Anandamide as an intracellular messenger regulating ion channel activity

The endocannabinoid anandamide (N-arachidonoylethanolamine) was proposed to be an extracellular retrograde messenger, which regulates excitability of neurons by cannabinoid CB1 receptor-dependent inhibition of neurotransmitter release. Recent findings indicate that the neuromodulatory actions of anandamide might be more complex. Anandamide has been shown to directly modulate various ion channels, such as alpha7-nicotinic acetylcholine receptors, T-type Ca2+ channels, voltage-gated and background K+-channels and Transient Receptor Potential Vanilloid type 1 (TRPV1) channels. The binding site of anandamide at some of these ion channels appears to be intracellular or at the bilayer interface. This rises the intriguing possibility that anandamide, prior to its release into the synaptic cleft, may regulate ion homeostasis and excitability of neurons as an intracellular modulator of ion channels independent of its action at cannabinoid CB1 receptors. This possibility might extend the concept of anandamide as an endocannabinoid retrograde messenger and may have profound implications for its role in neurotransmission and neuronal function. Here, we will review the evidence for this hypothesis.     

The challenge of brain lipidomics

After many years backstage, lipids have made a come back in the limelight of neuroscience. This renewed excitement was sparked by a series of convergent discoveries in the fields of neural development, synaptic physiology and receptor pharmacology, which have begun to reveal the roles played by lipid messengers and their receptors in brain function. Such roles extend from the development of the neocortex to the processing of complex behaviors, encompassing a territory as vast as those traditionally assigned to growth factors, neurotransmitters and neuropeptides. Along with these basic discoveries, technical advances have simplified the identification and quantification of neural lipids, achieving a degree of sensitivity and selectivity that was unthinkable only 10 years ago. Thanks to this progress, we can now resolve complex mixtures of lipid molecules and quantify each of their components, which are often present in tissues at vanishingly low concentrations. In this review, I outline several key features of brain lipid signaling and discuss the opportunities and challenges that such features impose on future lipidomic approaches.     

Pterostilbene and 3'-hydroxypterostilbene are effective apoptosis-inducing agents in MDR and BCR-ABL-expressing leukemia cells

Pterostilbene and 3,5-hydroxypterostilbene are the natural 3,5-dimethoxy analogs of trans-resveratrol and piceatannol, two compounds which can induce apoptosis in tumor cells. In previous studies we demonstrated the importance of a 3,5-dimethoxy motif in conferring pro-apoptotic activity to stilbene based compounds so we now wanted to evaluate the ability of pterostilbene and 3,5-hydroxypterostilbene in inducing apoptosis in sensitive and resistant leukemia cells. When tested in sensitive cell lines, HL60 and HUT78, 3'-hydroxypterostilbene was 50-97 times more potent than trans-resveratrol in inducing apoptosis, while pterostilbene appeared barely active. However, both compounds, but not trans-resveratrol and piceatannol, were able to induce apoptosis in the two Fas-ligand resistant lymphoma cell lines, HUT78B1 and HUT78B3, and the multi drug-resistant leukemia cell lines HL60-R and K562-ADR (a Bcr-Abl-expressing cell line resistant to imatinib mesylate). Of note, pterostilbene-induced apoptosis was not inhibited by the pancaspase-inhibitor Z-VAD-fmk, suggesting that this compound acts through a caspase-independent pathway. On the contrary, 3'-hydroxypterostilbene seemed to trigger apoptosis through the intrinsic apoptotic pathway: indeed, it caused a marked disruption of the mitochondrial membrane potential delta psi and its apoptotic effects were inhibited by Z-VAD-fmk and the caspase-9-inhibitor Z-LEHD-fmk. Moreover, pterostilbene and 3'-hydroxypterostilbene, when used at concentrations that elicit significant apoptotic effects in tumor cell lines, did not show any cytotoxicity in normal hemopoietic stem cells. In conclusion, our data show that pterostilbene and particularly 3'-hydroxypterostilbene are interesting antitumor natural compounds that may be useful in the treatment of resistant hematological malignancies, including imatinib, non-responsive neoplasms.     

