T cell cytoskeletal forces shape synapse topography for targeted lysis via membrane curvature bias of perforin.

Cytotoxic T lymphocytes (CTLs) lyse target cells by delivering lytic granules that contain the pore former perforin to the cytotoxic immunological synapse. Here, we establish that opposing cytoskeletal forces drive lytic granule polarization and simultaneously shape T cell synapse topography to enhance target perforation. At the cell rear, actomyosin contractility drives the anterograde movement of lytic granules toward the nucleus. At the synapse, dynein-derived forces induce negatively curved membrane pockets to which granules are transported around the nucleus. These highly concave degranulation pockets are located directly opposite positively curved bulges on the target cell membrane. We identify a curvature bias in the action of perforin, which preferentially perforates positively curved tumor cell membrane. Together, these findings demonstrate murine and human T cell-mediated cytotoxicity to be a highly tuned mechano-biochemical system, in which the forces that polarize lytic granules locally bend the synaptic membrane to favor the unidirectional perforation of the target cell.

Modulating the Mechanical Activation of TRPV4 at the Cell-Substrate Interface.

Ion channels activated by mechanical inputs are important force sensing molecules in a wide array of mammalian cells and tissues. The transient receptor potential channel, TRPV4, is a polymodal, nonselective cation channel that can be activated by mechanical inputs but only if stimuli are applied directly at the interface between cells and their substrate, making this molecule a context-dependent force sensor. However, it remains unclear how TRPV4 is activated by mechanical inputs at the cell-substrate interface, which cell intrinsic and cell extrinsic parameters might modulate the mechanical activation of the channel and how mechanical activation differs from TRPV4 gating in response to other stimuli. Here we investigated the impact of substrate mechanics and cytoskeletal components on mechanically evoked TRPV4 currents and addressed how point mutations associated with TRPV4 phosphorylation and arthropathy influence mechanical activation of the channel. Our findings reveal distinct regulatory modulation of TRPV4 from the mechanically activated ion channel PIEZO1, suggesting the mechanosensitivity of these two channels is tuned in response to different parameters. Moreover, our data demonstrate that the effect of point mutations in TRPV4 on channel activation are profoundly dependent on the gating stimulus.

A tetrapeptide class of biased analgesics from an Australian fungus targets the µ-opioid receptor.

An Australian estuarine isolate of Penicillium sp. MST-MF667 yielded 3 tetrapeptides named the bilaids with an unusual alternating LDLD chirality. Given their resemblance to known short peptide opioid agonists, we elucidated that they were weak (Ki low micromolar) µ-opioid agonists, which led to the design of bilorphin, a potent and selective µ-opioid receptor (MOPr) agonist (Ki 1.1 nM). In sharp contrast to all-natural product opioid peptides that efficaciously recruit ß-arrestin, bilorphin is G protein biased, weakly phosphorylating the MOPr and marginally recruiting ß-arrestin, with no receptor internalization. Importantly, bilorphin exhibits a similar G protein bias to oliceridine, a small nonpeptide with improved overdose safety. Molecular dynamics simulations of bilorphin and the strongly arrestin-biased endomorphin-2 with the MOPr indicate distinct receptor interactions and receptor conformations that could underlie their large differences in bias. Whereas bilorphin is systemically inactive, a glycosylated analog, bilactorphin, is orally active with similar in vivo potency to morphine. Bilorphin is both a unique molecular tool that enhances understanding of MOPr biased signaling and a promising lead in the development of next generation analgesics.

Analysis of Mechanically Activated Ion Channels at the Cell-Substrate Interface: Combining Pillar Arrays and Whole-Cell Patch-Clamp.

Ionic currents can be evoked by mechanical inputs applied directly at the cell-substrate interface. These ionic currents are mediated by mechanically activated ion channels, where the open probability increases with increasing mechanical input. In order to study mechanically activated ion channels directly at the interface between cells and their environment, we have developed a technique to simultaneously monitor ion channel activity whilst stimuli are applied via displacement of cell-substrate contacts. This technique utilizes whole-cell patch-clamp electrophysiology and elastomeric pillar arrays, it is quantitative and appropriate for studying channels that respond to stimuli that are propagated to an adherent cell via the physical substrate. The mammalian channels PIEZO1, PIEZO2 have been shown to be activated by substrate deflections, using this technique. In addition, TRPV4 mediated currents can be evoked by substrate deflections, in contrast to alternate stimulation methods such as membrane stretch or cellular indentation. The deflections applied at cell-substrate points mimic the magnitude of physical stimuli that impact cells in situ.

