L-Carnitine Reduces in Human Conjunctival Epithelial Cells Hypertonic- Induced Shrinkage through Interacting with TRPV1 Channels

Background/Aims: Ocular surface health depends on conjunctival epithelial (HCjE) layer integrity since it protects against pathogenic infiltration and contributes to tissue hydration maintenance. As the same increases in tear film hyperosmolarity described in dry eye disease can increase corneal epithelial transient receptor potential vanilloid type-1 (TRPV1) channel activity, we evaluated its involvement in mediating an osmoprotective effect by L-carnitine against such stress. Methods: Using siRNA gene silencing, Ca2+imaging, planar patch- clamping and relative cell volume measurements, we determined if the protective effects of this osmolyte stem from its interaction with TRPV1. Results: TRPV1 activation by capsaicin (CAP) and an increase in osmolarity to≈450 mOsM both induced increases in Ca2+levels. In contrast, blocking TRPV1 activation with capsazepine (CPZ) fully reversed this response. Similarly, L-carnitine (1 mM) also reduced underlying whole-cell currents. In calcein-AM loaded cells, hypertonic-induced relative cell volume shrinkage was fully blocked during exposure to L-carnitine. On the other hand, in TRPV1 gene-silenced cells, this protective effect by L-carnitine was obviated. Conclusion: The described L-carnitine osmoprotective effect is elicited through suppression of hypertonic-induced TRPV1 activation leading to increases in L-carnitine uptake through a described Na+-dependent L-carnitine transporter

Vascular endothelial growth factor (VEGF) induced downstream responses to transient receptor potential vanilloid 1 (TRPV1) and 3-lodothyronamine (3-T1AM) in human corneal keratocytes

This study was undertaken to determine if crosstalk among the transient receptor potential (TRP) melastatin 8 (TRPM8), TRP vanilloid 1 (TRPV1), and vascular endothelial growth factor (VEGF) receptor triad modulates VEGF-induced Ca2+ signaling in human corneal keratocytes. Using RT-PCR, qPCR and immunohistochemistry, we determined TRPV1 and TRPM8 gene and protein coexpression in a human corneal keratocyte cell line (HCK) and human corneal cross sections. Fluorescence Ca2+ imaging using both a photomultiplier and a single cell digital imaging system as well as planar patch-clamping measured relative intracellular Ca2+ levels and underlying whole-cell currents. The TRPV1 agonist capsaicin increased both intracellular Ca2+ levels and whole-cell currents, while the antagonist capsazepine (CPZ) inhibited them. VEGF-induced Ca2+ transients and rises in whole-cell currents were suppressed by CPZ, whereas a selective TRPM8 antagonist, AMTB, increased VEGF signaling. In contrast, an endogenous thyroid hormone-derived metabolite 3-lodothyronamine (3-T(1)AM) suppressed increases in the VEGF-induced current. The TRPM8 agonist menthol increased the currents, while AMTB suppressed this response. The VEGF-induced increases in Ca2+ influx and their underlying ionic currents stem from crosstalk between VEGFR and TRPV1, which can be impeded by 3-T(1)AM-induced TRPM8 activation. Such suppression in turn blocks VEGF-induced TRPV1 activation. Therefore, crosstalk between TRPM8 and TRPV1 inhibits VEGFR-induced activation of TRPV1

3-Iodothyronamine Activates a Set of Membrane Proteins in Murine Hypothalamic Cell Lines

