Luzia da Silva Sampaio

The endocannabinoid system in renal cells: regulation of Na+ transport by CB1 receptors through distinct cell signalling pathways

The function of the endocannabinoid system (ECS) in renal tissue is not completely understood. Kidney function is closely related to ion reabsorption in the proximal tubule, the nephron segment responsible for the re‐absorption of 70–80% of the filtrate. We studied the effect of compounds modulating the activity of cannabinoid (CB) receptors on the active re‐absorption of Na+ in LLC‐PK1 cells.

Membrane Cholesterol Depletion by Methyl-β-Cyclodextrin Enhances the Expression of Cardiac Differentiation Markers

Cholesterol is a sterol lipid that plays pleiotropic roles in the plasma membrane; it is involved in maintaining membrane fluidity and permeability and the structure of lipid microdomains. Despite its importance, the consequences of membrane cholesterol depletion during cardiac differentiation have not been described. Therefore, we investigated the cellular and molecular mechanisms associated with cholesterol depletion in cultures of chick cardiac cells. We used methyl-β-cyclodextrin (MCD) to deplete membrane cholesterol and investigate its role in cardiac differentiation by following the expression of several markers including the transcriptional factor Nkx2.5, the myofibrillar protein tropomyosin, the cytoskeletal intermediate filament protein desmin, the caveolar protein caveolin-3, the cadherin/β-catenin adhesion complex, and the junctional protein connexin 43. Confocal microscopy showed that desmin-positive cells were located more externally in the aggregates in relation to the more internally located caveolin-3-positive cells. Desmin and caveolin-3 were co-localized in filamentous structures in the subsarcolemmal region of well-spread cells outside the aggregates. β-Catenin was concentrated in regions of cell-cell contact, and tropomyosin in sarcomeric structures. Western blot tests showed that immediately following cholesterol depletion, there was a slight decrease in the expression of caveolin-3 and desmin, and at the same time there was a sharp increase in the expression of cadherin, tropomyosin, Nkx2.5 and connexin 43. Further, we found an increase in the expression of cardiac β-myosin heavy chain 7, a marker of the cardiac hypertrophic phenotype. These observations suggest that membrane cholesterol plays a significant role in regulating cardiomyocyte differentiation.

Early Double-Negative Thymocyte Export in Trypanosoma cruzi Infection Is Restricted by Sphingosine Receptors and Associated with Human Chagas Disease

The protozoan parasite Trypanosoma cruzi is able to target the thymus and induce alterations of the thymic microenvironmental and lymphoid compartments. Acute infection results in severe atrophy of the organ and early release of immature thymocytes into the periphery. To date, the pathophysiological effects of thymic changes promoted by parasite-inducing premature release of thymocytes to the periphery has remained elusive. Herein, we show that sphingosine-1-phosphate (S1P), a potent mediator of T cell chemotaxis, plays a role in the exit of immature double-negative thymocytes in experimental Chagas disease. In thymuses from T. cruzi-infected mice we detected reduced transcription of the S1P kinase 1 and 2 genes related to S1P biosynthesis, together with increased transcription of the SGPL1 sphingosine-1-lyase gene, whose product inactivates S1P. These changes were associated with reduced intrathymic levels of S1P kinase activity. Interestingly, double-negative thymocytes from infected animals expressed high levels of the S1P receptor during infection, and migrated to lower levels of S1P. Moreover, during T. cruzi infection, this thymocyte subset expresses high levels of IL-17 and TNF-α cytokines upon polyclonal stimulation. In vivo treatment with the S1P receptor antagonist FTY720 resulted in recovery the numbers of double-negative thymocytes in infected thymuses to physiological levels. Finally, we showed increased numbers of double-negative T cells in the peripheral blood in severe cardiac forms of human Chagas disease.

Membrane cholesterol depletion reduces breast tumor cell migration by a mechanism that involves non-canonical Wnt signaling and IL-10 secretion

BackgroundMigration and invasion are hallmarks of cancer cells that allow their dissemination to other tissues. Studying the cellular and molecular basis of cancer cell migration can help us to understand and control cancer metastasis. Many membrane molecules have been described as being involved in tumor cell migration, and among them is cholesterol.MethodIn the present study we investigated the role of membrane cholesterol in the migration of breast tumor cells. The human mammary gland/breast epithelial cell line MDA-MB 231 was used and membrane cholesterol was depleted with methyl-β-cyclodextrin (MbCD). Cell migration was measured in a cell-based scratch assay and the involvement of the Wnt signaling was tested using Lef-1/TCF reporter activation in permanently transfected MDA-MB 231 cells. Cell morphology was analyzed using fluorescent phalloidin to label F-actin structures.ResultsOur results show that membrane cholesterol depletion reduces cell migration, induces changes in cell morphology and in membrane protrusions. Cholesterol depletion also induces an increase in the secretion of IL-10, a cytokine involved in the control of cell migration. In addition, we show that these effects do not involve the activation of the canonical Wnt/beta-catenin pathway. Interestingly, Wnt5a inhibited cell migration in similar levels as MbCD, pointing to a role for the non-canonical Wnt pathway and IL-10 in this process.ConclusionTaken together, our results suggest a correlation between membrane cholesterol, non-canonical Wnt signaling, IL-10 secretion and breast tumor cell migration. These results could contribute to the understanding of breast tumor cell migration and may be used in the development of new therapeutic strategies targeted to breast cancer metastasis.