Role of Phosphorylation Sites in Desensitization of µ-Opioid Receptor.

Phosphorylation of residues in the C-terminal tail of the µ-opioid receptor (MOPr) is thought to be a key step in desensitization and internalization. Phosphorylation of C-terminal S/T residues is required for internalization (Just et al., 2013), but its role in desensitization is unknown. This study examined the influence of C-terminal phosphorylation sites on rapid desensitization of MOPr. Wild-type MOPr, a 3S/T-A mutant (S363A, T370A, S375A) that maintains internalization, 6S/T-A (S363A, T370A, S375A, T376A, T379A, T383A) and 11S/T-A (all C-terminal S/T residues mutated) mutants not internalized by MOPr agonists were stably expressed in AtT20 cells. Perforated patch-clamp recordings of MOPr-mediated activation of G-protein-activated inwardly rectifying potassium channel (Kir3.X) (GIRK) conductance by submaximal concentrations of Met(5)-enkephalin (ME) and somatostatin (SST; coupling to native SST receptor [SSTR]) were used to examine desensitization induced by exposure to ME and morphine for 5 minutes at 37°C. The rates of ME- and morphine-induced desensitization did not correlate with phosphorylation using phosphorylation site-specific antibodies. ME-induced MOPr desensitization and resensitization did not differ from wild-type for 3S/T-A and 6S/T-A but was abolished in 11S/T-A. Morphine-induced desensitization was unaffected in all three mutants, as was heterologous desensitization of SSTR. Morphine-induced desensitization (but not ME) was reduced by protein kinase C inhibition in wild-type MOPr and abolished in the 11S/T-A mutant, as was heterologous desensitization. These findings establish that MOPr desensitization can occur independently of S/T phosphorylation and internalization; however, C-terminal phosphorylation is necessary for some forms of desensitization because mutation of all C-terminal sites (11S/T-A) abolishes desensitization induced by ME.

Chronic lithium impairs skin tolerance to ischemia in random-pattern skin flap of rats.

BACKGROUND: Despite its apparent anti-apoptotic effect, lithium impairs endothelium-dependent vasorelaxation in various tissues. In this study, we assessed the effect of lithium treatment on ischemic skin flap survival and its interaction with nitric oxide pathway. MATERIALS AND METHODS: Seventy-six male Sprague-Dawley rats were randomly assigned into 13 groups. For skin flap surgery, dorsal skin flap measuring 8 × 2 cm was elevated on the midline. After local injections (if needed), the cranial pedicle was cut and flap was sutured back. Flap survival was assessed after 7 d. Animals in the chronic lithium group received lithium chloride in tap water for 4 wk preoperatively and 7 d postoperatively. Acute lithium groups received 3 nmol, 10 nmol and 3 µmol/flap lithium locally. In another experiment, interaction with nitric oxide synthase inhibitor L-NAME as well as nitric oxide precursor L-arginine was studied, and the effect of ischemic preconditioning on skin flap survival in lithium treated rats was investigated. RESULTS: Chronic lithium group had mean flap survival value of 32.6% ± 5.2% (mean ± SD), which was significantly lower than normal subjects (52.7% ± 6.1%, P < 0.001), while acute local lithium treatment had no effect. In chronic lithium group, systemic L-NAME (10 mg/kg, 30 min before flap elevation) failed to significantly decrease the survival, while sub-effective systemic L-arginine (100 mg/kg) and ischemic preconditioning significantly increased flap survival (P < 0.001 and P < 0.01, respectively). CONCLUSIONS: We conclude that long-term lithium treatment impairs the skin tolerance to ischemia in rats, which seems to be nitric oxide mediated. This effect is prevented by ischemic preconditioning or L-arginine treatment. The results suggest that skin-involving interventions should be applied more cautiously in patients who are on lithium treatment.