3-Iodothyronamine (3-T1AM) is an endogenous thyroid hormone metabolite. The profound pharmacological effects of 3-T1AM on energy metabolism and thermal homeostasis have raised interest to elucidate its signaling properties in tissues that pertain to metabolic regulation and thermogenesis. Previous studies identified G protein-coupled receptors (GPCRs) and transient receptor potential channels (TRPs) as targets of 3-T1AM in different cell types. These two superfamilies of membrane proteins are largely expressed in tissue which influences energy balance and metabolism. As the first indication that 3-T1AM virtually modulates the function of the neurons in hypothalamus, we observed that intraperitoneal administration of 50 mg/kg bodyweight of 3-T1AM significantly increased the c-FOS activation in the paraventricular nucleus (PVN) of C57BL/6 mice. To elucidate the underlying mechanism behind this 3-T1AM-induced signalosome, we used three different murine hypothalamic cell lines, which are all known to express PVN markers, GT1-7, mHypoE-N39 (N39) and mHypoE-N41 (N41). Various aminergic GPCRs, which are the known targets of 3-T1AM, as well as numerous members of TRP channel superfamily, are expressed in these cell lines. Effects of 3-T1AM on activation of GPCRs were tested for the two major signaling pathways, the action of Gαs/adenylyl cyclase and Gi/o. Here, we demonstrated that this thyroid hormone metabolite has no significant effect on Gi/o signaling and only a minor effect on the Gαs/adenylyl cyclase pathway, despite the expression of known GPCR targets of 3-T1AM. Next, to test for other potential mechanisms involved in 3-T1AM-induced c-FOS activation in PVN, we evaluated the effect of 3-T1AM on the intracellular Ca2+ concentration and whole-cell currents. The fluorescence-optic measurements showed a significant increase of intracellular Ca2+ concentration in the three cell lines in the presence of 10 μM 3-T1AM. Furthermore, this thyroid hormone metabolite led to an increase of whole-cell currents in N41 cells. Interestingly, the TRPM8 selective inhibitor (10 μM AMTB) reduced the 3-T1AM stimulatory effects on cytosolic Ca2+ and whole-cell currents. Our results suggest that the profound pharmacological effects of 3-T1AM on selected brain nuclei of murine hypothalamus, which are known to be involved in energy metabolism and thermoregulation, might be partially attributable to TRP channel activation in hypothalamic cells

TRPV6 modulates proliferation of human pancreatic neuroendocrine BON-1 tumour cells

Highly Ca2+ permeable receptor potential channel vanilloid type 6 (TRPV6) modulates a variety of biological functions including calcium-dependent cell growth and apoptosis. So far, the role of TRPV6 in controlling growth of pancreatic neuroendocrine tumour (NET) cells is unknown. In the present study, we characterize the expression of TRPV6 in pancreatic BON-1 and QGP-1 NET cells. Furthermore, we evaluate the impact of TRPV6 on intracellular calcium, the activity of nuclear factor of activated T-cells (NFAT) and proliferation of BON-1 cells. TRPV6 expression was assessed by real-time PCR and Western blot. TRPV6 mRNA expression and protein production were down-regulated by siRNA. Changes in intracellular calcium levels were detected by fluorescence calcium imaging (fura-2/AM). NFAT activity was studied by NFAT reporter assay; cell proliferation by bromodeoxyuridine (BrdU), MTT and propidium iodine staining. TRPV6 mRNA and protein are present in BON-1 and QGP-1 NET-cells. Down-regulation of TRPV6 attenuates BON-1 cell proliferation. TRPV6 down- regulation is associated with decreased Ca2+ response pattern and reduced NFAT activity. In conclusion, TRPV6 is expressed in pancreatic NETs and modulates cell proliferation via Ca2+-dependent mechanism, which is accompanied by NFAT activation

Somatic mutations and progressive monosomy modify SAMD9-related phenotypes in humans

It is well established that somatic genomic changes can influence phenotypes in cancer, but the role of adaptive changes in developmental disorders is less well understood. Here we have used next-generation sequencing approaches to identify de novo heterozygous mutations in sterile α motif domain–containing protein 9 (SAMD9, located on chromosome 7q21.2) in 8 children with a multisystem disorder termed MIRAGE syndrome that is characterized by intrauterine growth restriction (IUGR) with gonadal, adrenal, and bone marrow failure, predisposition to infections, and high mortality. These mutations result in gain of function of the growth repressor product SAMD9. Progressive loss of mutated SAMD9 through the development of monosomy 7 (–7), deletions of 7q (7q–), and secondary somatic loss-of-function (nonsense and frameshift) mutations in SAMD9 rescued the growth-restricting effects of mutant SAMD9 proteins in bone marrow and was associated with increased length of survival. However, 2 patients with –7 and 7q– developed myelodysplastic syndrome, most likely due to haploinsufficiency of related 7q21.2 genes. Taken together, these findings provide strong evidence that progressive somatic changes can occur in specific tissues and can subsequently modify disease phenotype and influence survival. Such tissue-specific adaptability may be a more common mechanism modifying the expression of human genetic conditions than is currently recognized

Expressionsanalysen und Funktion von temperatur-sensitiven Transient Rezeptor Potenzial Kanälen in kultivierten humanen konjunktivalen Epithel- und humanen uvealen Melanomzellen

