Hemopressin as a breakthrough for the cannabinoid field.

Hemopressin (PVNFKFLSH in rats, and PVNFKLLSH in humans and mice), a fragment derived from the α-chain of hemoglobin, was the first peptide described to have type 1 cannabinoid receptor activity. While hemopressin was shown to have inverse agonist/antagonistic activity, extended forms of hemopressin (i.e. RVD-hemopressin, also called pepcan-12) exhibit type 1 and type 2 cannabinoid receptor agonistic/allosteric activity, and recent studies suggest that they can activate intracellular mitochondrial cannabinoid receptors. Therefore, hemopressin and hemopressin-related peptides could have location-specific and biased pharmacological action, which would increase the possibilities for fine-tunning and broadening cannabinoid receptor signal transduction. Consistent with this, hemopressins were shown to play a role in a number of physiological processes including antinociceptive and anti-inflammatory activity, regulation of food intake, learning and memory. The shortest active hemopressin fragment, NFKF, delays the first seizure induced by pilocarpine, and prevents neurodegeneration in an experimental model of autoimmune encephalomyelitis. These functions of hemopressins could be due to engagement of both cannabinoid and non-cannabinoid receptor systems. Self-assembled nanofibrils of hemopressin have pH-sensitive switchable surface-active properties, and show potential as inflammation and cancer targeted drug-delivery systems. Upon disruption of the self-assembled hemopressin nanofibril emulsion, the intrinsic analgesic and anti-inflammatory properties of hemopressin could help bolster the therapeutic effect of anti-inflammatory or anti-cancer formulations. In this article, we briefly review the molecular and behavioral pharmacological properties of hemopressins, and summarize studies on the intricate and unique mode of generation and binding of these peptides to cannabinoid receptors. Thus, the review provides a window into the current status of hemopressins in expanding the repertoire of signaling and activity by the endocannabinoid system, in addition to their new potential for pharmaceutic formulations.

Hemopressin as a breakthrough for the cannabinoid field

Hemopressin (PVNFKFLSH in rats, and PVNFKLLSH in humans and mice), a fragment derived from the α-chain of hemoglobin, was the first peptide described to have type 1 cannabinoid receptor activity. While hemopressin was shown to have inverse agonist/antagonistic activity, extended forms of hemopressin (i.e. RVD-hemopressin, also called pepcan-12) exhibit type 1 and type 2 cannabinoid receptor agonistic/allosteric activity, and recent studies suggest that they can activate intracellular mitochondrial cannabinoid receptors. Therefore, hemopressin and hemopressin-related peptides could have location-specific and biased pharmacological action, which would increase the possibilities for fine-tunning and broadening cannabinoid receptor signal transduction. Consistent with this, hemopressins were shown to play a role in a number of physiological processes including antinociceptive and anti-inflammatory activity, regulation of food intake, learning and memory. The shortest active hemopressin fragment, NFKF, delays the first seizure induced by pilocarpine, and prevents neurodegeneration in an experimental model of autoimmune encephalomyelitis. These functions of hemopressins could be due to engagement of both cannabinoid and non-cannabinoid receptor systems. Self-assembled nanofibrils of hemopressin have pH-sensitive switchable surface-active properties, and show potential as inflammation and cancer targeted drug-delivery systems. Upon disruption of the self-assembled hemopressin nanofibril emulsion, the intrinsic analgesic and anti-inflammatory properties of hemopressin could help bolster the therapeutic effect of anti-inflammatory or anti-cancer formulations. In this article, we briefly review the molecular and behavioral pharmacological properties of hemopressins, and summarize studies on the intricate and unique mode of generation and binding of these peptides to cannabinoid receptors. Thus, the review provides a window into the current status of hemopressins in expanding the repertoire of signaling and activity by the endocannabinoid system, in addition to their new potential for pharmaceutic formulations.

Capsaicin inhibits lipopolysaccharide-induced adrenal steroidogenesis by raising intracellular calcium levels.

