens are important for the expression of IS-enriched genes, whereas the expression of distal caput-enriched genes is typically regulated by androgens. Identical data were obtained by independent clustering analyses performed for the expression data of 3626 epididymal genes. Several novel genes with putative involvement in epididymal sperm maturation, such as a disintegrin and metallopeptidase domain 28 (Adam28) and a solute carrier organic anion transporter family, member 4C1 (Slco4c1), were identified, indicating that this approach is successful for identifying novel epididymal genes.

We have previously demonstrated that male transgenic (TG) mice overexpressing human chorionic gonadotropin (hCG þ ) develop reproductive organ defects, but no tumors, in adult age. In this study, the effects of persistently elevated hCG were followed in TG males between day 5 postpartum and adulthood. Leydig cell (LC) adenomas were found in prepubertal mice, most prominently at the age of 10 days, but not in adult age. Serum testosterone concentrations were significantly increased in TG males at all ages studied. The phenotype of the prepubertal hCG þ males resembled that found in boys upon expression of constitutively activating luteinizing hormone (LH) receptor mutations. The temporal expression patterns of the fetal LC marker gene, thrombospondin 2, and those of adult LCs, hydroxysteroid dehydrogenase-6, delta 5 -3-beta and prostaglandin D synthase, were similar in wild-type and hCG þ males. Hence, the postnatal adenomas resemble functionally fetal LCs, and only these cells are susceptible to hCG-induced tumorigenesis. Our findings demonstrate a novel intriguing difference between the fetal and adult LC populations and provide further insight into the potential tumorigenic effects of gonadotropins.

Sphingomyelin derivatives modulate a multitude of cellular processes, including the regulation of [Ca2+]i (the intracellular free calcium concentration). Previous studies have shown that these metabolites often inhibit calcium entry through VOCCs (voltage-operated calcium channels). In the present study, we show that, in pituitary GH4C1 cells, C1P (C2-ceramide 1-phosphate) enhances calcium entry in a dose-dependent manner. The phospholipase C inhibitor U73122 attenuated the response. C1P invoked a small, but significant, increase in the formation of inositol phosphates. Pre-treatment of the cells with pertussis toxin was without an effect on the C1P-evoked increase in [Ca2+]i. The effect of C1P was critically dependent on extracellular calcium, since no increase in [Ca2+]i was observed when cells in a calcium-free buffer were stimulated with C1P. Furthermore, if the cells were retreated with 300 nM of the VOCC inhibitor nimodipine, the effect of C1P was almost totally abolished. In addition, ceramide C8-1-phosphate evoked an increase in [Ca2+]i, but the onset of the response was slow compared with that of C1P. In cells treated with 1 mM thapsigargin for 15 min, C1P still evoked an increase in [Ca2+]i. In patch-clamp experiments in the whole-cell mode, C1P enhanced calcium entry through the VOCCs compared with vehicle-treated cells. Dialysis of the cells with C1P did not enhance the calcium current. On-cell patch-clamp experiments showed an enhanced probability of the VOCCs being open (P(open)) in the presence of C1P. Inhibition of PKC (protein kinase C) with GF109203X and down-regulation of PKC with PMA attenuated the C1P-evoked increase in [Ca2+]i. Furthermore, down-regulation of PKC abolished the effect of C1P on P(open). This is the first report showing that a sphingomyelin derivative enhances calcium entry through VOCCs.