Role of receptor internalization in the agonist-induced desensitization of cannabinoid type 1 receptors

Agonist-induced internalization of G protein-coupled receptors (GPCRs) is an important mechanism for regulating signaling transduction of functional receptors at the plasma membrane. We demonstrate here that both caveolae/lipid-rafts- and clathrin-coated-pits-mediated pathways were involved in agonist-induced endocytosis of the cannabinoid type 1 receptor (CB1R) in stably transfected human embryonic kidney (HEK) 293 cells and that the internalized receptors were predominantly sorted into recycling pathway for reactivation. The treatment of CB1 receptors with the low endocytotic agonist Δ⁹-THC induced a faster receptor desensitization and slower resensitization than the high endocytotic agonist WIN 55,212-2. In addition, the blockade of receptor endocytosis or recycling pathway markedly enhanced agonist-induced CB1 receptor desensitization. Furthermore, co-expression of phospholipase D2, an enhancer of receptor endocytosis, reduced CB1 receptor desensitization, whereas co-expression of a phospholipase D2 negative mutant significantly increased the desensitization after WIN 55,212-2 treatment. These findings provide evidences for the importance of receptor endocytosis in counteracting CB1 receptor desensitization by facilitating receptor reactivation. Moreover, in primary cultured neurons, the low endocytotic agonist Δ⁹-THC or anandamide exhibited a greater desensitization of endogenous CB1 receptors than the high endocytotic agonist WIN 55,212-2, CP 55940 or 2-arachidonoyl glycerol, indicating that cannabinoids with high endocytotic efficacy might cause reduced development of cannabinoid tolerance to some kind cannabinoid-mediated effects.     

Release of ( 3 H)noradrenaline and ( 3 H)dopamine from field stimulated cerebral cortex slices. Effect of tyrosine hydroxylase and dopamine- -hydroxylase inhibition

Rat cerebral cortex slices were incubated in vitro with [³H]dopamine (DA) or [³H]noradrenaline (NA) (10^?7M), superfused by fresh buffer and stimulated by an electric field. The stimulation-induced overflow of [³H]DA and [³H]NA was determined. In slices from untreated rats about 16 ng [³H]NA/g tissue was formed from [³H]DA, corresponding to about 5 per cent of the endogenous NA concentration. Stimulation markedly enhanced the overflow of [³H]NA. The [³H]NA newly formed from [³H]DA was overflowing to a greater extent than [³H]NA previously taken up from the incubation medium, indicating a preferential release of newly synthesized transmitter. The stimulation-induced overflow of [³H]DA and [³H]NA was increased in slices of rats pretreated with a tyrosine hydroxylase inhibitor (H44/68). It seems that depletion of the endogenous NA stores of central NA neurons by tyrosine hydroxylase inhibition makes the [³H]cate-cholamines more available for release. Pretreatment of the rats with the DA-β-hydroxylase inhibitors FLA63 or FLA69 considerably diminished the formation of [³H]NA from [³H]DA. Stimulation markedly enhanced the overflow of [³H]DA indicating that DA can act as a ‘false transmitter’ in central NA neurons after DA-β-hydroxylase inhibition.     

Interleukin-18 acts as an angiogenesis and tumor suppressor.

Interleukin-18 (IL-18), also called interferon-gamma (IFN-gamma)-inducing factor, has recently been characterized as a potent IFN-gamma-inducing cytokine. We now report that IL-18 is a novel antiangiogenic and antitumor cytokine. In vitro, IL-18 specifically inhibits fibroblast growth factor-2-stimulated proliferation of capillary endothelial cells. In vivo, IL-18 is sufficiently potent to suppress the fibroblast growth factor-induced corneal neovascularization by systemic administration in mice. This cytokine also inhibits embryonic angiogenesis in the chick chorioallantoic membrane assay. Systemic and intralesional administrations of IL-18 produce a significant suppression of the growth of murine T241 fibrosarcoma in syngeneic C57Bl6/J and immunodeficient SCID mice. The antitumor effect appears to be potent because an average of >75% inhibition of primary tumor growth was observed at a dose of 50 microg/kg/day. In cell culture, murine T241 fibrosarcoma cells are insensitive to recombinant IL-18 at concentrations that significantly inhibit endothelial cell proliferation. Immunohistochemical studies of tumor tissues reveal hypovascularization of the IL-18-treated tumors. These results suggest that IL-18 may participate in the regulation of a switch of tumor angiogenesis.-Cao, R., Farnebo, J., Kurimoto, M., Cao, Y. Interleukin-18 acts as an angiogenesis and tumor suppressor.     

Importance of high quality early-successional habitats in managed forest landscapes to rare beetle species

Species adapted to early-successional forest habitats are in managed landscapes largely confined to clearcuts. To improve habitat quality on clearcuts, green tree and dead wood retention is widely applied in forestry; however, its effects on rare early-successional species have rarely been shown. We repeatedly surveyed two red-listed beetle species (Upis ceramboides and Platysoma minus) on clearcuts in a managed boreal forest landscape. We found that U. ceramboides decreased its occupancy over time while P. minus increased, indicating that red-listed species vary in their ability to successfully utilise managed habitats. We found no effect of connectivity on probability of occurrence, colonisation or extinction per clearcut. Trees retained alive improved habitat quality of clearcuts, since both species were more frequent in dead wood of such trees, in comparison to logging residues. We suggest that retention can be improved by protecting and creating dead wood as intact trees during harvesting. Rare specialist species require habitat of high quality, and consequently it is impossible to meet the requirements of these species on every clearcut. To preserve all early-successional species at a regional scale, we recommend focusing retention of green trees and dead wood to one or a few trees species on each clearcut and in each landscape.     