INTRODUCTION: Glucocorticoid release by adrenals has been described as significant to survive sepsis. The activation of transient receptor potential vanilloid type 1 (TRPV1) inhibited ACTH-induced glucocorticoid release by adrenal glands in vitro. OBJECTIVE: The aim of this study was to investigate if capsaicin, an activator of TRPV1, would prevent LPS-induced glucocorticoid production by adrenals. METHODS: Male Swiss-Webster mice were treated with capsaicin intraperitoneally (0.2 or 2 mg/kg) 30 min before LPS injection. All analyses were performed 2 h after the LPS stimulation, including plasma corticosterone and peritoneal IL-1ß and TNF-α levels. Furthermore, murine adrenocortical Y1 cells were used to assess the effects of capsaicin on LPS-induced corticosterone production in vitro. RESULTS: Capsaicin (2 mg/kg, i.p.) significantly reduced plasma corticosterone levels and adrenal hypertrophy induced by LPS without alter the levels of pro-steroidogenic cytokines IL-1ß and TNF-α in peritoneal cavity of mice, while the dose of 0.2 mg/kg of capsaicin did not interfere with adrenal steroidogenesis, attested by RIA and ELISA, respectively. Y1 cells express TRPV1, measured by immunofluorescence and western blot, and capsaicin decreased LPS-induced corticosterone production by these cells in vitro. Capsaicin also induces calcium mobilization in Y1 cells in vitro. CONCLUSIONS: These findings suggest that capsaicin inhibits corticosterone production induced by LPS by acting directly on adrenal cells producing glucocorticoids, in a mechanism probably associated with induction of a cytoplasmic calcium increase in these cells.

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

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.

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

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.

P2X7 receptor large pore signaling in avian Müller glial cells.

ATP is a pleiotropic molecule that promotes extra- and intracellular signaling to regulate numerous functions. This nucleotide activates purine and pyrimidine receptors at the plasma membrane, categorized as ionotropic P2X or G-protein-coupled receptor (GPCR) P2Y receptors. P2X are ligand-gated ion channel receptors, expressed in both retinal neurons and Müller cells leading to neuron-glia communication, calcium waves and neurovascular coupling. However, how P2X pore is formed upon ATP activation and how signaling pathways regulates the complex is still a matter of controversy. Here we studied the properties of the P2X7 receptor (P2X7R) using electrophysiology, single cell Ca2+ imaging, and dye uptake assay in purified avian Müller glia in culture. Our data show that ATP (or benzoyl-benzoyl ATP, BzATP) evoked large inward currents in patch-clamp studies while addition of P2X7R antagonist such as brilliant Blue G (BBG), abolished these currents. Ruthenium red (RU-2), a general transient receptor potential (TRP) inhibitor, reduced currents induced by ATP. Our data also point to the involvement of mitogen activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), Ca2+-calmodulin kinase II (CAMKII), microtubules or protein kinase C (PKC) modulating ATP-induced ionic current in Müller cells. We show that ATP induced Ca2+ influx, partially inhibited by P2X7R antagonists (oxidized ATP or BBG), and totally inhibited by blockers of other pores such as transient receptor potential (TRPs) or connexin hemichannel. Additionally, MAPK, PKC, PI3K or CAMKII inhibitors also are involved in the modulation of intracellular calcium signaling. Finally, ATP induced 80-90% of dye uptake in Muller glia cells, while oxidized ATP (oATP), BBG or A740003 inhibited this effect. We conclude that large conductance channel and other P2XRs are not involved in the ATP-induced dye uptake, but signaling pathways such as MAPK, PI3-K, microtubules or PKC are involved in pore formation.

P2X7R large pore is partially blocked by pore forming proteins antagonists in astrocytes.

The ATP-gated P2X7R (P2X7R) is a channel, which is involved in events, such as inflammation, cell death, and pain. The most intriguing event concerning P2X7R functions is the phenomenon of pore dilation. Once P2X7R is activated, the permeability of the plasma membrane becomes higher, leading to the permeation of 1000 Da-weight solutes. The mechanisms involved in this process remain unclear. Nevertheless, this event is not exclusively through P2X7R, as other proteins may form large pores in the plasma membrane. Recent evidence concerning pore formation reveals putative P2X7R and other pores-associated protein complexes, revealing cross-interactive pharmacological and biophysical issues. In this work, we showed results that corroborated with cross-interactive aspects with P2X7R and pores in astrocytes. These cells expressed most of the pores, including P2X7R. We discovered that different pore types open with peculiar characteristics, as both anionic and cationic charged solutes permeate the plasma membrane, following P2X7R activation. Moreover, we showed that both synergic and additive relationships are found within P2X7, cationic, and anionic large pores. Therefore, our data suggest that other protein-related pores are assembled following the formation of P2X7R pore.

