Characterization of new molecular targets involved in iodide flux in the thyroid gland: the anoctamins

Iodide transport is necessary for the synthesis of thyroid hormones following accumulation in the follicular lumen out of thyroid cells, via channels unknown with the exception of pendrin. According to our hypothesis, TMEM16A could be the main molecular identity of the channel mediating iodide efflux in the thyroid gland. TMEM16A is the prior candidate for calcium-activated chloride conductance (CaCC). TMEM16A belongs to the TMEM16/anoctamin family comprising ten members (TMEM16A-K). Higher affinity of TMEM16A for iodide and predicted expression in the thyroid gland suggest its mediation of iodide efflux. The aim of this project was to identify the role of TMEM16A in iodide transport in the thyroid gland, by characterizing its molecular expression and functional properties. We demonstrated that TMEM16F, H, K transcripts are expressed in FRTL-5 thyroid cells, as well as TMEM16A, which is TSH-independent. Tumor tissue from human thyroid maintains TMEM16A expression. Functional in vivo experiments in FRTL-5, stably expressing YFP-H148Q/I152L fluorescent protein as a biosensor, showed that iodide efflux is stimulated by agonists of purinergic receptors with an order of potency of ATP>UTP>ADP (compatible with an involvement of P2Y purinergic receptors), and by agonists of adrenergic receptors (epinephrine, norepinephrine and phenylephrine). Iodide efflux was blocked by α-receptor antagonists prazosin and phentolamine, consistent with a role of α1 adrenergic receptors. Iodide efflux was specifically dependent on calcium mobilized from intracellular compartments and induced by the calcium ionophore ionomycin. CaCC blockers suppressed ionomycin-/ATP-/epinephrine-stimulated iodide efflux. Heterologous expression of TMEM16A in CHO K1 cells induced calcium-activated iodide fluxes. All these results support the hypothesis of the involvement of TMEM16A in calcium-dependent iodide efflux induced by receptor agonists in thyroid cells. TMEM16A may represent a new pharmacological target for thyroid cancer therapy, since its blockade may enhance the retention of radioiodide by tumour cells enhancing the efficacy of radioablative therapy.

L'infiammazione intestinale nell'animale sperimentale come modello per lo sviluppo di nuovi farmaci: ruolo della via tachichininergica nelle malattie infiammatorie intestinali

Background: Several lines of evidence showed that inflammation is associated with changes in the expression of tachykinins both in human and animal models. Tachykinins, including substance P (SP), are small peptides expressed in the extrinsic primary afferent nerve fibres and enteric neurons of the gut: they exert their action through three distinct receptors, termed NK1, NK2 and NK3. SP modulates intestinal motility and enteric secretion, acting preferentially through the NK1 receptor. SP neural network and NK1 receptor expression are increased in patients with inflammatory bowel disease, and similar changes were observed in experimental models of inflammation. The 2,4 Dinitrobenzene Sulphonic Acid (DNBS) model of colitis is useful to study innate immunity, non-specific inflammation and wound healing; it has been suggested that the transmural inflammation seen in this model resembles that found in Crohn’s disease and can therefore be used to study what cells and mediators are involved in this type of inflammation. Aim: To test the possible protective effect of the NK1 receptor antagonist SSR140333 on: 1) acute model of intestinal inflammation; 2) reactivation of DNBS-induced colitis in rats. Methods: Acute colitis was induced in male SD rats by intrarectal administration of DNBS (15 mg/rat in 50% ethanol). Reactivation of colitis was induced by intrarectal injections of DNBS on day 28 (7.5 mg/rat in 35% ethanol). Animals were sacrificed on day 6 (acute colitis) and 29 (reactivation of colitis). SSR140333 (10 mg/kg) was administered orally starting from the day before the induction of colitis for 7 days (acute colitis) or seven days before the reactivation of colitis. Colonic damage was assessed by means of macroscopic and microscopic scores, myeloperoxidase activity (MPO) and TNF-α tissue levels. Enzyme immunoassay was used to measure colonic substance P levels. Statistical analysis was performed using analysis of variance (one-way or two-way, as appropriate) with the Bonferroni’s correction for multiple comparisons. Results: DNBS administration impaired body weight gain and markedly increased all inflammatory parameters (p<0.01). Treatment with SSR140333 10 mg/kg significantly counteracted the impairment in body weight gain, decreased macroscopic and histological scores and reduced colonic myeloperoxidase activity (p<0.01). Drug treatment counteracted TNF-α tissue levels and colonic SP concentrations (acute model). Similar results were obtained administering the NK1 receptor antagonist SSR140333 (3 and 10 mg/kg) for 5 days, starting the day after the induction of colitis. Intrarectal administration of DNBS four weeks after the first DNBS administration resulted in reactivation of colitis, with increases in macroscopic and histological damage scores and increase in MPO activity. Preventive treatment with SSR140333 10 mg/kg decreased macroscopic damage score, significantly reduced microscopic damage score but did not affect MPO activity. Conclusions: Treatment with SSR140333 significantly reduced intestinal damage in acute model of intestinal inflammation in rats. The NK1 receptor antagonist SSR140333 was also able to prevent relapse in experimental colitis. These results support the hypothesis of SP involvement in intestinal inflammation and indicate that NK receptor antagonists may have a therapeutic potential in inflammatory bowel disease.

