Phenylalanine hydroxylase contributes to serotonin synthesis in mice.

Serotonin is an important signaling molecule in the periphery and in the brain. The hydroxylation of tryptophan is the first and rate-limiting step of its synthesis. In most vertebrates, two enzymes have been described to catalyze this step, tryptophan hydroxylase (TPH) 1 and 2, with expression localized to peripheral and neuronal cells, respectively. However, animals lacking both TPH isoforms still exhibit about 10% of normal serotonin levels in the blood demanding an additional source of the monoamine. In this study, we provide evidence by the gain and loss of function approaches in in vitro and in vivo systems, including stable-isotope tracing in mice, that phenylalanine hydroxylase (PAH) is a third TPH in mammals. PAH contributes to serotonin levels in the blood, and may be important as a local source of serotonin in organs in which no other TPHs are expressed, such as liver and kidney.

Neuropeptide B promotes proliferation and differentiation of rat brown primary preadipocytes.

Neuropeptide B (NPB) is reported to regulate energy homeostasis and metabolism via the NPBWR1 and NPBWR2 receptors in various tissues. However, the molecular mechanisms triggered from their interaction are not well investigated in brown adipose tissue. In this study, we specifically analyzed the role of NPB in controlling brown adipogenesis in rat brown preadipocytes. We first detected the expression of NPBWR1 and NPB on mRNA and protein level in brown preadipocytes and observed that NPB increased viability and proliferation of preadipocytes. Moreover, NPB stimulated expression of adipogenic genes (Prdm16, Ucp1) and suppressed the expression of antiadipogenic preadipocyte factor 1 (Pref1) during the differentiation process. Altogether, this led to an increase in intracellular lipid accumulation during preadipocyte differentiation, coupled with an increase in adrenaline-induced oxygen consumption mediated by NPB. Furthermore, Ucp1 expression stimulated by NPB was attenuated by blockade of p38 kinase. In summary, we conclude that NPB promotes proliferation and differentiation of rat brown preadipocytes via p38-dependent mechanism and plays an important role in controlling brown adipose tissue formation.

Targeted esterase-induced dye (TED) loading supports direct calcium imaging in eukaryotic cell-free systems.

Calcium imaging is an important functional tool for analysing ion channels, transporters and pumps for drug screening in living cells. Depicted eukaryotic cell-free systems utilize microsomes, derived from the endoplasmic reticulum to incorporate the synthesized membrane proteins-like ion channels. Carboxylesterase is required to cleave the acetoxymethyl ester moiety of the chemical calcium indicators in order to ensure its immobility across the endoplasmic reticulum membrane. Absence or an inadequate amount of carboxylesterase in the endoplasmic reticulum of different eukaryotic cells poses a hindrance to perform calcium imaging in microsomes. In this work, we try to overcome this drawback and adapt the cell-based calcium imaging principle to a cell-free protein synthesis platform. Carboxylesterase synthesized in a Spodoptera frugiperda Sf21 lysate translation system is established as a viable calcium imaging tool in microsomes. Cell-free synthesized carboxylesterase inside microsomes is validated with esterase and dye loading assays. Native proteins from the endoplasmic reticulum, such as ryanodine channels and calcium ATPase, are analysed. Cell-free synthesized transient receptor potential channels are used as model proteins to demonstrate the realization of this concept.

TRPM8 Activation via 3-Iodothyronamine Blunts VEGF-Induced Transactivation of TRPV1 in Human Uveal Melanoma Cells.

In human uveal melanoma (UM), tumor enlargement is associated with increases in aqueous humor vascular endothelial growth factor-A (VEGF-A) content that induce neovascularization. 3-Iodothyronamine (3-T1AM), an endogenous thyroid hormone metabolite, activates TRP melastatin 8 (TRPM8), which blunts TRP vanilloid 1 (TRPV1) activation by capsaicin (CAP) in human corneal, conjunctival epithelial cells, and stromal cells. We compare here the effects of TRPM8 activation on VEGF-induced transactivation of TRPV1 in an UM cell line (92.1) with those in normal primary porcine melanocytes (PM) since TRPM8 is upregulated in melanoma. Fluorescence Ca2+-imaging and planar patch-clamping characterized functional channel activities. CAP (20 µM) induced Ca2+ transients and increased whole-cell currents in both the UM cell line and PM whereas TRPM8 agonists, 100 µM menthol and 20 µM icilin, blunted such responses in the UM cells. VEGF (10 ng/ml) elicited Ca2+ transients and augmented whole-cell currents, which were blocked by capsazepine (CPZ; 20 µM) but not by a highly selective TRPM8 blocker, AMTB (20 µM). The VEGF-induced current increases were not augmented by CAP. Both 3-T1AM (1 µM) and menthol (100 µM) increased the whole-cell currents, whereas 20 µM AMTB blocked them. 3-T1AM exposure suppressed both VEGF-induced Ca2+ transients and increases in underlying whole-cell currents. Taken together, functional TRPM8 upregulation in UM 92.1 cells suggests that TRPM8 is a potential drug target for suppressing VEGF induced increases in neovascularization and UM tumor growth since TRPM8 activation blocked VEGF transactivation of TRPV1.

