Pauli M. Turunen

OX1 Orexin/Hypocretin Receptor Signaling through Arachidonic Acid and Endocannabinoid Release

We showed previously that OX1 orexin receptor stimulation produced a strong 3H overflow response from [3H]arachidonic acid (AA)-labeled cells. Here we addressed this issue with a novel set of tools and methods, to distinguish the enzyme pathways responsible for this response. CHO-K1 cells heterologously expressing human OX1 receptors were used as a model system. By using selective pharmacological inhibitors, we showed that, in orexin-A-stimulated cells, the AA-derived radioactivity was released as two distinct components, i.e., free AA and the endocannabinoid 2-arachidonoyl glycerol (2-AG). Two orexin-activated enzymatic cascades are responsible for this response: cytosolic phospholipase A2 (cPLA2) and diacylglycerol lipase; the former cascade is responsible for part of the AA release, whereas the latter is responsible for all of the 2-AG release and part of the AA release. Essentially only diacylglycerol released by phospholipase C but not by phospholipase D was implicated as a substrate for 2-AG production, although both phospholipases were strongly activated. The 2-AG released acted as a potent paracrine messenger through cannabinoid CB1 receptors in an artificial cell-cell communication assay that was developed. The cPLA2 cascade, in contrast, was involved in the activation of orexin receptor-operated Ca2+ influx. 2-AG was also released upon OX1 receptor stimulation in recombinant HEK-293 and neuro-2a cells. The results directly show, for the first time, that orexin receptors are able to generate potent endocannabinoid signals in addition to arachidonic acid signals, which may explain the proposed orexin-cannabinoid interactions (e.g., in neurons).

Involvement of TRPC3 channels in calcium oscillations mediated by OX1 orexin receptors

Oscillations of intracellular Ca2+ provide a novel mechanism for sustained activation of cellular processes. Receptor-activated oscillations are mainly thought to occur through rhythmic IP3-dependent store discharge. However, as shown here in HEK293 cells 1 nM orexin-A (Ox-A) acting at OX1 receptors (OX1R) triggered oscillatory Ca2+ responses, requiring external Ca2+. These responses were attenuated by interference with TRPC3 channel (but not TRPC1/4) function using dominant negative constructs, elevated Mg2+ (a blocker of many TRP channels) or inhibition of phospholipase A2. These treatments did not affect Ca2+ oscillations elicited by high concentrations of Ox-A (100 nM) in the absence of external Ca2+. OX1R are thus able to activate TRPC(3)-channel-dependent oscillatory responses independently of store discharge.

Arachidonic acid release mediated by OX1 orexin receptors

Background and purpose:  We have previously shown that lipid mediators, produced by phospholipase D and C, are generated in OX1 orexin receptor signalling with high potency, and presumably mediate some of the physiological responses to orexin. In this study, we investigated whether the ubiquitous phospholipase A2 (PLA2) signalling system is also involved in orexin receptor signalling.

Autocrine Endocannabinoid Signaling through CB1 Receptors Potentiates OX1 Orexin Receptor Signaling

It has been proposed that OX1 orexin receptors and CB1 cannabinoid receptors can form heteromeric complexes, which affect the trafficking of OX1 receptors and potentiate OX1 receptor signaling to extracellular signal–regulated kinase (ERK). We have recently shown that OX1 receptor activity releases high levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG), suggesting an alternative route for OX1-CB1 receptor interaction in signaling, for instance, in retrograde synaptic transmission. In the current study, we set out to investigate this possibility utilizing recombinant Chinese hamster ovary K1 cells. 2-AG released from OX1 receptor–expressing cells acted as a potent paracrine messenger stimulating ERK activity in neighboring CB1 receptor–expressing cells. When OX1 and CB1 receptors were expressed in the same cells, OX1 stimulation–induced ERK phosphorylation and activity were strongly potentiated. The potentiation but not the OX1 response as such was fully abolished by specific inhibition of CB1 receptors or the enzyme responsible for 2-AG generation, diacylglycerol lipase (DAGL). Although the results do not exclude the previously proposed OX1-CB1 heteromerization, they nevertheless unequivocally identify DAGL-dependent 2-AG generation as the pivotal determinant of the OX1-CB1 synergism and thus suggest a functional rather than a molecular interaction of OX1 and CB1 receptors.

