Activation of the cell membrane angiotensin AT2 receptors in human leiomyosarcoma cells induces differentiation and apoptosis by a PPARγ - dependent mechanism.

Angiotensin II (Ang II), the main effector peptide of the renin-angiotensin system (RAS), acting on AT1 and AT2 receptors participates in the regulation of proliferation, differentiation and apoptosis in tumour cells. The peroxisome-proliferator activated receptor γ (PPARγ) and its ligands exert anti-tumour effects in various human cancer cell lines. The present study investigates the effects initiated by AT1- and AT2 receptor stimulation in SK-UT-1 cells, a human leiomyosarcoma cell line, and clarifies the role of the PPARγ in the AT2 receptor-induced differentiation and apoptosis.Selective stimulation of AT1- and AT2 receptors was achieved by incubation of the cells with Ang II (10-6 M) in the presence of the selective AT2 receptor antagonist, PD 123177 (10-6 M) and the AT1 receptor antagonist, losartan (10-5 M), respectively, the selective PPARγ antagonist, GW 9662, was used at concentration 10-6 M. The expression of smooth muscle cell differentiation markers, SM22α and calponin, was analysed at RNA- and protein levels using RT PCR and Western blot, which was also used to quantify Bcl-2-, Bax- and cleaved caspase-3 proteins. The translocation of the AT2-receptor interacting protein 1 (ATIP1) to the nuclei was studied by Western blot and immunofluorescence staining. The mitochondrial status and the metabolic activity in response to AT1- and AT2 receptor activation were assessed by the quantification of 99mTc - sestamibi and 2´-deoxy-2´-[18F]fluoro-D-glucose uptake.AT1 receptor stimulation did not exert any profound effects in quiescent SK-UT-1 cells. The effects induced by Ang II acting on AT2 receptors were time-dependent. A short, 3 - 6 h lasting stimulation promotes differentiation, i.e increases in the mRNA- and protein levels of SM22α and calponin, whereas a sustained stimulation for 48 h activates the intrinsic apoptotic pathway, as evidenced by reduced cell numbers, down-regulation of the anti-apoptotic Bcl-2 protein and increased levels of the Bax protein and cleaved caspase-3. The effects were reversed by the PPARγ antagonist, GW 9662, clearly implying a PPARγ-dependent mechanism. Our results also demonstrate a co-localisation of the AT2-receptor interacting protein, ATIP1, and the PPARγ in nuclei of SK-UT-1 cells and an accumulation of ATIP1 in the nuclear fraction in response to AT2 receptor stimulation. The regulation of the differentiation and apoptosis via the AT2 receptor favours an important functional role of this receptor in quiescent, slow-cycling SK-UT-1 cells and provides the rationale for the use of AT1 receptor antagonists for the treatment of human leiomyosarcomas.

Activation of cerebral peroxisome proliferator-activated receptors γ (PPARγ) reduces neuronal damage in the substantia nigra after transient focal cerebral ischaemia in the rat.

AIM: The function of brain (neuronal) peroxisome proliferator-activated receptor(s) γ (PPARγ) in the delayed degeneration and loss of neurones in the substantia nigra (SN) was studied in rats after transient occlusion of the middle cerebral artery (MCAO). METHODS: The PPARγ agonist, pioglitazone, or vehicle was infused intracerebroventricularly over a 5-day period before, during and 5 days after MCAO (90 min). The neuronal degeneration in the SN pars reticularis (SNr) and pars compacta (SNc), the analysis of the number of tyrosine hydroxylase-immunoreactive (TH-IR) neurones and the expression of the PPARγ in these neurones were studied by immunohistochemistry and immunofluorescence staining. The effects of PPARγ activation on excitotoxic and oxidative neuronal damage induced by glutamate and 6-hydroxydopamine were investigated in primary cortical neurones expressing PPARγ. RESULTS: Pioglitazone reduced the total and striatal infarct size, neuronal degeneration in both parts of the ipsilateral SN, the loss of TH-IR neurones in the SNc and increased the number of PPARγ-positive TH-IR neurones. Pioglitazone protected primary cortical neurones against oxidative and excitotoxic damage, prevented the loss of neurites and supported the formation of synaptic networks in neurones exposed to glutamate or 6-hydroxydopamine by a PPARγ-dependent mechanism. CONCLUSIONS: Activation of cerebral PPARγ confers neuroprotection after ischaemic stroke by preventing both, neuronal damage within the peri-infarct zone and delayed degeneration of neurones and neuronal death in areas remote from the site of ischaemic injury. Pioglitazone and other PPARγ agonists may be useful therapeutic agents to prevent progression of brain damage after cerebral ischaemia.