Upregulation of Transient Receptor Potential Vanilloid Type-1 Channel Activity and Ca2+ Influx Dysfunction in Human Pterygial Cells.

PURPOSE: The heat-sensitive transient receptor potential vanilloid type-1 (TRPV1) channel (i.e., capsaicin [CAP] receptor) is upregulated in numerous cancers. This study determined if this response occurs in fresh and cultured hyperplastic human pterygial epithelial tissues. METHODS: Reverse transcriptase PCR and quantitative real-time PCR, along with immunohistochemistry and Western blotting, characterized TRPV1 expression patterns in pterygial and healthy conjunctival tissue, primary and immortalized pterygial cells (hPtEC), and primary and immortalized conjunctival epithelial cells (HCjEC). Imaging of Ca2+ and planar whole-cell patch-clamping evaluated TRP channel activity. An MTS assay measured cell metabolic activity and a cell growth assay monitored proliferation. RESULTS: Capsaicin (20 µM) and elevating bath temperature above 43°C activated Ca2+ transients more in hPtEC than HCjEC. Capsaicin induced corresponding changes in inward currents that were inhibited by 20 µM capsazepine (CPZ). Vascular endothelial growth factor (VEGF) also increased Ca2+-influx and induced corresponding inward currents more in hPtEC than in HCjEC, whereas CPZ (20 µM), BCTC (20 µM), or La3+ (500 µM) reduced these responses, respectively. Whereas epidermal growth factor (EGF) increased proliferation more in hPtEC than in HCjEC, VEGF had no effect on this response. Capsazepine suppressed hPtEC proliferation induced by EGF and VEGF, whereas it was cytotoxic to HCjEC. CONCLUSIONS: Mitogenic responses to EGF and VEGF are mediated through TRPV1 transactivation. Only in hPtEC do the increases in proliferation induced by EGF exceed those in HCjEC. Therefore, TRPV1 is a potential drug target whose clinical relevance in treating pterygium warrants further assessment.

3-Iodothyronamine increases transient receptor potential melastatin channel 8 (TRPM8) activity in immortalized human corneal epithelial cells.

3-Iodothyronamine (3T1AM) is an endogenous thyroid hormone metabolite that interacts with the human trace amine-associated receptor 1 (hTAAR1), a G-protein-coupled receptor, to induce numerous physiological responses including dose-dependent body temperature lowering in rodents. 3T1AM also directly activates cold-sensitive transient receptor potential melastatin 8 (TRPM8) channels in human conjunctival epithelial cells (HCjEC) at constant temperature as well as reducing rises in IL-6 release induced by transient receptor potential vanilloid 1 (TRPV1) activation by capsaicin (CAP). Here, we describe that 3T1AM-induced TRPM8 activation suppresses through crosstalk TRPV1 activation in immortalized human corneal epithelial cells (HCEC). RT-PCR and immunofluorescent staining identified TRPM8 gene and protein expression. Increases in Ca(2+) influx induced by the TRPM8 agonists either 3T1AM (0.1-10 µM), menthol (500 µM), icilin (15-60 µM) or temperature lowering (either <17°C or >17°C) were all blocked by 10-20 µM BCTC, a mixed TRPV1/TRPM8 antagonist. BCTC blocked 3T1AM-induced recombinant TRPM8 activation of Ca(2+) transients in an osteosarcoma heterologous expression system. The effects of BCTC in HCEC were attributable to selective TRPM8 inhibition since whole-cell patch-clamp currents underlying Ca(2+) rises induced by 20 µM CAP were BCTC insensitive. On the other hand, Ca(2+) transients induced by activating TRPV1 with either CAP or a hyperosmolar medium were suppressed during exposure to either 1 µM 3T1AM or 15 µM icilin. All of these modulatory effects on intracellular Ca(2+) regulation induced by the aforementioned agents were attributable to changes in underlying inward and outward current. Taken together, TRPM8 activation by 3T1AM markedly attenuates and even eliminates hyperosmolar and CAP induced TRPV1 activation through crosstalk.