Orexin/hypocretin receptor chimaeras reveal structural features important for orexin peptide distinction

We wanted to analyze the basis for the distinction between OX1 and OX2 orexin receptors by the known agonists, orexin‐A, orexin‐B and Ala11, d‐Leu15‐orexin‐B, of which the latter two show some selectivity for OX2. For this, chimaeric OX1/OX2 and OX2/OX1 orexin receptors were generated. The receptors were transiently expressed in HEK‐293 cells, and potencies of the agonists to elicit cytosolic Ca2+ elevation were measured. The results show that the N‐terminal regions of the receptor are most important, and the exchange of the area from the C‐terminal part of the transmembrane helix 2 to the transmembrane helix 4 is enough to lead to an almost total change of the receptors ligand profile.

Calpain Activity Is Essential for ATP-Driven Unconventional Vesicle-Mediated Protein Secretion and Inflammasome Activation in Human Macrophages

Extracellular ATP is an endogenous danger signal that is known to activate inflammatory responses in innate immune cells, including macrophages. Activated macrophages start to secrete proteins to induce an immune response, as well as to recruit other immune cells to the site of infection and tissue damage. In this study, we characterized the secretome (i.e., the global pattern of secreted proteins) of ATP-stimulated human macrophages. We show that ATP stimulation activates robust vesicle-mediated unconventional protein secretion, including exosome release and membrane shedding, from human macrophages. Pathway analysis of the identified secreted proteins showed that calpain-related pathways were overrepresented in the secretome of ATP-stimulated cells. In accordance with this, calpains, which are calcium-dependent nonlysosomal cysteine proteases, were activated upon ATP stimulation through a P2X purinoceptor 7 receptor–dependent pathway. Functional studies demonstrated that calpain activity is essential for the P2X purinoceptor 7 receptor–mediated activation of unconventional protein secretion. Unconventional protein secretion was followed by cell necrosis and NLRP3 inflammasome–mediated secretion of the mature form of the proinflammatory cytokine IL-1β. Furthermore, ATP-driven NLRP3 inflammasome activation was also dependent on calpain activity. Interestingly, pro–IL-1β and inflammasome components ASC and caspase-1 were released by ATP-activated macrophages through a vesicle-mediated secretion pathway. In conclusion, to our knowledge, we provide the first global characterization of proteins secreted by ATP-activated human macrophages and show a pivotal role for calpains in the activation of the inflammatory response during ATP exposure.

Transient Receptor Potential Channels and Their Role in Modulating Radial Glial–Neuronal Interaction: A Signaling Pathway Involving mGluR5

The guidance of developing neurons to the right position in the central nervous system is of central importance in brain development. Canonical transient receptor potential (TRPC) channels are thought to mediate turning responses of growth cones to guidance cues through fine control of calcium transients. Proliferating and 1- to 5-day-differentiated neural progenitor cells (NPCs) showed expression of Trpc1 and Trpc3 mRNA, while Trpc4-7 was not clearly detected. Time-lapse imaging showed that the motility pattern of neuronal cells was phasic with bursts of rapid movement (>60 μm/h), changes in direction, and intermittent slow phases or stallings (<40 μm/h), which frequently occurred in close contact with radial glial processes. Genetic interference with the TRPC3 and TRPC1 channel enhanced the motility of NPCs (burst frequency/stalling frequency). TRPC3-deficient cells or cells treated with the TRPC3 blocker pyr3 infrequently changed direction and seldom contacted radial glial processes. TRPC channels are also activated by group I metabotropic glutamate receptors (mGluR1 and mGluR5). As shown here, pyr3 blocked the calcium response mediated through mGluR5 in radial glial processes. Furthermore, 2-methyl-6-(phenylethynyl)pyridine, a blocker of mGluR5, affected the motility pattern in a similar way as TRPC3/6 double knockout or pyr3. The results suggest that radial glial cells exert attractant signals to migrating neuronal cells, which alter their motility pattern. Our results suggest that mGluR5 acting through TRPC3 is of central importance in radial glial-mediated neuronal guidance.