Morphofunctional integration between skeletal myoblasts and adult cardiomyocytes in coculture is favored by direct cell-cell contacts and relaxin treatment

The success of cellular cardiomyoplasty, a novel therapy for the repair of postischemic myocardium, depends on the anatomical integration of the engrafted cells with the resident cardiomyocytes. Our aim was to investigate the interaction between undifferentiated mouse skeletal myoblasts (C₂C₁₂ cells) and adult rat ventricular cardiomyocytes in an in vitro coculture model. Connexin43 (Cx43) expression, Lucifer yellow microinjection, Ca²⁺ transient propagation, and electrophysiological analysis demonstrated that myoblasts and cardiomyocytes were coupled by functional gap junctions. We also showed that cardiomyocytes upregulated gap junctional communication and expression of Cx43 in myoblasts. This effect required direct cell-to-cell contact between the two cell types and was potentiated by treatment with relaxin, a cardiotropic hormone with potential effects on cardiac development. Analysis of the gating properties of gap junctions by dual cell patch clamping showed that the copresence of cardiomyocytes in the cultures significantly increased the transjunctional current and conductance between myoblasts. Relaxin enhanced this effect in both the myoblast-myoblast and myoblast-cardiomyocyte cell pairs, likely acting not only on gap junction formation but also on the electrical properties of the preexisting channels. Our findings suggest that myoblasts and cardiomyocytes interact actively through gap junctions and that relaxin potentiates the intercellular coupling. A potential role for gap junctional communication in favoring the intercellular exchange of regulatory molecules, including Ca²⁺, in the modulation of myoblast differentiation is discussed. gap junctions; connexin43     

Report on antibodies submitted to the stromal cell section of HLDA8

The paradigm for tissue specific homing of leukocytes is the "area code" hypothesis, which predicts that a specific combination of adhesive interactions and chemokine signals from the endothelium directs leukocyte migration into specific tissue sites. This area code hypothesis has been supported by studies from previous HLDA workshops where endothelial specific cell antigens have been studied. Similarly, a clear haematopoietic "stem cell code" comprising the chemokine SDF-1 (CXCL12) and the adhesion receptor VCAM-1 (CD106) has been shown to contribute to the stem cell niche within bone marrow [K. Tokoyoda, T. Egawa, T. Sugiyama, B.I. Chai, T. Nagasawa, Cellular niches controlling B lymphocyte behaviour within bone marrow during development, Immunity 20 (2004) 707-718]. HLDA 7 included a section devoted to stem cell antigens, which began to define additional antigens important in these processes. During the course of HLDA 8 we have extended these observations to determine whether a more global stromal address code defined by fibroblasts, exists in variety of different tissues [G. Parsonage, A.D. Filer, O. Haworth, G.B. Nash, G.E. Rainger, M. Salmon, C.D. Buckley, A stromal area postcode defined by fibroblasts, Trends Immunol. 26 (2005) 150-156]. The stromal cell section in HLDA 8 was designed to complement the malignant cell, endothelial cell, and stem cell/progenitor cell sections. Seven new CD numbers were assigned to antibodies included in this section at the HLDA 8 Workshop meeting held during December 2004.     

Dissolved oxygen and nutrient budgets in a phytotreatment pond colonised by <Emphasis Type="Italic">Ulva</Emphasis> spp.