Blockade of tachykinin NK1/NK2 receptors in the brain attenuates the activation of corticotrophin-releasing hormone neurones in the hypothalamic paraventricular nucleus and the sympathoadrenal and pituitary-adrenal responses to formalin-induced pain in the rat.

Evidence from pharmacological studies has implicated substance P (SP), a natural ligand of tachykinin NK(1) receptors which can also interact with NK(2) receptors, in the generation of pressor and tachycardic responses to stress. Using selective blockade of brain NK(1) and NK(2) receptors, we tested in conscious rats the hypothesis that SP initiates, within the neuronal brain circuits, the sympathoadrenal, hypothalamic-pituitary-adrenal (HPA) and behavioural responses to noxious stimuli. Formalin injected s.c. through a chronically implanted catheter in the area of the lower leg was used as a pain stimulus. Rats were pretreated i.c.v. with vehicle or the selective, nonpeptide antagonists of tachykinin NK(1) and NK(2) receptors, RP 67580 and SR 48968, respectively. Ten minutes thereafter, formalin was injected s.c. and the cardiovascular responses were recorded, plasma concentrations of catecholamines, adrenocorticotrophic hormone (ACTH) and corticosterone were determined and the expression of the inducible transcription factor c-Fos in the paraventricular (PVN) and supraoptic nuclei was detected to identify neurones which were activated during pain stimulation. Blockade of NK(1) and NK(2) receptors attenuated the formalin-induced increases in mean arterial pressure and heart rate, adrenaline and ACTH concentrations in plasma, and completely abolished the pain-induced c-Fos expression in corticotrophin-releasing hormone neurones localised in the parvocellular division of the PVN. The results obtained provide pharmacological evidence that tachykinins, most probably SP, act as mediators within the neuronal circuits linked to the initiation and control of the cardiovascular, sympathoadrenal, HPA and behavioural responses to pain stimuli and provide an excitatory input to corticotrophin-releasing hormone neurones in the PVN to activate the HPA axis. Our data demonstrating the inhibition of the complex response pattern to noxious stimuli and stress are consistent with the proposed anxiolytic and antidepressant activity of NK(1) and NK(2) receptor antagonists.

New approach for the synthesis of [18F]fluoroethyltyrosine for cancer imaging: simple, fast, and high yielding automated synthesis.

O-(2-[(18)F]fluoroethyl)-L-tyrosine ([(18)F]FET) is one of the first (18)F-labeled amino acids for imaging amino acid metabolism in tumors. This tracer overcomes the disadvantages of [(18)F]fluorodeoxyglucose, [(18)F]FDG, and [(11)C]methionine, [(11)C]MET. Nevertheless, the various synthetic methods providing (18)F[FET] exhibit a big disadvantage concerning the necessity of two purification steps during the synthesis including HPLC purification, which causes difficulties in the automation, moderate yields, and long synthesis times >60 min. A new approach for the synthesis of [(18)F]FET is developed starting from 2-bromoethyl triflate as precursor. After optimization of the synthesis parameters including the distillation step of [(18)F]-FCH(2)CH(2)Br combined with the final purification of [(18)F]FET using a simple solid phase extraction instead of an HPLC run the synthesis [(18)F]FET could be significantly simplified, shortened, and improved. The radiochemical yield (RCY) was about 45% (not decay corrected and calculated relative to [(18)F]F(-) activity that was delivered from the cyclotron). Synthesis time was only 35 min from the end of bombardment (EOB) and the radiochemical purity was >99% at the end of synthesis (EOS). Thus, this simplified synthesis for [(18)F]FET offers a very good option for routine clinical use.

Simplified fast and high yielding automated synthesis of [18F]fluoroethylcholine for prostate cancer imaging.

(11)C- and (18)F-labeled choline analogues are successful tracers for prostate cancer imaging using positron emission tomography (PET). Due to the practical advantages of the longer-living radioisotope (18)F (t(1/2)=110 min) instead of (11)C (t(1/2)=20 min), [(18)F]fluoroethylcholine has been introduced to increase the opportunity of widespread clinical application. Nevertheless, the various known synthetic methods provide [(18)F]fluoroethylcholine for human use only in moderate overall yields of up to 30% so far. Here, a new simplified and high yield two-step-synthesis for [(18)F]fluoroethylcholine is described for potential clinical applications starting from 2-bromoethyl triflate (BETfO) using a modified, commercially available fully automated synthesis module. All synthesis parameters were subsequently optimized resulting in a total yield of 47+/-5% (not decay corrected) in only 40min. [(18)F]fluoroethylcholine could be obtained ready for human use as physiological solution after fixation on Sep-Pak Accell Light cartridges (waters((R))) and formulation with saline without the need of GC or HPLC purification. Radiochemical purity was >99.9% and no contamination of the sterile solution with chemicals used during the synthesis was detected.