Filtration assay for arachidonic acid release.

Arachidonic acid (AA) release is a central message in cell signaling. Fatty acid release is generally assessed by manual sampling of radioactivity release from cells prelabeled with a radiolabeled fatty acid. The assay is laborious, time-consuming, and susceptible to high noise. Here we present a fast and reproducible method for 96-well filter plates and cells in suspension, a method that is best suited for agonist concentration-response studies and, thus, for ligand screening. The method offers tremendous time and effort savings and enables execution of large experimental series previously unattainable for AA release studies.

Regulation of radial glial process growth by glutamate via mGluR5/TRPC3 and neuregulin/ErbB4

Radial glial cells play an essential role through their function as guides for neuronal migration during development. Disruption of metabotropic glutamate receptor 5 (mGluR5) function retards the growth of radial glial processes in vitro. Neuregulins (NRG) are activated by proteolytic cleavage and regulate (radial) glial maintenance via ErbB3/ErbB4 receptors. We show here that blocking ErbB4 disrupts radial process extension. Soluble NRG acting on ErbB4 receptors is able to promote radial process extension in particular where process elongation has been impeded by blockade of mGluR5, the nonselective cation channel canonical transient receptor potential 3 (TRPC3), or matrix metalloproteases (MMP). NRG does not restore retarded process growth caused by ErbB4 blockade. Stimulation of muscarinic receptors restores process elongation due to mGluR5 blockade but not that caused by TRPC3, MMP or ErbB4 blockade suggesting that muscarinic receptors can replace mGluR5 with respect to radial process extension. Additionally, NRG/ErbB4 causes Ca2+ mobilization in a population of cells through cooperation with ErbB1 receptors. Our results indicate that mGluR5 promotes radial process growth via NRG activation by a mechanism involving TRPC3 channels and MMPs. Thus neurotransmitters acting on G‐protein coupled receptors could play a central role in the maintenance of the radial glial scaffold through activation of NRG/ErbB4 signaling.

Stapled truncated orexin peptides as orexin receptor agonists

HIGHLIGHTSPeptide stapling of the neuropeptide orexin‐A15–33 lowers the potency significantly.The peptide C‐terminus is crucial for activity, and modifications were not allowed.Central and N‐terminal modifications yielded bioactive peptides. ABSTRACT The peptides orexin‐A and −B, the endogenous agonists of the orexin receptors, have similar 19‐amino‐acid C‐termini which retain full maximum response as truncated peptides with only marginally reduced potency, while further N‐terminal truncations successively reduce the activity. The peptides have been suggested to bind in an &agr;‐helical conformation, and truncation beyond a certain critical length is likely to disrupt the overall helical structure. In this study, we set out to stabilize the &agr;‐helical conformation of orexin‐A15–33 via peptide stapling at four different sites. At a suggested hinge region, we varied the length of the cross‐linker as well as replaced the staple with two &agr;‐aminoisobutyric acid residues. Modifications close to the peptide C‐terminus, which is crucial for activity, were not allowed. However, central and N‐terminal modifications yielded bioactive peptides, albeit with decreased potencies. This provides evidence that the orexin receptors can accommodate and be activated by &agr;‐helical peptides. The decrease in potency is likely linked to a stabilization of suboptimal peptide conformation or blocking of peptide backbone–receptor interactions at the hinge region by the helical stabilization or the modified amino acids.