Net daily budgets of dissolved oxygen (O₂), dissolved inorganic carbon (DIC), dissolved inorganic nitrogen (DIN = NH₄⁺+NO₂⁻+NO₃⁻) and soluble reactive phosphorus (SRP) were determined in a pond colonised by Ulva spp. This pond received wastewater from a land-based fish farm and was used as a phytotreatment plant. Three consecutive 24-h cycles of measurements were performed with 8–14 samplings per day. Water samples were collected at the inlet and outlet of the pond and budgets were estimated from differences between inlet and outlet loadings. The first cycle was started when Ulva biomass was 8 kg m⁻², as wet weight. The second cycle was performed after the harvest of ~20% of the macroalgal biomass and the third after the harvest of another ~20% of the remaining biomass. Ulva removal was very fast (<1 h) and samplings for cycles 2 and 3 were started two hours after harvesting, so that the whole experiment lasted ~80 h. When Ulva biomass was at its maximum, the aquatic system was heterotrophic with an O₂ demand of 519 mol d⁻¹ and a net regeneration of DIC (2686 mol d⁻¹), NH₄⁺ (49 mol d⁻¹) and SRP (2.5 mol d⁻¹). The DIC to O₂ ratio was an indicator of persistent anaerobic metabolism. Following the first harvest intervention, this system displayed a prompt response and shifted toward a lower O₂ demand (from −519 to −13 mol d⁻¹), with a lesser regeneration degree of NH₄⁺ (11.4 mol d⁻¹) and DIC (1066 mol d⁻¹). After the second Ulva removal the net budget of SRP became negative (−1.0 mol d⁻¹). By integrating these results over the three days cycle we estimated that in order to operate an efficient nutrient control and maintain macroalgal mats in a healthy status the optimal Ulva biomass should be well below ~4 kg m⁻² as wet weight. Above this threshold, self-limitation would render most of the algal mat unable to exploit light and nutrients. An efficient removal of nitrogen and phosphorus could be attained through the management of macroalgal biomass only with an optimisation of recipient surface to nutrient loading ratio.     

Myocardial protection from ischemic preconditioning is not blocked by sub-chronic inhibition of carnitine palmitoyltransferase I

Ischemic preconditioning (IP) triggers cardioprotection via a signaling pathway that converges on mitochondria. The effects of the inhibition of carnitine palmitoyltransferase I (CPT-I), a key enzyme for transport of long chain fatty acids (LCFA) into the mitochondria, on ischemia/reperfusion (I/R) injury are unknown. Here we investigated, in isolated perfused rat hearts, whether sub-chronic CPT-I inhibition (5 days i.p. injection of 25 mg/kg/day of Etomoxir) affects I/R-induced damages and whether cardioprotection by IP can be induced after this inhibition. Effects of global ischemia (30 min) and reperfusion (120 min) were examined in hearts harvested from Control (untreated), Vehicle- or Etomoxir-treated animals. In subsets of hearts from the three treated groups, IP was induced by three cycles of 3 min ischemia followed by 10 min reperfusion prior to I/R. The extent of I/R injury under each condition was assessed by changes in infarct size as well as in myocardial contractility. Postischemic contractility, as indexed by developed pressure and dP/dt(max), was similarly affected by I/R, and was similarly improved with IP in Control, Vehicle or Etomoxir treated animals. Infarct size was also similar in the three subsets without IP, and was significantly reduced by IP regardless of CPT-I inhibition. We conclude that CPT-I inhibition does not affect I/R damages. Our data also show that IP affords myocardial protection in CPT-I inhibited hearts to a degree similar to untreated animals, suggesting that a long-term treatment with the metabolic anti-ischemic agent Etomoxir does not impede the possibility to afford cardioprotection by ischemic preconditioning.     

Searching for intermediates in the carbon skeleton rearrangement of 2-methyleneglutarate to (R)-3-methylitaconate catalyzed by coenzyme B12-dependent 2-methyleneglutarate mutase from Eubacterium barkeri