The Sub-Cellular Localization of WRAP53 Has Prognostic Impact in Breast Cancer

WRAP53 protein controls intracellular trafficking of DNA repair proteins, the telomerase enzyme, and splicing factors. Functional loss of the protein has been linked to carcinogenesis, premature aging and neurodegeneration. The aim of this study was to investigate the prognostic significance of WRAP53 protein expression in breast cancer. A tissue microarray was constructed from primary breast tumors and immunostained by a polyclonal WRAP53 antibody to assess the protein expression pattern. Two different patient cohorts with long term follow-up were studied; a test- and a validation set of 154 and 668 breast tumor samples respectively. Breast cancer patients with tumor cells lacking the expression of WRAP53 in the nucleus had a significantly poorer outcome compared to patients with tumor cells expressing this protein in the nuclei (HR = 1.95, 95%CI = 1.09–3.51, p = 0.025). Nuclear localization of WRAP53 was further shown to be an independent marker of prognosis in multivariate analysis (HR = 2.57, 95%CI = 1.27–5.19, p = 0.008), and also significantly associated with better outcome in patients with TP53 mutation. Here we show that the sub-cellular localization of the WRAP53 protein has a significant impact on breast cancer survival, and thus has a potential as a clinical marker in diagnostics and treatment.     

Annual rhythms of swimming behaviour and seawater adaptation in young baltic salmon,Salmo salar,associated with smolting

Synopsis Sexually immature and sexually mature precocious male Baltic salmon,Salmo salar, parr from Umeälven (Ume river) were tested for rheotactic behaviour and adaptation to seawater before, during, and after the time period for smolt migration. Size of fish at the beginning of the experiment in January was on average 13.5 cm. Rheotactic behaviour was tested in annular stream tanks with photocells to measure upstream and downstream movements. Samples of fish were given a Seawater challenge test at monthly intervals in order to determine their ability to adapt to 20%. saltwater. During spring, both immature and sexually precocious parr became silvery and showed progressive development of downstream-directed movements. In early June the fish exhibited good hypoosmoregulatory ability in 20%. saltwater and swimming was predominantly downstream. During late June and early July there was a marked reversal in swimming behaviour, accompanied by a dramatic change in saltwater adaptation. The fish moved mainly upstream and showed decreasing ability to meet the seawater challenge test. This was accompanied by a loss of silvery coloration. The annual cycle of swimming behaviour and seawater adaptation is discussed in relation to the appearance of a smolt-window, i.e., a critical interval for smolt migration.     

Time Learning and Anticipatory Activity in Groups of Arctic Charr

Time learning and anticipatory activity were studied in five groups of 16–17 Arctic charr (Salvelinus alpinus) using self‐feeding devices with individual recognition (PIT (Passive Integrated Transponders)‐tags). The fish were kept under a LD 12:12 h cycle and periods of free access to the food were alternated with periods of time‐limited (2 h) access to food. Self‐feeding activity was significantly related to the light period in unrestricted conditions while related to the feeding periods during time‐limited access to food. The fish learned to concentrate their feeding activity to the restricted mealtime (>50% of the daily self‐feeding activity) within 10 d. The food anticipatory activity, measured as an increased self‐feeding activity before the feeding time and as aggressive interactions close to the trigger, was significant in both cases. The dominant individuals increased the trigger activity in advance of the time‐restricted reward period and subdominant individuals approached the trigger also in advance, inducing aggressive interactions. Thus, anticipating and learning a temporally predictable food source was pronounced in groups of self‐feeding Arctic charr.     

ADP-ribosylation factor 6 regulates mu-opioid receptor trafficking and signaling via activation of phospholipase D2

Endocytosis of the mu-opioid receptor (MOPr) has been shown to play a protective role against the development of tolerance to opioid drugs by facilitating receptor reactivation and recycling. It has been further demonstrated, that the opioid-mediated and ADP-ribosylation factor (ARF)-dependent activation of phospholipase D2 (PLD2) is a prerequisite for MOPr endocytosis. In this study, we investigated which particular ARF protein is involved in opioid-mediated PLD2 activation and what are the mechanisms of ARF function in MOPr trafficking and signaling. By coexpressing the MOPr and dominant negative or constitutively active ARF mutants in human embryonic kidney (HEK) 293 cells and primary cultured cortical neurons as well as by using siRNA technology, we identified the ARF6 protein to be involved in the regulation of MOPr endocytosis. We also found that expression of an effector domain mutant of ARF6, which is incapable of activating PLD, blocked agonist-induced endocytosis suggesting that ARF6 function in MOPr trafficking is PLD2-mediated. Analogously, opioid-mediated activation of PLD2 is blocked in the presence of dominant negative ARF6 mutants. Finally, we also showed that ARF6 protein influences the recycling/reactivation of internalized MOPr and thus modulates agonist-induced MOPr desensitization. Together, these results provide evidence that ARF6 protein regulates MOPr trafficking and signaling via PLD2 activation and hence affects the development of opioid receptor desensitization and tolerance.