Study of morphine-induced dependence in gonadectomized male and female mice.

In this study we evaluated the effects of sex difference and also sex hormones on the naloxone-precipitated morphine withdrawal in both orchidectomized (ORC) male and ovariectomized (OVX) female mice. Morphine (50, 50 and 75 mg/kg/day for 4 days, s.c.) was administered to animals and at 5th day naloxone (4 mg/kg, i.p.)-precipitated morphine withdrawal signs, jumpings and the percentage of weight loss, were measured. There was no significant alteration in withdrawal jumpings between male and female mice, though weight loss was significantly higher in male ones. Jumpings was significantly lower in both OVX and ORC mice and percentage of weight loss was significantly higher in OVX mice than corresponding non-operated or sham animals. In OVX mice, E(2)V (10 mg/kg, s.c.) increased number of jumpings and decreased percentage of weight loss. Progesterone (25 mg/kg, s.c.) had no effect on jumpings, whereas it decreased weight loss in OVX mice. Testosterone (2.5 mg/kg, s.c.) increased jumpings in ORC mice while it had no effect on percentage of weight loss. Our results demonstrated that sex hormones could play a role in the morphine withdrawal syndrome in both ORC male and OVX female mice.

The nonadrenergic noncholinergic-mediated relaxation of corpus cavernosum was impaired in chronic lithium-treated rats: improvement with l-arginine.

One-third of lithium-treated men complain from sexual dysfunction, although the exact mechanisms of which are not yet known. In this study we investigated the effect of chronic lithium (LiCl, 600 mg/l for 30 days) administration on the neurogenic relaxation of isolated rat corpus cavernosum. The corporal strips were precontracted with phenylephrine and electrical field stimulation (EFS) was applied to obtain relaxation. Relaxation to EFS was significantly (P<0.001) impaired in LiCl-treated rats. The nitric oxide (NO) synthase inhibitor Nomega-nitro-l-arginine methyl ester (l-NAME; 100 microM) inhibited the relaxation to EFS in both LiCl-treated and control rats. The NO precursor l-arginine, at per se noneffective concentration (0.1 mM), significantly (P<0.001) enhanced the EFS-induced relaxation of LiCl-treated corporal strips. The relaxation responses to the NO donor sodium nitroprusside were similar between two groups. These data demonstrate that chronic lithium treatment could impair the NO-mediated neurogenic relaxation of rat corpus cavernosum which could be prevented by l-arginine.

Modulation by female sex hormones of the cannabinoid-induced catalepsy and analgesia in ovariectomized mice.

Cannabinoids are psychoactive compounds with many pharmacological properties such as analgesia, sedation and catalepsy most of which are mediated by cannabinoid CB1 receptors. In the present study, we evaluated whether the ovarian sex hormones are involved in the cannabinoid-induced catalepsy and analgesia in ovariectomized female mice. Female NMRI mice (weighing 25-30 g) were divided into 3 main groups: unoperated, sham-operated and ovariectomized. Both the catalepsy and analgesia induced by different doses of the synthetic cannabinoid WIN 55,212-2 (2 and 4 mg/kg, i.p.) were examined in the groups in the presence or absence of the cannabinoid CB1 antagonist AM251 (0.5 mg/kg). We also evaluated effects of estradiol valerate (10 mg/kg) and progesterone (25 mg/kg) on catalepsy and analgesia induced by WIN 55,212-2 in ovariectomized mice. The antinociceptive effect of WIN 55,212-2 was significantly (P<0.01) enhanced in ovariectomized mice, which was prevented by pretreatment with estradiol but not by progesterone. There was no significant difference in the cannabinoid-induced catalepsy between control and ovariectomized mice. However, pretreatment with progesterone but not estradiol potentiated the cataleptic effect of low dose of WIN 55,212-2 (2 mg/kg) in ovariectomized mice (P<0.01). The present data demonstrated for the first time that ovarian sex steroids could modulate both cannabinoid-induced catalepsy and analgesia in female ovariectomized mice.