Coenzyme B(12)-dependent 2-methyleneglutarate mutase from the strict anaerobe Eubacterium barkeri catalyzes the equilibration of 2-methyleneglutarate with (R)-3-methylitaconate. Proteins with mutations in the highly conserved coenzyme binding-motif DXH(X)(2)G(X)(41)GG (D483N and H485Q) exhibited decreased substrate turnover by 2000-fold and >4000-fold, respectively. These findings are consistent with the notion of H485 hydrogen-bonded to D483 being the lower axial ligand of adenosylcobalamin in 2-methyleneglutarate mutase. (E)- and (Z)-2-methylpent-2-enedioate and all four stereoisomers of 1-methylcyclopropane-1,2-dicarboxylate were synthesized and tested, along with acrylate, with respect to their inhibitory potential. Acrylate and the 2-methylpent-2-enedioates were noninhibitory. Among the 1-methylcyclopropane-1,2-dicarboxylates only the (1R,2R)-isomer displayed weak inhibition (noncompetitive, K(i) = 13 mM). Short incubation (5 min) of 2-methyleneglutarate mutase with 2-methyleneglutarate under anaerobic conditions generated an electron paramagnetic resonance (EPR) signal (g(xy) approximately 2.1; g(z) approximately 2.0), which by analogy with the findings on glutamate mutase from Clostridium cochlearium [Biochemistry, 1998, 37, 4105-4113] was assigned to cob(II)alamin coupled to a carbon-centered radical. At longer incubation times (>1 h), inactivation of the mutase occurred concomitant with the formation of oxygen-insensitive cob(II)alamin (g(xy) approximately 2.25; g(z) approximately 2.0). In order to identify the carbon-centered radical, various (13)C- and one (2)H-labeled substrate/product molecules were synthesized. Broadening (0.5 mT) of the EPR signal around g = 2.1 was observed only when C2 and/or C4 of 2-methyleneglutarate was labeled. No effect on the EPR signals was seen when [5'-(13)C]adenosylcobalamin was used as coenzyme. The inhibition and EPR data are discussed in the context of the addition-elimination and fragmentation-recombination mechanisms proposed for 2-methyleneglutarate mutase.     

Depression by relaxin of neurally induced contractile responses in the mouse gastric fundus

The peptide hormone relaxin, which attains high circulating levels during pregnancy, has been shown to depress small-bowel motility through a nitric oxide (NO)-mediated mechanism. In the present study we investigated whether relaxin also influences gastric contractile responses in mice. Female mice in proestrus or estrus were treated for 18 h with relaxin (1 microg s.c.) or vehicle (controls). Mechanical responses of gastric fundal strips were recorded via force-displacement transducers. Evaluation of the expression of nitric oxide synthase (NOS) isoforms was performed by immunohistochemistry and Western blot. In control mice, neurally induced contractile responses elicited by electrical field stimulation (EFS) were reduced in amplitude by addition of relaxin to the organ bath medium. In the presence of the NO synthesis inhibitor l-NNA, relaxin was ineffective. Direct smooth muscle contractile responses were not influenced by relaxin or l-NNA. In strips from relaxin-pretreated mice, the amplitude of neurally induced contractile responses was also reduced in respect to the controls, while that of direct smooth muscle contractions was not. Further addition of relaxin to the bath medium did not influence EFS-induced responses, whereas l-NNA did. An increased expression of NOS I and NOS III was observed in gastric tissues from relaxin-pretreated mice. In conclusion, the peptide hormone relaxin depresses cholinergic contractile responses in the mouse gastric fundus by up-regulating NO biosynthesis at the neural level.     

Activated primary and memory CD8 T cells migrate to nonlymphoid tissues regardless of site of activation or tissue of origin

Following activation within secondary lymphoid tissue, CD8 T cells must migrate to targets, such as infected self tissue, allografts, and tumors, to mediate contact-dependent effector functions. To test whether the pattern of migration of activated CD8 T cells was dependent on the site of Ag encounter, we examined the distribution of mouse Ag-specific CD8 T cells following local challenges. Our findings indicated that activated CD8 T cells migrated pervasively to all nonlymphoid organs irrespective of the site of initial Ag engagement. Using an adoptive transfer system, migration of nonlymphoid memory cells was also examined. Although some limited preference for the tissue of origin was noted, transferred CD8 memory T cells from various nonlymphoid tissues migrated promiscuously, except to the intestinal mucosa, supporting the concept that distinct memory pools may exist. However, regardless of the tissue of origin, reactivation of transferred memory cells resulted in widespread dissemination of new effector cells. These data indicated that recently activated primary or memory CD8 T cells were transiently endowed with the ability to traffic to all nonlymphoid organs, while memory cell trafficking was more restricted. These observations will help refine our understanding of effector and memory CD8 T cell migration patterns.