α-Cyclodextrin enhances myoblast fusion and muscle differentiation by the release of IL-4.

Muscle fibers are formed during embryonic development by the fusion of mononucleated myoblasts. The spatial structure and molecular composition of the sarcolemma are crucial for the myoblast recognition and fusion steps. Cyclodextrins are a group of substances that have the ability to solubilize lipids through the formation of molecular inclusion complexes. Previously, we have shown that methyl-β-cyclodextrin (MbCD) enhances muscle differentiation. Here, we analyzed the effects of α-cyclodextrin (aCD) during myogenesis. Myogenic cultures treated with aCD showed an increase in myoblast fusion and in the expression of myogenin, sarcomeric tropomyosin and desmin. aCD-conditioned media accelerates myogenesis in a similar way as aCD does, and increased levels of IL-4 were found in aCD-conditioned media. aCD-induced effects on myogenesis were inhibited by an anti-IL4 antibody. These results show that α-cyclodextrin induces myogenic differentiation by the release of IL-4.

Neuro-glial cannabinoid receptors modulate signaling in the embryonic avian retina

&NA; Endocannabinoids are endogenous lipids that activate selective G protein coupled receptors (CB1 and CB2), mostly found at neuronal presynaptic sites in the nervous system. One of the main consequences of the activation of CB receptors is a decrease in GABA or glutamate release, controlling cell excitability. Here we studied the expression of CB1 and CB2 receptors in E8C8 cultured retina cells (embryonic day 8 and 8 days in vitro) using immunocytochemistry and western blot analysis. We also evaluated their functions in terms of cyclic AMP (cAMP) production, single cell calcium imaging (SCCI) and GABA release induced in basal conditions or activated by l‐Aspartate (L‐ASP) in cell cultures or under ischemia in young chick retina. We show that both cannabinoid receptors are expressed in retinal neurons and glial cells. WIN 55,212‐2 (WIN, a CB1/CB2 agonist) decreased cAMP production in cultured avian embryonic retinal cells in basal conditions. WIN also led to a decrease in the number of glial cells that increased Ca2+ levels evoked by ATP, but had no effect in Ca2+ shifts in neuronal cells activated by KCl. Finally, WIN inhibited [3H]‐GABA release induced by KCl or L‐ASP, accumulated in amacrine cells, but had no effect in the amount of GABA released in an oxygen glucose deprivation (OGD) condition. Altogether, our data indicate that cannabinoid receptors function as regulators of avian retina signaling at critical embryonic stages during synapse formation. HighlightsBoth CB1 and CB2 are expressed in retinal neurons and glial cells in culture.WIN 55,212‐2 decreases cAMP production in retinal cells in basal conditions.WIN decreases the number of glial cells that increased Ca2+ evoked by ATP.WIN inhibited [3H]‐GABA release induced by KCl or L‐Aspartate.WIN had no effect in GABA released in an oxygen glucose deprivation condition in chick retina.

Beta-adrenergic receptor activation increases GABA uptake in adolescent mice frontal cortex: Modulation by cannabinoid receptor agonist WIN55,212-2

&NA; GABA transporters regulate synaptic GABA levels and dysfunctions in this system might result in psychiatric disorders. The endocannabinoid system (ECS) is the main circuit breaker in the nervous system and may alter noradrenaline (NA) communication, which in turn modulates the release of GABA. However, a close relationship between these systems has not been recognized. We asked whether NA and ECS might control extracellular GABA levels in slices of frontal cortex (FC) of adolescent Swiss mice with 40 days after birth (PN40). Here we show that NA and isoproterenol (ISO), a beta‐adrenergic agonist, increased [3H]‐GABA uptake in mice FC, while alpha1‐adrenergic agonist phenylephrine had no effect. As GAT‐1 is expressed and fully functional at the FC, addition of NO‐711, a GAT‐1 inhibitor, dose dependently blocked [3H]‐GABA uptake. The increase of [3H]‐GABA uptake induced by ISO was also blocked by NO‐711. [3H]‐GABA release induced by 80 mM KCl was reduced by NO‐711, but not by removal of Ca2+. ISO also increased cyclic AMP (cAMP) levels and addition of WIN 55,212‐2, a mixed CB1/CB2 receptor agonist, inhibited the effect of ISO in GABA uptake increase, GAT‐1 expression and cAMP levels compared to control. Our data show that GABA transport increased by NA and ISO is negatively regulated by cannabinoid receptor agonist WIN55,212‐2. HighlightsNoradrenaline increases [3H]‐GABA uptake in adolescent mice Frontal Cortex.The increase of [3H]‐GABA uptake induced by isoproterenol (ISO) is blocked by NO‐711.[3H]‐GABA release induced by 80 mM KCl is reduced by NO‐711, but not by removal of Ca2+.WIN 55,212‐2 inhibited the effect of ISO in GABA uptake, GAT‐1 expression and cAMP levels.