Functional plasticity of GAT-3 in avian Müller cells is regulated by neurons via a glutamatergic input.

GABA (γ-amino butyric acid) is the major inhibitory transmitter in the central nervous system and its action is terminated by specific transporters (GAT), found in neurons and glial cells. We have previously described that GAT-3 is responsible for GABA uptake activity in cultured avian Müller cells and that it operates in a Na(+) and Cl(-) dependent manner. Here we show that glutamate decreases [(3)H] GABA uptake in purified cultured glial cells up to 50%, without causing cell death. This effect is mediated by ionotropic glutamatergic receptors. Glutamate inhibition on GABA uptake is not reverted by inhibitors of protein kinase C or modified by agents that modulate cyclic AMP/PKA. Biotinylation experiments demonstrate that this reduction in GABA uptake correlates with a decrease in GAT-3 plasma membrane levels. Interestingly, both GAT-1 and GAT-3 mRNA levels are also decreased by glutamate. Conditioned media (CM) prepared from retinal neurons could also decrease GABA influx, and glutamate receptor antagonists (MK-801 + CNQX) were able to prevent this effect. However, glutamate levels in CM were not different from those found in fresh media, indicating that a glutamatergic co-agonist or modulator could be regulating GABA uptake by Müller cells in this scenario. In the whole avian retina, GAT-3 is present from embryonic day 5 (E5) increasing up to the end of embryonic development and post-hatch period exclusively in neuronal layers. However, this pattern may change in pathological conditions, which drive GAT-3 expression in Müller cells. Our data suggest that in purified cultures and upon extensive neuronal lesion in vivo, shown as a Brn3a reduced neuronal cells and an GFAP increased gliosis, Müller glia may change its capacity to take up GABA due to GAT-3 up regulation and suggests a regulatory interplay mediated by glutamate between neurons and glial cells in this process.

Bone marrow-derived fibroblast growth factor-2 induces glial cell proliferation in the regenerating peripheral nervous system.

BACKGROUND: Among the essential biological roles of bone marrow-derived cells, secretion of many soluble factors is included and these small molecules can act upon specific receptors present in many tissues including the nervous system. Some of the released molecules can induce proliferation of Schwann cells (SC), satellite cells and lumbar spinal cord astrocytes during early steps of regeneration in a rat model of sciatic nerve transection. These are the major glial cell types that support neuronal survival and axonal growth following peripheral nerve injury. Fibroblast growth factor-2 (FGF-2) is the main mitogenic factor for SCs and is released in large amounts by bone marrow-derived cells, as well as by growing axons and endoneurial fibroblasts during development and regeneration of the peripheral nervous system (PNS). RESULTS: Here we show that bone marrow-derived cell treatment induce an increase in the expression of FGF-2 in the sciatic nerve, dorsal root ganglia and the dorsolateral (DL) region of the lumbar spinal cord (LSC) in a model of sciatic nerve transection and connection into a hollow tube. SCs in culture in the presence of bone marrow derived conditioned media (CM) resulted in increased proliferation and migration. This effect was reduced when FGF-2 was neutralized by pretreating BMMC or CM with a specific antibody. The increased expression of FGF-2 was validated by RT-PCR and immunocytochemistry in co-cultures of bone marrow derived cells with sciatic nerve explants and regenerating nerve tissue respectivelly. CONCLUSION: We conclude that FGF-2 secreted by BMMC strongly increases early glial proliferation, which can potentially improve PNS regeneration.

Secreted human amyloid precursor protein binds semaphorin 3a and prevents semaphorin-induced growth cone collapse.