Corneal transparency maintenance is essential for normal vision. In dry eye disease (DED), which afflicts increasing number of individuals, patients experience discomfort due to ocular surface pain and inflammation. Hyperosmolarity of the tear film is one crucial pathogenic factor in the development of ocular surface inflammation in DES. Osmoprotective agents or modulation of putative osmosensitive receptors may help to prevent a hyperosmolar tear film from damaging the ocular surface. Promising receptor targets whose selective modulation may reduce inflammation and pain are transient receptor potential channels (TRPs) such as TRP vanilloid 1 (TRPV1; capsaicin receptor) and TRP melastatin 8 (TRPM8; menthol receptor). TRPs are non-selective cation channels and play a substantial role in Ca2+ regulation. This PhD thesis was undertaken to investigate functional TRPM8 and TRPV1 expression in human conjunctival epithelial cells (HCjEC) and ocular tumor cells such as uveal melanoma (UM). The specific aims are: 1) characterization of the osmoprotective agents through compatible solutes such as L-carnitine on Ca2+ regulation in HCjEC since previous data indicated a link to TRPs. 2) Similar investigations should be carried out using the thyroxine metabolites, so-called thyronamine (3-T1AM). As a result, gene/protein and functional expression of TRPV1/TRPM8 could be detected in HCjEC and uveal melanoma cells. The functional expression of TRPM8 and TRPV1 was demonstrated by using the super cooling agent icilin (20 µM) and specific agonist capsaicin (CAP) (20 µM), respectively. During exposure to L-carnitine (1 mM), TRPV1 activation by hyperosmotic challenge (450 mOsM) or CAP suppressed Ca2+ influx and whole-cell currents in HCjEC. Furthermore, TRPV1 mediated shrinkage induced by exposure to a hyperosmotic challenge was blocked during L-carnitine exposure. In another set of experiments, extracellular application of 3-T1AM (1 µM) led to an increase of intracellular Ca2+ in HCjEC which could be blocked by TRPM8 antagonist indicating TRPM8 activation by 3-T1AM. Interestingly, the increases in TRPV1 functional activity induced by CAP were markedly blunted by pre- stimulating TRPM8 with 3-T1AM. Furthermore, an endpoint of such signaling activation induced by CAP, namely, an approximate 2-fold increase in interleukin-6 (IL-6) release was clearly attenuated during exposure to 3-T1AM. Therefore, inhibition of TRPV1 activity by possible endogenous modulators such as 3-T1AM may suppress inflammation in DES as well as in UM. Taken together, TRPM8, TRPV1 as well as 3-T1AM are interesting targets for the development of (adjuvant) therapies for DED and tumor diseases since (neoplastic) properties of (tumor) cells are determined by Ca2+-dependent cellular mechanisms, which are regulated by TRPs.Die Erhaltung der Hornhauttransparenz ist essenziell für den Sehvorgang. Beim „Trockenen Auge“, mit wachsender Prävalenz, haben die Patienten Beschwerden, die auf Schmerzen und Entzündung am Auge zurückzuführen sind. Angesichts limitierter Therapieoptionen, die in der Regel nicht die Ursachen beheben, sind besondere Maßnahmen erforderlich um Rezeptoren zu identifizieren, welche die Symptome hervorrufen. Vielversprechende Rezeptoren sind transient Rezeptor Potenzial Kanäle wie beispielsweise TRP Vanilloid 1 Rezeptor (TRPV1; Capsaicinrezeptor) und TRP Melastatin 8 Rezeptor (TRPM8; Mentholrezeptor), dessen Modulation Entzündung und Schmerz reduzieren können. TRP-Kanäle sind nicht-selektive Kationenkanäle und spielen eine substantielle Rolle bei der Calciumregulation. In dieser Doktorarbeit soll die funktionelle Expression von TRPM8 und TRPV1 sowohl in humanen Konjunktiva Epithelzellen (HCjEC) als auch in Augentumorzellen wie uvealen Melanomzellen untersucht werden. Die speziellen Ziele dieses Projekts sind: 1) Charakterisierung des Effekts von Osmoprotektiva mit kompatiblen Soluten wie L-Carnitin auf die Calciumregulation in HCjEC da Voruntersuchungen eine Verbindung zu den TRP- Kanälen hinweisen. 2) Ähnliche Untersuchungen sollen unter Verwendungvon Schilddrüsenhormonmetaboliten wie den sogenannten Thryronaminen (T1AM) durchgeführt werden. Daraufhin konnte die Gen-, Protein- und funktionelle Expression von TRPV1 und TRPM8 in HCjEC und uvealen Melanomzellen nachgewiesen werden. Die funktionelle TRPM8 Expression konnte speziell mit dem pharmakologisch künstlich kühlenden Icilin (20 µM) und dem TRPM8-Blocker BCTC (10 µM) gezeigt werden. Bezüglich des Nachweises der funktionellen TRPV1 Expression wurde Capsaicin (CAP; 20 µM) verwendet. Die Vorbehandlung der Zellen mit L-Carnitin (1 mM) unterdrückte die über hypertonen Stress (450 mOsM) oder über CAP (20 µM) ausgelöste TRPV1 Aktivität. Außerdem blockte die Behandlung mit L-Carnitin die TRPV1-vemittelte Senkung des relativen Zellvolumens nach hyperosmolaren Stress. In einer weiteren Versuchsserie führte die extrazellulare Applikation von 1 µM T1AM zu einer Erhöhung des intrazellulären Calciums in HCjEC, die durch BCTC geblockt werden konnte. Dies weist auf eine TRPM8 Aktivierung über T1AM hin. Interessanterweise konnten die durch CAP aktivierten TRPV1-Kanäle über eine Vorbehandlung der Zellen über T1AM-induzierte TRPM8 Aktivierung unterdrückt werden. Außerdem konnte die TRPV1-induzierte Interleukin-6 (IL-6) Freisetzung durch T1AM deutllich abgeschwächt werden. Die Unterdrückung der TRPV1 Aktivität über mögliche endogene Modulatoren wie T1AM könnten Entzündungsprozesse beim Trockenen Auge unterdrücken als auch das Wachstum von Tumorzellen wie den UM-Zellen inhibieren. TRPM8, TRPV1 sowie 3-T1AM sind interessante Targets für die Entwicklung von (begleitenden) Therapien des Trockenen Auges und auch Tumorerkrankungen da zell- und tumorbiologische Eigenschaften durch Kalzium- abhängige Regulationsmechanismen geprägt sind, die von TRP-Kanäle reguliert werden