Cannabinoid Receptor Type 1 Expression in the Developing Avian Retina: Morphological and Functional Correlation With the Dopaminergic System

The avian retina has been used as a model to study signaling by different neuro- and gliotransmitters. It is unclear how dopaminergic and cannabinoid systems are related in the retina. Here we studied the expression of type 1 and 2 cannabinoid receptors (CB1 and CB2), as well as monoacylglycerol lipase (MAGL), the enzyme that degrades 2-arachidonoylglycerol (2-AG), during retina development. Our data show that CB1 receptor is highly expressed from embryonic day 5 (E5) until post hatched day 7 (PE7), decreasing its levels throughout development. CB1 is densely found in the ganglion cell layer (GCL) and inner plexiform layer (IPL). CB2 receptor was also found from E5 until PE7 with a decrease in its contents from E9 afterwards. CB2 was mainly present in the lamination of the IPL at PE7. MAGL is expressed in all retinal layers, mainly in the IPL and OPL from E9 to PE7 retina. CB1 and CB2 were found both in neurons and glia cells, but MAGL was only expressed in Müller glia. Older retinas (PE7) show CB1 positive cells mainly in the INL and co-expression of CB1 and tyrosine hydroxylase (TH) are shown in a few cells when both systems are mature. CB1 co-localized with TH and was heavily associated to D1 receptor labeling in primary cell cultures. Finally, cyclic AMP (cAMP) was activated by the selective D1 agonist SKF38393, and inhibited when cultures were treated with WIN55, 212–2 (WIN) in a CB1 dependent manner. The results suggest a correlation between the endocannabinoid and dopaminergic systems (DSs) during the avian retina development. Activation of CB1 limits cAMP accumulation via D1 receptor activation and may influence embryological parameters during avian retina differentiation.

Experimental ischemia/reperfusion model impairs endocannabinoid signaling and Na+/K+ ATPase expression and activity in kidney proximal tubule cells

Graphical abstract Figure. No Caption available. ABSTRACT LLC‐PK1 cells, an immortalized epithelial cell line derived from pig renal proximal tubules, express all the major players of the endocannabinoid system (ECS) such as CB1, CB2 and TRPV1 receptors, as well as the main enzymes involved in the biosynthesis and degradation of the major endocannabinoids named 2‐arachidonoylglycerol, 2‐AG and anandamide, AEA. Here we investigated whether the damages caused by ischemic insults either in vitro using LLC‐PK1 cells exposed to antimycin A (an inductor of ATP‐depletion) or in vivo using Wistar rats in a classic renal ischemia and reperfusion (IR) protocol, lead to changes in AEA and 2‐AG levels, as well as altered expression of genes from the main enzymes involved in the regulation of the ECS. Our data show that the mRNA levels of the CB1 receptor gene were downregulated, while the transcript levels of monoacylglycerol lipase (MAGL), the main 2‐AG degradative enzyme, were upregulated in LLC‐PK1 cells after IR model. Accordingly, IR was accompanied by a significant reduction in the levels of 2‐AG and AEA, as well as of the two endocannabinoid related molecules, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) in LLC‐PK1 cells. In kidney cortex homogenates, only AEA levels were significantly decreased. In addition, we found that in both the in vitro and in vivo model IR caused a reduction in the expression and activity of the Na+/K+ ATPase. These changes were reversed by the CB1/CB2 agonist WIN55,212, in a CB1‐receptor dependent manner in the LLC‐PK1 IR model. In conclusion, the ECS and Na+/K+ ATPase are down‐regulated following IR in LLC‐PK1 cells and rat kidney. We suggest that CB1 agonists might represent a potential strategy to reverse the consequences of IR injury in kidney tissues.

In vitro effect of isoschaftoside isolated from Syngonium podophyllum on pig kidney Na+, K+-ATPase

The present study aimed to investigate the in vitro effects of isoschaftoside isolated from Syngonium podophyllum on pig kidney Na+,K+-ATPase. The Na+, K+-ATPase activity was determined by colorimetric measurement of inorganic phosphate (Pi), resulting from ATP hydrolysis. Isoschaftoside significantly decreased the renal Na+, K+-ATPase activity at the highest concentration as well as at a lower concentration. Our work suggests that isoschaftoside is a promising compound for the treatment of hypertension.