The amyloid precursor protein (APP) is well known for giving rise to the amyloid-ß peptide and for its role in Alzheimer's disease. Much less is known, however, on the physiological roles of APP in the development and plasticity of the central nervous system. We have used phage display of a peptide library to identify high-affinity ligands of purified recombinant human sAPPα(695) (the soluble, secreted ectodomain from the main neuronal APP isoform). Two peptides thus selected exhibited significant homologies with the conserved extracellular domain of several members of the semaphorin (Sema) family of axon guidance proteins. We show that sAPPα(695) binds both purified recombinant Sema3A and Sema3A secreted by transfected HEK293 cells. Interestingly, sAPPα(695) inhibited the collapse of embryonic chicken (Gallus gallus domesticus) dorsal root ganglia growth cones promoted by Sema3A (K(d)≤8·10(-9) M). Two Sema3A-derived peptides homologous to the peptides isolated by phage display blocked sAPPα binding and its inhibitory action on Sema3A function. These two peptides are comprised within a domain previously shown to be involved in binding of Sema3A to its cellular receptor, suggesting a competitive mechanism by which sAPPα modulates the biological action of semaphorins.

Plasticidade sináptica: natureza e cultura moldando o Self / Synaptic plasticity: nature and nurture shaping the self

O cérebro em desenvolvimento segue inicialmente um plano genético, estabelecido pela história evolutiva da espécie humana, porém é muito sensível ao ambiente. Estímulos ambientais modificam a estrutura dos circuitos neurais, refinando e tornando as sinapses, alvo de ação dos neurotransmissores, mais eficientes por meio de atividade elétrica e mensageiros químicos. O objetivo desse estudo é discutir teoricamente como a plasticidade e a especificação prévia de sistemas cerebrais coexistem no cérebro. Conclui-se destacando a importância de integrar aspectos de aprendizagem social e da biologia na construção e refinamento das variadas habilidades humanas.

The developing brain requires a genetic plan, established by the evolutionary history of human species, but it is also very sensitive to the surrounding environment. Environmental stimuli can modify the structure of neural circuits, refining and making synapses, which are the target of neurotransmitters, more efficient by electrical activity and chemical messengers. The aim of the present study is to discuss theoretically how both plasticity and previous specification of brain systems coexist in the brain. The conclusion shows the importance to integrate aspects of social learning and biology in building and refining the various humane skills.

Plasticidade sináptica: natureza e cultura moldando o Self

O cérebro em desenvolvimento segue inicialmente um plano genético, estabelecido pela história evolutiva da espécie humana, porém é muito sensível ao ambiente. Estímulos ambientais modificam a estrutura dos circuitos neurais, refinando e tornando as sinapses, alvo de ação dos neurotransmissores, mais eficientes por meio de atividade elétrica e mensageiros químicos. O objetivo desse estudo é discutir teoricamente como a plasticidade e a especificação prévia de sistemas cerebrais coexistem no cérebro. Conclui-se destacando a importância de integrar aspectos de aprendizagem social e da biologia na construção e refinamento das variadas habilidades humanas.(AU)

The developing brain requires a genetic plan, established by the evolutionary history of human species, but it is also very sensitive to the surrounding environment. Environmental stimuli can modify the structure of neural circuits, refining and making synapses, which are the target of neurotransmitters, more efficient by electrical activity and chemical messengers. The aim of the present study is to discuss theoretically how both plasticity and previous specification of brain systems coexist in the brain. The conclusion shows the importance to integrate aspects of social learning and biology in building and refining the various humane skills.(AU)

Expression of functional dopaminergic phenotype in purified cultured Müller cells from vertebrate retina.

Purified retina glial Müller cells can express the machinery for dopamine synthesis and release when maintained in culture. Dopamine is detected in cell extracts of cultures exposed to its precursor, L-DOPA. A large portion of synthesized dopamine is recovered in the superfusing medium showing the tendency of the accumulated dopamine to be released. Müller cells purified from developing chick and mouse retinas express L-DOPA decarboxylase (DDC; aromatic-L-amino-acid decarboxylase; EC 4.1.1.28) and the dopamine transporter DAT. The synthesis of dopamine from L-DOPA supplied to Müller cultures is inhibited by m-hydroxybenzylhydrazine, a DDC inhibitor. Dopamine release occurs via a transporter-mediated process and can activate dopaminergic D(1) receptors expressed by the glia population. The synthesis and release of dopamine were also observed in Müller cell cultures from mouse retina. Finally, cultured avian Müller cells display increased expression of tyrosine hydroxylase, under the influence of agents that increase cAMP levels, which results in higher levels of dopamine synthesized from tyrosine. A large proportion of glial cells in culture do express Nurr1 transcription factor, consistent with the dopaminergic characteristics displayed by these cells in culture. The results show that Müller cells, deprived of neuron influence, differentiate dopaminergic properties thought to be exclusive to neurons.