3-Iodothyronamine, a Novel Endogenous Modulator of Transient Receptor Potential Melastatin 8?

The decarboxylated and deiodinated thyroid hormone (TH) derivative, 3-iodothyronamine (3-T1AM), is suggested to be involved in energy metabolism and thermoregulation. G protein-coupled receptors (GPCRs) are known as the main targets for 3-T1AM; however, transient receptor potential channels (TRPs) were also recently identified as new targets of 3-T1AM. This article reviews the current knowledge of a putative novel role of 3-T1AM in the modulation of TRPs. Specifically, the TRP melastatin 8 (TRPM8) was identified as a target of 3-T1AM in different cell types including neoplastic cells, whereby 3-T1AM significantly increased cytosolic Ca2+ through TRPM8 activation. Similarly, the β-adrenergic receptor is involved in 3-T1AM-induced Ca2+ influx. Therefore, it has been suggested that 3-T1AM-induced Ca2+ mobilization might be due to β-adrenergic receptor/TRPM8 channel interaction, which adds to the complexity of GPCR regulation by TRPs. It has been revealed that TRPM8 activation leads to a decline in TRPV1 activity, which may be of therapeutic benefit in clinical circumstances such as treatment of TRPV1-mediated inflammatory hyperalgesia, colitis, and dry eye syndrome. This review also summarizes the inverse association between changes in TRPM8 and TRPV1 activity after 3-T1AM stimulation. This finding prompted further detailed investigations of the interplay between 3-T1AM and the GPCR/TRPM8 axis and indicated the probability of additional GPCR/TRP constellations that are modulated by this TH derivative

L-Carnitine Reduces in Human Conjunctival Epithelial Cells Hypertonic-Induced Shrinkage through Interacting with TRPV1 Channels

Background/Aims: Ocular surface health depends on conjunctival epithelial (HCjE) layer integrity since it protects against pathogenic infiltration and contributes to tissue hydration maintenance. As the same increases in tear film hyperosmolarity described in dry eye disease can increase corneal epithelial transient receptor potential vanilloid type-1 (TRPV1) channel activity, we evaluated its involvement in mediating an osmoprotective effect by L-carnitine against such stress. Methods: Using siRNA gene silencing, Ca2+ imaging, planar patch-clamping and relative cell volume measurements, we determined if the protective effects of this osmolyte stem from its interaction with TRPV1. Results: TRPV1 activation by capsaicin (CAP) and an increase in osmolarity to ≈ 450 mOsM both induced increases in Ca2+ levels. In contrast, blocking TRPV1 activation with capsazepine (CPZ) fully reversed this response. Similarly, L-carnitine (1 mM) also reduced underlying whole-cell currents. In calcein-AM loaded cells, hypertonic-induced relative cell volume shrinkage was fully blocked during exposure to L-carnitine. On the other hand, in TRPV1 gene-silenced cells, this protective effect by L-carnitine was obviated. Conclusion: The described L-carnitine osmoprotective effect is elicited through suppression of hypertonic-induced TRPV1 activation leading to increases in L-carnitine uptake through a described Na+-dependent L-carnitine transporter