Molecular analysis of Sigma-1 receptor modulation of the dopamine transporter

Sigma (σ) receptors are well established as a non-opioid, non-phencyclidine, and haloperidol-sensitive receptor family with its own binding profile and a characteristic distribution in the central nervous system (CNS) as well as in endocrine, immune, and some peripheral tissues. Two σ receptors subtypes, termed σ1 and σ2, have been pharmacologically characterized, but, to date, only the σ1 has also been cloned. Activation of σ1 receptors alter several neurotransmitter systems and dopamine (DA) neurotrasmission has been often shown to constitute an important target of σ receptors in different experimental models; however the exact role of σ1 receptor in dopaminergic neurotransmission remains unclear. The DA transporter (DAT) modulates the spatial and temporal aspects of dopaminergic synaptic transmission and interprer the primary mechanism by wich dopaminergic neurons terminate the signal transmission. For this reason present studies have been focused in understanding whether, in cell models, the human subtype of σ1 (hσ1) receptor is able to directly modulate the human DA transporter (hDAT). In the first part of this thesis, HEK-293 and SH-SY5Y cells were permanently transfected with the hσ1 receptor. Subsequently, they were transfected with another plasmid for transiently expressing the hDAT. The hDAT activity was estimated using the described [3H]DA uptake assay and the effects of σ ligands were evaluated by measuring the uptaken [3H]DA after treating the cells with known σ agonists and antagonists. Results illustrated in this thesis demonstrate that activation of overexpressed hσ1 receptors by (+)-pentazocine, the σ1 agonist prototype, determines an increase of 40% of the extracellular [3H]DA uptake, in comparison to non-treated controls and the σ1 antagonists BD-1047 and NE-100 prevent the positive effect of (+)-pentazocine on DA reuptake DA is likely to be considered a neurotoxic molecule. In fact, when levels of intracellular DA abnormally invrease, vescicles can’t sequester the DA which is metabolized by MAO (A and B) and COMT with consequent overproduction of oxygen reactive species and toxic catabolites. Stress induced by these molecules leads cells to death. Thus, for the second part of this thesis, experiments have been performed in order to investigate functional alterations caused by the (+)-pentazocine-mediated increase of DA uptake; particularly it has been investigated if the increase of intracellular [DA] could affect cells viability. Results obtained from this study demonstrate that (+)-pentazocine alone increases DA cell toxicity in a concentration-dependent manner only in cells co-expressing hσ1 and hDAT and σ1 antagonists are able to revert the (+)-pentazocine-induced increase of cell susceptibility to DA toxicity. In the last part of this thesis, the functional cross-talking between hσ1 receptor and hDAT has been further investigated using confocal microscopy. From the acquired data it could be suggested that, following exposure to (+)-pentazocine, the hσ1 receptors massively translocate towards the plasma membrane and colocalize with the hDATs. However, any physical interaction between the two proteins remains to be proved. In conclusion, the presented study shows for the first time that, in cell models, hσ1 receptors directly modulate the hDAT activity. Facilitation of DA uptake induced by (+)-pentazocine is reflected on the increased cell susceptibility to DA toxicity; these effects are prevented by σ1 selective antagonists. Since numerous compounds, including several drugs of abuse, bind to σ1 receptors and activating them could facilitate the damage of dopaminergic neurons, the reported protective effect showed by σ1 antagonists would represent the pharmacological basis to test these compounds in experimental models of dopaminergic neurodegenerative diseases (i.e. Parkinson’s Disease).