Dopaminergic signaling in the developing retina.

The role of dopamine in the retina has been studied for the last 30 years and there is now increasing evidence that dopamine is used as a developmental signal in the embryonic retina. Dopamine is the main catecholamine found in the retina of most species, being synthesized from the L-amino acid tyrosine. Its effects are mediated by G protein coupled receptors constituting the D(1) (D(1) and D(5)) and D(2) (D(2), D(3) and D(4)) receptor subfamilies that can be coupled to adenylyl cyclase in opposite manners. Dopamine-mediated cyclic AMP (cAMP) accumulation, via D(1)-like receptors, is observed very early during retina ontogeny, before synaptogenesis and, in some species, before the expression of tyrosine hydroxylase (TH), the enzyme that characterizes the neuronal dopaminergic phenotype. D(2)-like receptors appear in the tissue days after D(1)-like activity is detected. In the embryonic avian retina, before the tissue is capable of synthesizing its own dopamine via TH, dopamine synthesis is observed from L-DOPA supplied to the neuroretina from retina pigmented epithelium which results in dopaminergic communication in the embryonic tissue before TH expression. Müller cells, the main glia type found in the retina, seem to actively contribute to dopaminergic activity in the retinal tissue. Understanding the dopaminergic role during retina development may contribute to novel strategies approaching certain visual dysfunctions such as those found in ocular albinism.

Regulation of acetylcholine synthesis and storage.

Acetylcholine is one of the major modulators of brain functions and it is the main neurotransmitter at the peripheral nervous system. Modulation of acetylcholine release is crucial for nervous system function. Moreover, dysfunction of cholinergic transmission has been linked to a number of pathological conditions. In this manuscript, we review the cellular mechanisms involved with regulation of acetylcholine synthesis and storage. We focus on how phosphorylation of key cholinergic proteins can participate in the physiological regulation of cholinergic nerve-endings.

Sympathetic neuronal survival induced by retinal trophic factors.

Neuronal survival in the vertebrate peripheral nervous system depends on neurotrophic factors available from target tissues. In an attempt to identify novel survival factors, we have studied the effect of secreted factors from retinal cells on the survival of chick sympathetic ganglion neurons. Embryonic day 10 sympathetic neurons undergo programmed cell death after 48 h without appropriate levels of nerve growth factor (NGF). Retina Conditioned Media (RCM) from explants of embryonic day 11 retinas maintained for 4 days in vitro supported 90% of E10 chick sympathetic neurons after 48 h. Conditioned medium from purified chick retinal Muller glial cells supported nearly 100% of E10 chick sympathetic neurons. Anti-NGF (1 microg/mL) blocked the survival effect of NGF, but did not block the trophic effect of RCM. Neither BDNF nor NT4 (0.1-50 ng/mL) supported E10 sympathetic neuron survival. Incubation of chimeric immunoglobulin-receptors TrkA, TrkB, or TrkC had no effect on RCM-induced sympathetic neuron survival. The survival effects were not blocked by anti-GDNF, anti-TGFbeta, and anti-CNTF and were not mimicked by FGFb (0.1-10 nM). LY294002 at 50 microM, but not PD098059 blocked sympathetic survival induced by RCM. Further, the combination of RCM and NGF did not result in an increase in neuronal survival compared with NGF alone (82% survival after 48 h). The secreted factor in RCM is retained in subfractions with a molecular weight above 100 kDa, binds to heparin, and is unaffected by dialysis, but is heat sensitive. Our results indicate the presence of a high-molecular weight retinal secreted factor that supports sympathetic neurons in culture.