3-Iodothyronamine Activates a Set of Membrane Proteins in Murine Hypothalamic Cell Lines

3-Iodothyronamine (3-T1AM) is an endogenous thyroid hormone metabolite. The profound pharmacological effects of 3-T1AM on energy metabolism and thermal homeostasis have raised interest to elucidate its signaling properties in tissues that pertain to metabolic regulation and thermogenesis. Previous studies identified G protein-coupled receptors (GPCRs) and transient receptor potential channels (TRPs) as targets of 3-T1AM in different cell types. These two superfamilies of membrane proteins are largely expressed in tissue which influences energy balance and metabolism. As the first indication that 3-T1AM virtually modulates the function of the neurons in hypothalamus, we observed that intraperitoneal administration of 50 mg/kg bodyweight of 3-T1AM significantly increased the c-FOS activation in the paraventricular nucleus (PVN) of C57BL/6 mice. To elucidate the underlying mechanism behind this 3-T1AM-induced signalosome, we used three different murine hypothalamic cell lines, which are all known to express PVN markers, GT1-7, mHypoE-N39 (N39) and mHypoE-N41 (N41). Various aminergic GPCRs, which are the known targets of 3-T1AM, as well as numerous members of TRP channel superfamily, are expressed in these cell lines. Effects of 3-T1AM on activation of GPCRs were tested for the two major signaling pathways, the action of Gαs/adenylyl cyclase and Gi/o. Here, we demonstrated that this thyroid hormone metabolite has no significant effect on Gi/o signaling and only a minor effect on the Gαs/adenylyl cyclase pathway, despite the expression of known GPCR targets of 3-T1AM. Next, to test for other potential mechanisms involved in 3-T1AM-induced c-FOS activation in PVN, we evaluated the effect of 3-T1AM on the intracellular Ca2+ concentration and whole-cell currents. The fluorescence-optic measurements showed a significant increase of intracellular Ca2+ concentration in the three cell lines in the presence of 10 μM 3-T1AM. Furthermore, this thyroid hormone metabolite led to an increase of whole-cell currents in N41 cells. Interestingly, the TRPM8 selective inhibitor (10 μM AMTB) reduced the 3-T1AM stimulatory effects on cytosolic Ca2+ and whole-cell currents. Our results suggest that the profound pharmacological effects of 3-T1AM on selected brain nuclei of murine hypothalamus, which are known to be involved in energy metabolism and thermoregulation, might be partially attributable to TRP channel activation in hypothalamic cells

Role of Somatic Testicular Cells during Mouse Spermatogenesis in Three-Dimensional Collagen Gel Culture System

Objective: Spermatogonial stem cells (SSCs) are the only cell type that can restore fertility to an infertile recipient following transplantation. Much effort has been made to develop a protocol for differentiating isolated SSCs in vitro. Recently, three-dimensional (3D) culture system has been introduced as an appropriate microenvironment for clonal expansion and differentiation of SSCs. This system provides structural support and multiple options for several manipulation such as addition of different cells. Somatic cells have a critical role in stimulating spermatogenesis. They provide complex cell to cell interaction, transport proteins and produce enzymes and regulatory factors. This study aimed to optimize the culture condition by adding somatic testicular cells to the collagen gel culture system in order to induce spermatogenesis progression. Materials and Methods: In this experimental study, the disassociation of SSCs was performed by using a two-step enzymatic digestion of type I collagenase, hyaluronidase and DNase. Somatic testicular cells including Sertoli cells and peritubular cells were obtained after the second digestion. SSCs were isolated by Magnetic Activated Cell Sorting (MACS) using GDNF family receptor alpha-1 (Gfrα-1) antibody. Two experimental designs were investigated. 1. Gfrα-1 positive SSCs were cultured in a collagen solution. 2. Somatic testicular cells were added to the Gfrα-1 positive SSCs in a collagen solution. Spermatogenesis progression was determined after three weeks by staining of synaptonemal complex protein 3 (SCP3)-positive cells. Semi-quantitative Reverse Transcription PCR was undertaken for SCP3 as a meiotic marker and, Crem and Thyroid transcription factor-1 (TTF1) as post meiotic markers. For statistical analysis student t test was performed. Results: Testicular supporter cells increased the expression of meiotic and post meiotic markers and had a positive effect on extensive colony formation. Conclusion: Collagen gel culture system supported by somatic testicular cells provides a microenvironment that mimics seminiferous epithelium and induces spermatogenesis in vitro