High-dose treatment with (186)Re-HEDP or (153)Sm-EDTMP combined with amifostine in a rabbit model.

UNLABELLED: The aim of this experimental study was to investigate the myeloprotective potential of amifostine in rabbits receiving high-dose treatment with either (153)Sm-ethylenediaminetetramethylene phosphonate (EDTMP) or (186)Re-hydroxyethylidene diphosphonate (HEDP) and to check for drug interactions impairing the skeletal uptake of these radiopharmaceuticals by amifostine. METHODS: To a total of 24 rabbits, we administered 1,000 MBq of either (153)Sm-EDTMP (n = 12) or (186)Re-HEDP (n = 12). Six animals of each group received 500 mg amifostine intravenously 10-15 min before injection of the radiopharmaceutical, whereas the other 6 animals served as controls. Up to 8 wk after treatment, blood samples were collected every 3-5 d to measure platelet and leukocyte counts. Furthermore, whole-body images were acquired at 3 min, 3 h, and 24 h after injection of the radiopharmaceutical to quantify the skeletal uptake. RESULTS: For (186)Re-HEDP, the mean decrease in platelets was significantly less in the amifostine group (35.5% +/- 2.4%) than in the control group (61.3% +/- 5.4%, P < 0.001). Similar results were found for (153)Sm-EDTMP (36.5% +/- 8.3% vs. 52.3% +/- 14.0%, P < 0.05). No significant differences in leukocyte counts were found for (186)Re-HEDP (75.3% +/- 12.3% in the amifostine group and 72.5% +/- 4.1% in the control group, P > 0.05), whereas rabbits treated with (153)Sm-EDTMP plus amifostine showed a significantly greater decrease in leukocytes (69.2% +/- 10.8%) than did the control group (56.6% +/- 4.0%, P < 0.05). Bone uptake in percentage of initial total whole-body activity was significantly decreased in animals treated with amifostine compared with the control groups for both (186)Re-HEDP (15.8% +/- 3.1% vs. 30.9% +/- 1.9%, P < 0.001) and (153)Sm-EDTMP (31.7% +/- 8.9% vs. 44.0% +/- 6.5%, P < 0.05). CONCLUSION: For amifostine, we found a highly significant cytoprotective effect on platelets but no leukoprotective effect. The latter probably relies on the intrinsic myelotoxicity of high-dose amifostine, which seemed to potentiate the leukodepression of the radiopharmaceuticals. The lower bone uptake in amifostine-treated animals may be caused by the chemical structure of amifostine, which is a potentially complex-forming compound that may be able to displace bisphosphonates from the rhenium- and samarium-bisphosphonate complexes, resulting in altered biodistribution patterns.

Age- and sex-related bone uptake of Tc-99m-HDP measured by whole-body bone scanning.

UNLABELLED: AIM of this study was to validate a recently introduced new and easy-to-perform method for quantifying bone uptake of Tc-99m-labelled diphosphonate in a routine clinical setting and to establish a normal data base for bone uptake depending on age and gender. METHODS: In 49 women (14-79 years) and 47 men (6-89 years) with normal bone scans as well as in 49 women (33-81 years) and 37 men (27-88 years) with metastatic bone disease whole-body bone scans were acquired at 3 min and 3-4 hours p.i. to calculate bone uptake after correction for both urinary excretion and soft tissue retention. RESULTS: Bone uptake values of various age-related subgroups showed no significant differences between men and women (p > 0.05). Furthermore, no differences could be proven between age-matched subgroups of normals and patients with less than 10 metastatic bone lesions, while patients with wide-spread bone metastases revealed significantly increased uptake values. In both men and women highest bone uptake was obtained (p < 0.05) in subjects younger than 20 years with active epiphyseal growth plates. In men, bone uptake slowly decreased with age up to 60 years and then showed a tendency towards increasing uptake values. In women, the mean uptake reached a minimum in the decade 20-29 years and then slowly increased with a positive linear correlation of age and uptake in subjects older than 55 years (r = 0.57). CONCLUSION: Since the results proposed in this study are in good agreement with data from literature, the new method used for quantification could be validated in a large number of patients. Furthermore, age- and sex-related normal bone uptake values of Tc-99m-HDP covering a wide range of age could be presented for this method as a basis for further studies on bone uptake.